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WO2021013061A1 - Anticorps anti-vegfr2 humanisé et son utilisation - Google Patents

Anticorps anti-vegfr2 humanisé et son utilisation Download PDF

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Publication number
WO2021013061A1
WO2021013061A1 PCT/CN2020/102556 CN2020102556W WO2021013061A1 WO 2021013061 A1 WO2021013061 A1 WO 2021013061A1 CN 2020102556 W CN2020102556 W CN 2020102556W WO 2021013061 A1 WO2021013061 A1 WO 2021013061A1
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antibody
vegfr2
antigen
binding fragment
seq
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Chinese (zh)
Inventor
谢良志
孙春昀
郭二红
代雅琪
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Sinocelltech Ltd
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Sinocelltech Ltd
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Priority to CN202080045497.2A priority Critical patent/CN114341170B/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K14/475—Growth factors; 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the invention belongs to the field of tumor immunotherapy, and relates to a humanized anti-VEGFR2 antibody and its application.
  • Vascular endothelial growth factor receptor 2 (vascular endothelial growth factor receptor, VEGFR2, also known as FLK-1 or KDR), is mainly distributed on the surface of the endothelial cell membrane of blood vessels and lymphatic vessels. It is a variety of vascular endothelial growth factors. VEGF, including the main functional receptors of VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E), can induce the proliferation and migration of endothelial cells and changes in vascular permeability, and promote new blood vessels Form and maintain its integrity. VEGFR2 is the main member of the VEGFR family and belongs to the type III receptor tyrosine kinase.
  • VEGFR2 The extracellular region of VEGFR2 contains 7 immunoglobulin-like domains (ie D1-D7), including the ligand VEGF binding domain (D2-D3). ) And dimerization domains (D4-D5 and D7), the intracellular region contains tyrosine kinase domain [1, 19-20].
  • VEGFR2 ligand VEGF (including VEGF-A, C, D, E, etc.), the concentration in normal human plasma is about 137 ⁇ 7.7pg/mL[2], while the concentration of VEGF in serum of patients with pancreatic cancer and ovarian cancer Significantly up-regulated, 200-400pg/mL, while the concentration of VEGF in tumor tissue or tumor cyst effusion is as high as 0.5-20ng/mL[2-4].
  • 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[5 ].
  • PLC phospholipase C
  • Drugs that target angiogenesis can block the nutrient supply of tumors, thereby achieving the effect of "starving" the tumor.
  • VEGFR2 antibody drugs can block the binding of VEGFR2 to VEGF.
  • the first anti-VEGFR2 tumor treatment drug ramucirumab (Cyramza) developed by Eli Lilly can inhibit angiogenesis and reduce vascular communication. Permeability, the tumor microenvironment can be rebalanced in a short time, so as to obtain a good anti-tumor effect [6].
  • ramucirumab has been used in gastric/gastroesophageal junction cancer after chemotherapy/radiotherapy failure [7], non-small cell lung cancer after chemotherapy failure [8], chemotherapy/radiotherapy combined with Avastin (VEGF antibody) failure
  • Colorectal cancer [9] and metastatic breast cancer [10] have shown good therapeutic effects in a variety of tumors, especially in the second-line treatment of gastric cancer, showing better efficacy than the VEGF monoclonal antibody Avastin.
  • 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 [11], 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.
  • the drug mainly inhibits VEGF-A and VEGFR2.
  • This mechanism is limited in its tumor suppressor effect at high VEGF concentrations.
  • the antibody disclosed in the present invention binds to the sixth domain of VEGFR2 independently of the ligand VEGF-A. Play a role, can directly inhibit the formation of dimers of VEGFR2, thereby blocking the phosphorylation of tyrosine residues and downstream signal pathways caused by the dimerization of the intracellular region of VEGFR2.
  • the antibody also retains the ability to partially block VEGF-C and VEGF-D, so it has a better inhibitory effect on 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 an amino acid sequence shown in SEQ ID NO: 14
  • the heavy chain CDR2 domain and the heavy chain variable region 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 shown in SEQ ID NO: 10 have SEQ ID The light chain CDR2 domain of the amino acid sequence shown in NO: 11 and the light chain variable region of the light chain CDR3 domain having the amino acid sequence of SEQ ID NO: 12.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein comprises an amino acid sequence as shown in SEQ ID NO: 8 or at least 90%, 92%, 95%, 98% with SEQ ID NO: 8
  • the variable region of the heavy chain with an amino acid sequence of% or 99% sequence identity and an amino acid sequence as shown in SEQ ID NO: 9 or at least 90%, 92%, 95%, 98% or at least with SEQ ID NO: 9 The light chain variable region of an amino acid sequence with 99% sequence identity.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein is a humanized antibody or a chimeric antibody.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein has an amino acid sequence as shown in SEQ ID NO: 22 or at least 85%, 90%, 95% or 99% with SEQ ID NO: 22.
