WO2021013065A1 - 人源化抗VEGF Fab抗体片段及其用途 - Google Patents

人源化抗VEGF Fab抗体片段及其用途 Download PDF

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WO2021013065A1
WO2021013065A1 PCT/CN2020/102560 CN2020102560W WO2021013065A1 WO 2021013065 A1 WO2021013065 A1 WO 2021013065A1 CN 2020102560 W CN2020102560 W CN 2020102560W WO 2021013065 A1 WO2021013065 A1 WO 2021013065A1
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seq
antibody
vegf
amino acid
acid sequence
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PCT/CN2020/102560
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French (fr)
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谢良志
孙春昀
赵俊
孔德生
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神州细胞工程有限公司
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Priority to JP2022503863A priority Critical patent/JP7229419B2/ja
Priority to MX2022000780A priority patent/MX2022000780A/es
Priority to BR112022001021A priority patent/BR112022001021A2/pt
Priority to CA3150046A priority patent/CA3150046C/en
Priority to CN202080051385.8A priority patent/CN114127106B/zh
Priority to AU2020316495A priority patent/AU2020316495A1/en
Priority to KR1020227004898A priority patent/KR20220034868A/ko
Priority to EP20844600.5A priority patent/EP4043490A4/en
Publication of WO2021013065A1 publication Critical patent/WO2021013065A1/zh
Priority to US17/578,424 priority patent/US20220135665A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention belongs to the field of tumor immunotherapy, and specifically relates to humanized anti-VEGF Fab antibody fragments.
  • Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) is a group of growth factors with important pro-angiogenic activity. It has the functions of promoting endothelial cell mitosis and anti-apoptosis, increasing vascular permeability, and promoting cell migration.
  • the human VEGF gene is located on the 6p21.3 chromosome and belongs to the VEGF/PDGF supergene family. The encoded VEGF is connected by disulfide bonds to form a dimer.
  • the VEGF family includes multiple members with different functions: VEGFA (VEGF, with a variety of different spliced forms), VEGFB, VEGFC, VEGFD, VEGFE, VEGFF, and Placenta Growth Factor (PIGF).
  • VEGFA VEGF, with a variety of different spliced forms
  • VEGFB VEGF, with a variety of different spliced forms
  • VEGFC VEGFD
  • VEGFE VEGFF
  • PIGF Placenta Growth Factor
  • PIGF Placenta Growth Factor
  • endocrine-derived endothelial growth factor Endocrine gland-derived vascular endothelial growth factor, EG-VEGF
  • VEGF is widely distributed in human tissues and organs, among which retinal pigment epithelial cells, vascular endothelial cells, nerve cells, etc.
  • VEGFR1 and VEGFR2 are mainly expressed in vascular endothelial cells, while VEGFR3 is mainly expressed in lymphatic endothelial cells.
  • VEGF plays an important role in the regulation of normal and pathological angiogenesis (Melincovici C S et al., Rom J Morphol Embryol. 2018; 59(2): 455-467).
  • VEGF is overexpressed in a variety of tumors that can cause malignant ascites, and the expression of VEGF in tumors is related to the migration ability of tumor cells.
  • concentration of VEGF in patients with solid tumors such as gastrointestinal cancer, ovarian cancer, breast cancer and lung cancer is positively correlated with the stage of the disease, and has a low survival rate (Sebastian, K et al., Oncologist. 2009; 14(12):1242 -1251).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • VEGF monoclonal antibody drugs inhibit the interaction of VEGF with endothelial cell surface receptors VEGFR2 and VEGFR1, block downstream signal pathway transduction, and inhibit endothelial cell expansion and angiogenesis.
  • the FDA approved VEGF-targeting antibody drugs for the treatment of ophthalmic diseases are Lucentis (Ranibizumab, approved in 2006), EYLEA (Aflibercept, approved in 2004), and Conbercept (Compaxi general).
  • Lucentis is a human-derived VEGFA antibody Fab fragment, which can bind all active forms of VEGFA and inhibit its binding to VEGFR1 and VEGFR2, thereby inhibiting the proliferation and migration of vascular endothelial cells and reducing blood vessel permeability, and inhibiting choroidal neovascularization. form.
  • the antibody of the Fab fragment is easier to penetrate the retina to the subretinal space, reach the target tissue and bind tightly to VEGF, thereby inhibiting the formation of choroidal neovascularization.
  • Fab fragment antibodies that penetrate into the systemic system through blood circulation are cleared in only 0.09 days or about 2 hours, which can minimize the impact on normal VEGF physiological functions, and reduce gastrointestinal perforation, high blood pressure and bleeding.