  • the antibody described herein 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 An amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 25, and/or the heavy chain constant region is an IgG1 with the amino acid sequence of SEQ ID NO: 24 The amino acid sequence of the heavy chain constant region or the amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 24.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein is an IgG antibody, preferably an IgG1 antibody.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein is a monoclonal antibody.
  • the binding affinity K D of the anti-VEGFR2 antibody or antigen-binding fragment thereof to recombinant human VEGFR2 protein is 5-150 pM, preferably 10-70 pM, and more preferably 38.8 pM.
  • the antigen-binding fragments described herein are Fv, Fab, Fab', Fab'-SH, F(ab')2, Fd fragment, Fd' fragment, single chain antibody molecule or single domain antibody; wherein
  • the single chain antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  • the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein has a binding epitope of Y565, L567, V576, L579, W591, K592, D620, V622, V637, R638 of the VEGFR2 molecule.
  • 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 as described above.
  • the present invention provides an antibody-drug conjugate comprising the anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein and another therapeutic agent, preferably the anti-VEGFR2 antibody or antigen-binding fragment thereof It is connected with another therapeutic agent through a connector.
  • the present invention provides a nucleic acid encoding the anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein.
  • the nucleic acid described herein 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: The nucleotide sequence shown in 30 and/or the nucleotide sequence shown in SEQ ID NO: 31.
  • the present invention provides an expression vector comprising a nucleic acid as described herein.
  • the present invention provides a host cell comprising a nucleic acid as described herein or an expression vector as described herein.
  • the present invention provides a method for producing an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein, comprising culturing a host cell as described herein under conditions suitable for antibody expression, and from culturing The expressed antibody is recovered from the base.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein or an antibody-drug conjugate as described herein or a nucleic acid as described herein or as described herein Said expression vector and pharmaceutically acceptable carrier.
  • the present invention provides the anti-VEGFR2 antibody or antigen-binding fragment thereof described herein or the antibody-drug conjugate described herein or the pharmaceutical composition described herein for use in the treatment of melanoma.
  • the present invention provides a method for treating melanoma, which comprises administering to a subject in need a therapeutically effective amount of an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein or An antibody-drug conjugate or a pharmaceutical composition as described herein to treat the melanoma.
  • the present invention provides that the anti-VEGFR2 antibody or antigen-binding fragment or antigen-binding fragment thereof described herein or the antibody-drug conjugate described herein or the pharmaceutical composition described herein are prepared for the treatment of melanin. Tumor use in medicine.
  • the present invention provides a pharmaceutical combination comprising an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein or an antibody-drug conjugate as described herein or as described herein
  • the pharmaceutical composition of and one or more additional therapeutic agents comprising an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein or an antibody-drug conjugate as described herein or as described herein.
  • the present invention provides a kit comprising an anti-VEGFR2 antibody or antigen-binding fragment thereof as described herein or an antibody-drug conjugate as described herein or as described herein
  • the pharmaceutical composition preferably further comprises a device for administration.
  • the present invention provides an epitope of a VEGFR2 molecule, which is Y565, L567, V576, L579, W591, K592, D620, V622, V637, R638 of the VEGFR2 molecule.
  • 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 does not block the binding of recombinant human VEGFR2-His to human VEGF-165 recombinant protein.
  • Figure 4 shows that the anti-VEGFR2 chimeric antibody blocks the proliferation effect of VEGF-165 on HUVEC cells.
  • Figure 5 shows the binding of VEGFR2-HS76 to recombinant human VEGFR2-His detected by ELSIA.
  • FIG. 6 shows the binding of VEGFR2-HS76 on the 293FT-VEGFR2 cell line detected by FACS.
  • Figure 7 shows the cross-binding of VEGFR2-HS76 species detected by ELISA.
  • Figure 8 shows that the detection of VEGFR2-HS76 by FACS does not block the binding of VEGF-165 to 293FT-VEGFR2 cells.
  • Figure 9 shows that VEGFR2-HS76 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-C detected by ELISA.
  • FIG. 10 shows that VEGFR2-HS76 blocked the binding of VEGFR2-Fc recombinant protein to VEGF-D detected by ELISA.
  • FIG. 11-1 shows that VEGFR2-HS76 blocks the proliferation effect of high concentration VEGF-165 on HUVEC cells.
  • FIG. 11-2 shows that VEGFR2-HS76 blocks the proliferation of HUVEC cells at low concentrations of VEGF-165.
  • FIG. 12 shows that VEGFR2-HS76 blocks the proliferation effect of VEGF-C on HUVEC cells.
  • Figure 13 shows the ADCC effect of VEGFR2-HS76 detected by CD16A recombinant reporter gene.
  • Figure 14 shows the effect of VEGFR2-HS76 on the body weight of B16-F1 melanoma transplanted C57BL/6J mice.
  • Figure 15 shows the effect of VEGFR2-HS76 on the tumor volume of B16-F1 melanoma transplanted in C57BL/6J mice.
  • Figure 16-1 shows the serum drug concentration-time curve of a single intravenous injection of VEGFR2-HS76 in CD1 mice.
  • Figure 16-2 shows the serum drug concentration-time curve of a single subcutaneous injection of VEGFR2-HS76 in CD1 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 its antigen-binding fragment, a pharmaceutical composition comprising the anti-VEGFR2 antibody or its antigen-binding fragment, and the use of the anti-VEGFR2 antibody or its antigen-binding fragment to treat melanoma The method and the pharmaceutical combination and kit containing the anti-VEGFR2 antibody or 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 may 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 antibodies of non-human origin 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 a 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 the target antigen, such as a tumor-associated polypeptide antigen target (herein, PD-1), that is, binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent to target the expression of the Cells or tissues of the antigen, and have no significant cross-reactivity with other proteins or with the exception of homologs and variants (such as mutant forms, splice variants, or truncated forms of proteolysis) of the antigen target mentioned above There is no significant cross-reaction of the protein.
  • PD-1 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 within 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 first uses recombinant human VEGFR2 protein to immunize mice, and then obtains the high binding force scFv antibody clone VEGFR2-MS76 that specifically binds to recombinant human VEGFR2 protein through phage display library screening. Afterwards, PCR was used to insert the nucleotide sequences encoding the heavy chain and light chain variable regions of the VEGFR2-MS76 scFv antibody into the pSTEP2 vector with the human IgG1 heavy chain constant region or human kappa light chain constant region nucleotide sequence. Perform culture expression. Purified by a protein A purification column to obtain high-purity human-mouse chimeric antibody VEGFR2-mhS76. The ELISA test showed that the anti-VEGFR2 chimeric antibody has a good binding to recombinant protein VEGFR2-His, and it has a strong blocking effect of VEGF-165 on HUVEC cell proliferation.
  • 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 for maintaining the stability of the CDR spatial structure and antibody activity, the key points were backmutated to the sequence of the mouse antibody to finally obtain the humanized antibody VEGFR2-HS76.
  • 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 36-37.
  • 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-HS76 The characteristics and functions of the humanized VEGFR2-HS76 antibody of the present invention were analyzed. The analysis results show that the antibody of the present invention has the following advantages: (1) The ability of VEGFR2-HS76 to bind recombinant human VEGFR2-His protein is significantly higher than that of ramucirumumab; the binding of VEGFR2-HS76 antibody on 293FT-VEGFR2 cells is slightly better VEGFR2-HS76 has a maximum binding MFI of 1.3 times that of ramucirumab; VEGFR2-HS76 has a strong ability to bind recombinant human VEGFR2 protein and has a higher affinity than ramucirumab; (implementation) Example 4.1) (2) VEGFR2-HS76 retains the ability to partially block the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D.
  • VEGFR2-HS76 has the same effect on blocking the proliferation of HUVEC cells by VEGF-C.
  • Murcirumab is equivalent; (4) VEGFR2-HS76 is significantly better than ramucirumab in blocking the effects of high concentrations of VEGF-165 (100ng/mL, 1000ng/mL) on the proliferation of HUVEC cells; (Example 4.3 (5) The ability of ramucirumab to mediate ADCC effect is weak, while VEGFR2-HS76 has strong ADCC effect; (Example 4.4) (6) B16-F1 melanoma subcutaneously in C57BL/6J mice The transplantation model showed obvious tumor suppressive effect; (Example 5) and (7) VEGFR2-HS76 antibody has weak immunogenicity in CD-1 mice. (Example 6)
  • 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.
  • Recombinant human VEGFR2 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.10012-H08H) was used to immunize Balb/c mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.).
  • the amino acid sequence of the extracellular region of the recombinant human VEGFR2 protein (UniProt KB-P35968) is Met1-Glu764 (SEQ ID NO: 1).
  • the mouse spleen tissue was extracted with TriPure Isolation Reagent reagent (Roche, Cat. No. 11 667 165 001), and the RNA was reverse transcribed using reverse transcription reagent to obtain a cDNA library.