  • Ferrara N et al., Retina. 2006; 26(8): 859-870; Van Wijngaarden et al., Clin Exp Optom. 2008; 91(5): 427-437).
  • Studies have shown that AMD has a certain relationship with the inflammatory response caused by the complement effect.
  • Fab antibody fragments do not contain Fc fragments and cannot stimulate the complement cascade reaction, thereby reducing the risk of causing endophthalmitis and autoimmune inflammation (Ferrara, N et al. ., Retina.
  • Bevacizumab (bevacizumab) is a recombinant human monoclonal antibody approved by the FDA for the treatment of metastatic colon cancer, non-small cell lung cancer and other solid tumors. It is currently used as an outside approved drug for the treatment of AMD.
  • Aflibercept and Conbercept are humanized recombinant fusion proteins that contain specific domains on VEGFR that bind to ligands, which can bind to VEGF with specific high affinity and block the binding of VEGF to receptors.
  • aflibercept Compared with bevacizumab and Lucentis, aflibercept has a higher affinity for VEGF165 and shows better efficacy in the treatment of DME.
  • Aflibercept has been approved for the treatment of wet AMD, branch retinal vein occlusion, central retinal vein occlusion, CNV, DME and diabetic retinopathy.
  • Conbercept has been approved in China for the treatment of wet AMD.
  • the present invention provides new humanized anti-VEGF Fab antibody fragments, which can be used to treat ophthalmic diseases characterized by choroidal neovascularization, including but not limited to age-related age-related macular degeneration (AMD), diabetic macular edema (DME), and retina Occurrence of edema, degenerative myopia, choroidal neovascularization (CNV).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • the present invention provides an isolated anti-VEGF 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 shown in SEQ ID NO: 14.
  • the heavy chain CDR2 domain and 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 The light chain CDR2 domain of the amino acid sequence shown in ID 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-VEGF antibody or antigen-binding fragment thereof comprises an amino acid sequence shown in SEQ ID NO: 22 or at least 90%, 92%, 95%, 98%, or 99% of SEQ ID NO: 22.
  • the heavy chain variable region of the amino acid sequence of sequence identity, and the amino acid sequence shown in SEQ ID NO: 23 or at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 23 The amino acid sequence of the light chain variable region.
  • the anti-VEGF antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region, preferably the heavy chain constant region is an IgG1 heavy chain constant with an amino acid sequence of SEQ ID NO: 38
  • the heavy chain constant region is an IgG1 heavy chain constant with an amino acid sequence of SEQ ID NO: 38
  • the light chain constant region has an amino acid sequence of SEQ ID NO:
  • the anti-VEGF antibody or antigen-binding fragment thereof is a humanized antibody or a chimeric antibody.
  • the present invention provides an isolated anti-VEGF antibody or antigen-binding fragment thereof, which comprises a heavy chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain CDR1 domain having the amino acid sequence shown in SEQ ID NO: 28.
  • 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: 29, and the light chain CDR1 domain with the amino acid sequence of SEQ ID NO: 24, have The light chain CDR2 domain of the amino acid sequence shown in SEQ ID NO: 25 and the light chain variable region of the light chain CDR3 domain having the amino acid sequence of SEQ ID NO: 26.
  • the anti-VEGF antibody or antigen-binding fragment thereof comprises an amino acid sequence shown in SEQ ID NO: 36 or at least 90%, 92%, 95%, 98%, or 99% of SEQ ID NO: 36.
  • the heavy chain variable region of the amino acid sequence of sequence identity, and the amino acid sequence shown in SEQ ID NO: 37 or at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 37 The amino acid sequence of the light chain variable region.
  • the anti-VEGF antibody or antigen-binding fragment thereof is a Fab fragment
  • the Fab fragment further comprises a heavy chain constant region CH1 and a light chain constant region.
  • the heavy chain constant region CH1 has an amino acid sequence of The amino acid sequence of the IgG1 heavy chain constant region of SEQ ID NO: 40 or an amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 40, and/or the light
  • the chain constant region is the amino acid sequence of the light chain constant region of SEQ ID NO: 39 or an amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 39.
  • the anti-VEGF antibody or antigen-binding fragment thereof further comprises a heavy chain signal peptide and a light chain signal peptide, preferably the heavy chain signal peptide is the amino acid sequence of SEQ ID NO: 34 or is SEQ ID NO: 34 has an amino acid sequence with at least 90%, 92%, 95%, 98% or 99% sequence identity, and/or the light chain signal peptide is the amino acid sequence of SEQ ID NO: 35 or An amino acid sequence having at least 90%, 92%, 95%, 98%, or 99% sequence identity with SEQ ID NO: 35.