  • the overlap extension PCR method [13-14] was used to amplify the light chain variable region sequence of the mouse antibody with two pairs of primers in the reference, and another pair of primers was used to amplify the heavy chain variable region sequence.
  • the nucleotide sequence of the variable region sequence of the chain and the heavy chain is spliced into a nucleotide sequence encoding the scFv.
  • variable region of the light and heavy chain is connected by a linker (SEQ ID NO: 2), and the resulting DNA fragment is subjected to the restriction enzyme Sfi After digestion with enzyme I, it was connected to the phage vector pComb3x (Beijing Yiqiao Shenzhou Technology Co., Ltd.), and electrotransformed with X-Blue competence to obtain the mouse phage display scFv antibody library.
  • phage antibody panning method and process [12] coat the recombinant human VEGFR2 protein on the ELISA plate, add the antibody phage to incubate, wash away the unbound phage, recover the bound phage, repeat this process, and enrich through multiple rounds of screening
  • a phage library of anti-VEGFR2 positive antibodies was obtained.
  • Monoclonal phages were selected from the enriched library for expression, and the binding to recombinant human VEGFR2 protein was detected by ELISA.
  • a high-affinity scFv antibody VEGFR2-MS76 that specifically binds to recombinant human VEGFR2 was screened and obtained by sequencing.
  • the nucleotide sequence of the scFv antibody (SEQ ID NO: 3).
  • the nucleotide sequence of the heavy chain variable region of the VEGFR2-MS76 scFv antibody was amplified by PCR method, and inserted into the signal peptide with human heavy chain signal peptide (SEQ ID NO: 28) and human IgG1 heavy chain constant region (
  • the chimeric antibody heavy chain (SEQ ID NO: 35) expression vector was obtained from the pSTEP2 vector digested with ScaI+NheI (Fermentas) of the nucleotide sequence of SEQ ID NO: 6).
  • the nucleotide sequence of the light chain variable region of the VEGFR2-MS76 scFv antibody was amplified by PCR, and inserted into the coding human light chain signal peptide (SEQ ID NO: 29) and human kappa constant region (SEQ ID NO: 7)
  • the chimeric antibody light chain (SEQ ID NO: 36) expression vector was obtained from the pSTEP2 vector digested with ScaI+BsiWI (Fermentas) of the nucleotide sequence.
  • the chimeric antibody heavy/light chain expression vector plasmid was extracted, and HEK-293 cells were co-transfected for culture and expression for 7 days.
  • the culture supernatant was purified by a protein A purification column to obtain high-purity chimeric antibody VEGFR2-mhS76.
  • Example 2 Function test of VEGFR2 chimeric antibody
  • Recombinant human VEGFR2-His protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.10012-H08H) at a concentration of 2 ⁇ g/mL was coated on a 96-well plate, 100 ⁇ L per well, and coated overnight at 4°C. Wash the plate the next day and block it at room temperature for 1 hour. Add 100 ⁇ L of different concentrations of VEGFR2-mhS76 chimeric antibody or isotype negative control antibody H7N9 (Beijing Yiqiao Shenzhou Technology Co., Ltd.) and incubate for 1 hour. Wash the plate to remove unbound antibody and add goat F.
  • VEGFR2 stable overexpression cell line 293FT-VEGFR2-13-13 (Shenzhou Cell Engineering Co., Ltd., hereinafter referred to as 293FT-VEGFR2) as the experimental material
  • 293FT-VEGFR2 cells were divided into 5 ⁇ 10 5 cells/tube with a volume of 50 ⁇ L, and 10 ⁇ L of different concentrations of VEGFR2-mhS76 or negative control antibody H7N9 were added. After mixing and incubating at 4°C, they were washed with PBS and centrifuged to remove unbound antibodies.
  • VEGF-165 is the most classic splicing body of VEGF-A at a concentration of 0.5 ⁇ g/mL on a 96-well plate, each well 100 ⁇ L, 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 VEGFR2-mhS76 or ramucirumab molecules (positive control antibody, in patent It is described in US2009/0306348 that it can block the binding of VEGF and VEGFR2, which is produced by Beijing Yiqiao Shenzhou Technology Co., Ltd.). The wells where only VEGFR2-His protein is added without antibody are set as positive wells. The plate is washed to remove unbound antibodies.
  • the ramucirumab molecule has a strong ability to block the binding of recombinant human VEGFR2-His protein to human VEGF-165 protein, while VEGFR2-mhS76 does not block VEGF-165 at all.
  • 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 the corresponding concentration of antibody VEGFR2-mhS76 or ramucirumab molecule (Beijing Yiqiao Shenzhou Technology Co., Ltd.) to each well, and then add VEGF-165 with a final concentration of 10ng/mL at a rate of 10 ⁇ L/well, and set detection blank well B( No cells), negative control group M (seeding cells, no sample, plus VEGF-165) and M'(seeding cells, no sample and VEGF-165).
  • 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 antibody VEGFR2-mhS76 has a strong blocking effect of VEGF-165 on the proliferation of HUVEC cells.
  • Example 1.3 the nucleotide sequence of the VEGFR2-MS76 scFv antibody was determined. Based on the nucleotide sequence, the heavy chain and light chain variable region amino acid sequences of the VEGFR2-MS76 scFv antibody were deduced, see SEQ ID NO: 8/ 9. Refer to the Kabat[15] and IMGT[16] numbering methods to determine the amino acid sequence of each of the 3 CDRs of the mouse antibody VEGFR2-MS76 scFv light chain and heavy chain, see Table 1. The above three CDRs of the light chain and the heavy chain were transplanted in the subsequent steps and retained in the finally obtained humanized antibody VEGFR2-HS76, see Examples 3.2 and 3.3.
  • Mouse antibody humanization adopts the classic CDR transplantation humanization method [17-18], the first choice is the human antibody light chain or heavy chain variable region that is closer to the mouse light chain or heavy chain variable region as the template, and the mouse antibody Each of the three CDRs of the light chain or the heavy chain (Table 1) was inserted into the variable region of the human antibody to obtain humanized light chain variable region (VL) and heavy chain variable region (VH) amino acid sequences.
  • VL humanized light chain variable region
  • VH heavy chain variable region
  • the human template of the light chain variable region of VEGFR2-MS76 used was IGKV1-12*01, which had 77.9% homology with the light chain of VEGFR2-MS76, and the human template of the heavy chain variable region was IGHV1-2 *02, the template has 63.3% homology with the heavy chain of VEGFR2-MS76.
  • the key points are backmutated to the corresponding amino acid sequence of the mouse antibody until an antibody with stable spatial structure is obtained.
  • the following mutations were made: According to Kabat numbering, the 11th position of the light chain was backmutated to L, the 42nd position was backmutated to N, the 43th position was backmutated to I, and the 83th position was backmutated to I;
  • the back mutation at position 48 is I
  • the back mutation at position 66 is K
  • the back mutation at position 67 is A
  • the back mutation at position 69 is L.
  • the modified antibody has similar affinities and is fully humanized. Its CDR sequence is completely consistent with the mouse CDR sequence. See Table 1 for details.
  • the heavy chain (SEQ ID NO: 26) expression vector was obtained from the pSTEP2 vector digested with ScaI+NsbI (Fermentas) with the nucleotide sequence of human IgG1 constant region (SEQ ID NO: 32) and human IgG1 constant region: 28).
  • the light chain variable region nucleotide sequence (SEQ ID NO: 31) was obtained by the method of whole gene synthesis using the primers in Table 3, and the in-fusion method was used to insert the nucleotide sequence with the light chain signal peptide (SEQ ID NO: 29) and human kappa constant region nucleotide sequence (SEQ ID NO: 7) were digested with ScaI+BsiWI (Fermentas) pSTEP2 vector to obtain the light chain (SEQ ID NO: 27) expression vector. After plasmid extraction, HEK-293 cells were cotransfected and cultured and expressed for 7 days. The culture supernatant was purified by a protein A purification column to obtain high-purity antibodies.
  • Recombinant human VEGFR2-His protein was coated on a 96-well plate, 100 ⁇ L per well, coated overnight at 4°C.
  • the plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of 2 ⁇ g/mL VEGFR2-HS76, ramucirumab (Eli Lilly, C839381C) and negative control antibody H7N9-R1 were added to incubate for 1 hour, and then the plate was washed to remove unbound Antibody, add the secondary antibody goat anti-human IgG Fc/HRP and incubate the plate repeatedly, wash the plate repeatedly, develop the color with the substrate coloring solution, and read the OD450 by the microplate reader after termination.
  • VEGFR2-HS76 and ramucirumab (Eli Lilly, C839381C) on VEGFR2 overexpressing 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-HS76, 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 The secondary antibody was finally added with 200 ⁇ L PBS to resuspend the cells, filtered with a 400-
  • VEGFR2-HS76 antibody As shown in Figure 6, the binding of VEGFR2-HS76 antibody on 293FT-VEGFR2 cells is slightly better than that of ramucirumab, and the maximum binding MFI of VEGFR2-HS76 is 1.3 times that of ramucirumab.
  • the Octet biomolecule interaction analysis system (model: Octet RED, manufacturer: Fortebio) was used to determine the VEGFR2-HS76 and ramucirumab (0.42nM, 0.90nM, 1.74nM, 3.47nM, 6.94nM) at multiple concentration points ( Eli Lilly, C839381C) and the affinity of biotinylated VEGFR2.
  • Table 4 shows, VEGFR2-HS76 to recombinant human VEGFR2 protein binding affinity KD values 38.8pM, association rate constant k on value of 4.30E + 05M -1 s -1, the dissociation rate constant k off value of 1.67E-05s - 1 .
  • the KD value of ramucirumumab binding to VEGFR2 protein is 45.8pM, the binding rate constant k on value is 3.86E+05M -1 s -1 , the dissociation rate constant k off value is 1.77E-05s -1 , from The results show that VEGFR2-HS76 has a strong ability to bind recombinant human VEGFR2 protein, and its affinity is slightly better than ramucirumab.
  • Recombinant mouse mKDR-His protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat:50998-M08H) at concentrations of 80 ⁇ g/mL and 20 ⁇ g/mL were coated on a 96-well plate, 100 ⁇ L per well, coated at 4°C overnight.
  • VEGFR2-HS76 and ramucirumab molecule (Beijing Yiqiao Shenzhou Technology Co., Ltd.) and incubate for 1 hour, wash the plate to remove unbound antibodies, and add goat anti- After incubating with the human IgG Fc/HRP secondary antibody, wash the plate repeatedly, add the substrate color developing solution for color development, and read the OD 450 with the microplate reader after termination. Take the protein concentration as the abscissa and the OD450 reading as the ordinate, and use GraphPad Prism 6.0 software to make a histogram. The results are shown in Fig. 7 that VEGFR2-HS76 has cross-binding with recombinant mouse mKDR-His protein, but ramucirumab molecule has no cross-binding.
  • VEGFR2-HS76 does not block the binding of VEGF-165 to 293FT-VEGFR2 cells
  • FACS was used to detect the effects of humanized antibodies VEGFR2-HS76 and ramucirumab (Eli Lilly, C839381C) on the binding of VEGFR2 overexpressing cells to recombinant protein VEGF-165.
  • 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-HS76 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 VEGF-165 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd., 11066-H27H-B) and mix and incubate.
  • the MFI sample is the MFI value of VEGF-165 protein binding to VEGFR2 cells after adding antibody. The results are shown in Figure 8.
  • the VEGFR2-HS76 antibody did not block the binding of VEGF-165 protein to VEGFR2 overexpressing cells.
  • VEGFR2-HS76 blocks the binding of VEGFR2 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-His (Beijing Yiqiao Shenzhou Technology Co., Ltd.) protein at a concentration of 0.5 ⁇ g/mL or 2 ⁇ g/mL of recombinant VEGF-D-His (Beijing Yiqiao Shenzhou Technology Co., Ltd.) protein, the protein wells are added at the same time with 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-HS76, ramucirumab (Eli Lilly, C839381C) and the negative control antibody H7N9-R1 were incubated together for 1h, and it was set to add only VEGF-C-His protein
  • the OD sample indicates the OD value of the test group with both VEGFR2-His protein and antibody added.
  • 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. The results are shown in Figure 9 and Figure 10.
  • VEGFR2-HS76 retains the ability to partially block the binding of VEGFR2-Fc recombinant protein to VEGF-C or VEGF-D, and can block 55% of VEGFR2-Fc recombination at an antibody concentration of 9000ng/mL
  • the protein binding to VEGF-C can block 44% of the binding of VEGFR2-Fc recombinant protein to VEGF-D.
  • VEGFR2-HS76 blocks the proliferation of HUVEC cells at different concentrations of VEGF-165 or VEGF-C
  • 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 Concentrations of VEGFR2-HS76, ramucirumab (Eli Lilly, C839381C) and negative control antibody H7N9-R1, then add 10 ⁇ L/well to a final concentration of 100ng/mL, 1000ng/mL VEGF-165 or a final concentration of 1000ng /mL of VEGF-C, set up detection blank well B (no cells), negative control group M (seeding cells, no sample, plus VEGF-165 or VEGF-C) and M'(seeding cells, no sample and VEGF -165 or VEGF-C).
  • 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 11, 12 and Table 5.
  • VEGFR2-HS76 is equivalent to ramucirumab in blocking the proliferation of HUVEC cells by VEGF-C, while blocking high concentrations of VEGF-165 (100ng/mL, 1000ng/mL ) VEGFR2-HS76 is significantly better than ramucirumab on the proliferation of HUVEC cells.
  • the recombinant CD16A reporter gene system method was used to determine the ADCC effect mediated by VEGFR2-HS76.
  • the effector cell is Jurkat-NFAT-Luc2p-CD16A, and the target cell is 293FT-VEGFR2.
  • VEGFR2 is added at the same time.
  • the Fab segment of VEGFR2-HS76 or ramucirumab binds to VEGFR2 overexpressed on the target cell, and its Fc segment can be combined with The effector cells overexpressing the Fc ⁇ III type receptor (CD16A) bind, thereby activating the effector cell Jurkat-NFAT-Luc2p-CD16A and promoting NFAT-RE-mediated bioluminescence.
  • CD16A Fc ⁇ III type receptor
  • 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 different concentrations of antibodies 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).
  • Example 4.2 shows that VEGFR2-HS76 cannot block the binding of ligand VEGF-165 to VEGFR2, indicating that the binding region of VEGFR2-HS76 is different from the ligand VEGF binding epitope.
  • proteins with different domains were designed and transferred to HEK293F cells instantaneously. The supernatant was purified by nickel column affinity chromatography to obtain a protein solution, and VEGFR2-HS76 was detected by ELISA. Binding with proteins of different domains in the extracellular domain of VEGFR2, the results are shown in Table 8.
  • the marketed antibodies ramucirumab and VEGF-A both bind to the domain 2-3 (domain 2-3) of the extracellular domain of VEGFR2, And VEGFR2-HS76 binds to domain 6 (domain 6).
  • VEGFR2-HS76 binds to the extracellular domain of VEGFR2
  • VEGFR2-HS76 In order to further confirm the key sites of VEGFR2-HS76 binding, a series of alanine single-point mutant proteins were designed on the extracellular domain 6 of VEGFR2, and the mutants were transferred to HEK293F cells instantaneously. The supernatant was measured by ELISA for VEGFR2 and The binding ability of VEGFR2-HS76, ramucirumab and ligand VEGF-A, the results showed that the binding of VEGFR2-HS76 and VEGFR2 mutants decreased to varying degrees (see Table 9 for details), while ramucirumumab, VEGFA combined It is not affected.
  • VEGFR2-HS76 binds on the domain 6 of VEGFR2
  • some of the sites that affect the binding are: N561A, K585A, E609A, L624A, K632A.
  • the main dimerization regions of VEGFR2 are the D4-D5 and D7 domains [19-20].
  • VEGFR2-HS76 After VEGFR2-HS76 binds to the D6 domain, it may inhibit the formation of dimers of VEGFR2 through steric hindrance, thereby blocking the phosphoric acid in the intracellular region of VEGFR2.
  • the downstream signal pathway of chemical conduction After VEGFR2-HS76 binds to the D6 domain, it may inhibit the formation of dimers of VEGFR2 through steric hindrance, thereby blocking the phosphoric acid in the intracellular region of VEGFR2.
  • the downstream signal pathway of chemical conduction The downstream signal pathway of chemical conduction.
  • VEGFR2-HS76 binds to alanine mutants in the extracellular region of VEGFR2
  • Example 5 Growth inhibitory effect of VEGFR2-HS76 antibody on B16-F1 melanoma subcutaneous transplantation model in C57BL/6J mice
  • mice Resuspend B16-F1 cells in logarithmic growth phase in PBS (Cell Bank of China Type Culture Collection), and inoculate C57BL/6J mice at 5 ⁇ 10 5 cells/0.1mL (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. A total of 46 mice were inoculated subcutaneously on the right rib of the company.
  • the tumor volume reached about 250 mm 3
  • the animals were randomly divided into 6 groups according to the tumor volume using excel software, with 5 animals in each group.
  • the first group is the solvent control group
  • the fourth and fifth groups are the experimental groups with different doses (VEGFR2-HS76)
  • the second, third, and sixth groups are other anti-VEGF antibodies not related to the application, so the relevant data are not presented.
  • the administration was started on the day of grouping.
  • the route of administration was intraperitoneal injection (IP).
  • IP intraperitoneal injection
  • the administration schedule is shown in Table 10.
  • the administration was administered twice a week for 4 consecutive administrations.
  • the mice were sacrificed 3 days after the last administration and the tumor tissues were collected. Detection.
  • the anti-tumor effect of the drug is evaluated by calculating the survival rate and tumor growth inhibition rate (TGI(%)): TGI(%) ⁇ 60% is invalid; TGI(%) ⁇ 60%, and the tumor volume of the treatment group is statistically processed Significantly lower than the solvent control group (P ⁇ 0.05) is effective, that is, it has a significant inhibitory effect on tumor growth.
  • TGI(%) The calculation method of TGI(%) is as follows:
  • TGI(%) [1-(Ti-T0)/(Vi-V0)] ⁇ 100, where:
  • T0 mean tumor volume of the treatment group on day 0 of administration
  • V0 The mean tumor volume of the solvent control group on day 0 of administration.
  • the administration volume is calculated as 10 ⁇ L/g based on the weight of the experimental animal.
  • the experimental end point of group 1 is 2 mice, the experimental end point of group 4 is 5 mice, and the experimental end point of group 5 is 4 mice.
  • the average tumor volume of the experimental high-dose group (VEGFR2-HS76-40mpk) on day 7 was 1394mm 3 and the TGI was 61%, suggesting that the antibody has anti-tumor effects.
  • HS76 partially binds to mouse KDR, its binding ability is significantly weaker than that of human KDR. It is expected that HS76 has a better anti-tumor effect in humans.
  • Group 1 has 4 experimental animals on the day;
  • Group 1 has 2 experimental animals on the day, and Group 5 has 2 experimental animals on the day;
  • VEGFR2-HS76 antibody was given to CD-1 mice by tail vein injection. It can be seen from the serum drug concentration that VEGFR2-HS76 antibody at doses of 1 mg/kg and 5 mg/kg basically showed linear pharmacokinetic characteristics in CD-1 mice. The drug concentration in the serum increases with the increase of the administered dose, and there is no significant difference between male and female. The serum drug concentration and the drug exposure in the serum are positively correlated with the administered dose ( Figure 16-1). VEGFR2-HS76 antibody at a dose of 5 mg/kg was subcutaneously administered to CD-1 mice. There was no significant difference between male and female animals, and the plasma concentration reached a peak at 32 hours ( Figure 16-2).
  • the C max for intravenous and subcutaneous administration were 87.47 and 29.62 ⁇ g/mL, and the AUC last were 10117.15 and 9804.09 h* ⁇ g/mL, respectively.
  • the absolute bioavailability of subcutaneous administration was 96.91%.
  • the parameters such as t 1/2 , Vz and Cl of the two administration methods are basically the same (Table 10).
  • ADA In the intravenous 1 mg/kg dose group, ADA was all negative; 1 animal in the 5 mg/kg dose group (1975) was weakly positive at 672h; 2 animals in the 5 mg/kg dose group (1951, 1948) were subcutaneously injected at 168h and 672h The ADA test result was weakly positive.
  • One animal (1951) only detected ADA at 168h, and the subsequent time points were all negative (see Table 11 for details); although the above individuals were positive for ADA, their values were close to SCP, so it is not ruled out The possibility of false positives, as a whole, the immunogenicity of VEGFR2-HS76 antibody in CD-1 mice is weak.
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • Ferrara N The role of vascular endothelial growth factor in pathological angiogenesis. [J]. Breast Cancer Res Treat,1995,36(2):127-137.

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Abstract

L'invention concerne un médicament à base d'anticorps humanisé anti-VEGFR2. L'invention concerne également une séquence d'acide nucléique (comprenant une région variable de chaîne lourde/légère) codant pour l'anticorps, un vecteur contenant la séquence d'acide nucléique, une composition pharmaceutique et un kit. L'anticorps peut se lier de manière spécifique au sixième domaine structural de VEGFR2, exerce une fonction indépendante du ligand VEGF-A, et peut directement inhiber VEGFR2 de former un dimère, bloquant ainsi la phosphorylation de résidus de tyrosine provoquée par la dimérisation d'un domaine intracellulaire de VEGFR2 et d'une voie de signalisation en aval. De plus, l'anticorps maintient une fonction de blocage partiel de VEGF-C et de VEGF-D, et présente ainsi une meilleure fonction d'inhibition de l'angiogenèse. L'anticorps peut également activer l'apparition d'une réaction de cytotoxicité à médiation cellulaire dépendante d'un anticorps (ADCC) dans des cellules NK, inhibe la croissance tumorale et les métastases tumorales, et peut être utilisé pour le traitement clinique de mélanomes.
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WO2025106118A3 (fr) * 2023-05-23 2025-07-10 Board Of Regents, The University Of Texas System Anticorps anti-cd138/syndécan1 et procédés d'utilisation associés
EP4403577A4 (fr) * 2021-09-15 2025-10-08 Pharmabcine Inc Anticorps anti-vegfr2 modifié (kdr) et son utilisation

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EP4403577A4 (fr) * 2021-09-15 2025-10-08 Pharmabcine Inc Anticorps anti-vegfr2 modifié (kdr) et son utilisation
WO2025106118A3 (fr) * 2023-05-23 2025-07-10 Board Of Regents, The University Of Texas System Anticorps anti-cd138/syndécan1 et procédés d'utilisation associés
CN120025456A (zh) * 2024-03-13 2025-05-23 寻济生物科技（北京）有限公司 一种抗vegfa融合构建体及其制备方法和应用

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