  • the anti-VEGF antibody or antigen-binding fragment thereof is a Fab antibody fragment.
  • the anti-VEGF antibody or antigen-binding fragment thereof is an IgG antibody, preferably an IgG1 antibody.
  • the anti-VEGF Fab antibody fragment is an IgG antibody-related Fab antibody fragment, preferably an IgG1 antibody-related Fab antibody fragment.
  • the anti-VEGF antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the anti-VEGF Fab antibody fragment is monoclonal.
  • the binding affinity K D of the anti-VEGF antibody or antigen-binding fragment thereof to the recombinant human VEGF165 protein is 0.01-8E-10M, preferably 0.1-5E-10M, more preferably 0.5-3E-10M, most preferably 1.54E-10M.
  • 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; wherein
  • the single chain antibody molecule is preferably scFv, di-scFv, tri-scFv, diabody or scFab.
  • the present invention provides an antibody-drug conjugate comprising the anti-VEGF antibody or antigen-binding fragment thereof of the present invention and another therapeutic agent, preferably the anti-VEGF antibody or antigen-binding fragment thereof and The additional therapeutic agent is connected by a linker.
  • the present invention provides a nucleic acid that encodes the anti-VEGF antibody or antigen-binding fragment thereof of the present invention.
  • the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 4 and/or the nucleotide sequence shown in SEQ ID NO: 5; or it comprises the nucleotide sequence shown in SEQ ID NO: 20
  • the nucleic acid further comprises the nucleotide sequence shown in SEQ ID NO: 49 and/or the nucleotide sequence shown in SEQ ID NO: 48. More preferably, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 41 and/or the nucleotide sequence shown in SEQ ID NO: 42.
  • the invention provides an expression vector comprising the nucleic acid of the invention.
  • the present invention provides a host cell comprising the nucleic acid of the present invention or the expression vector of the present invention.
  • the present invention provides a method for producing the anti-VEGF antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions suitable for antibody expression, and removing from the culture medium The expressed antibody is recovered in the process.
  • the present invention provides a method for producing the anti-VEGF antibody or antigen-binding fragment thereof of the present invention, which comprises culturing the host cell of the present invention under conditions suitable for expression of the Fab antibody fragment, and The expressed Fab antibody fragments are recovered from the culture medium.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-VEGF antibody or antigen-binding fragment thereof of the present invention, or the antibody-drug conjugate of the present invention, or the nucleic acid of the present invention, or Expression vector, and pharmaceutically acceptable carrier.
  • the present invention provides the anti-VEGF antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention for use in the treatment of disorders related to angiogenesis.
  • the angiogenesis-related disorder is ophthalmopathy.
  • the eye disease is an eye disease characterized by choroidal neovascularization, including age-related age-related macular degeneration (AMD), diabetic macular edema (DME), retinal edema, degenerative myopia, choroidal neovascularization (CNV ).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • the present invention provides a method for treating angiogenesis-related disorders, which comprises administering to a subject in need a therapeutically effective amount of the anti-VEGF antibody or antigen-binding fragment of the present invention or the present invention The antibody-drug conjugate of the invention or the pharmaceutical composition of the present invention, thereby treating the angiogenesis-related disorders.
  • the angiogenesis-related disorder is ophthalmopathy.
  • the eye disease is an eye disease characterized by choroidal neovascularization, including age-related age-related macular degeneration (AMD), diabetic macular edema (DME), retinal edema, degenerative myopia, choroidal neovascularization (CNV ).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • the present invention provides that the anti-VEGF antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention is prepared for the treatment of angiogenesis-related disorders. Use in medicine.
  • the angiogenesis-related disorder is ophthalmopathy.
  • the eye disease is an eye disease characterized by choroidal neovascularization, including age-related age-related macular degeneration (AMD), diabetic macular edema (DME), retinal edema, degenerative myopia, choroidal neovascularization (CNV ).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • the present invention provides a pharmaceutical combination comprising the anti-VEGF antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention and one or more Another therapeutic agent.
  • the present invention provides a kit comprising the anti-VEGF antibody or antigen-binding fragment thereof of the present invention or the antibody-drug conjugate of the present invention or the pharmaceutical composition of the present invention, preferably, still further Contains drug delivery device.
  • Figure 1 shows that the VEGF165 rabbit antibody VEGF-R988 blocks the binding of VEGF165 to VEGFR2 protein.
  • FIG. 2 shows that the VEGF165 rabbit antibody VEGF-R988 neutralizes the proliferation of HUVEC cells by VEGF165.
  • Figure 3 shows the binding of humanized antibody VEGF-H988 Fab to VEGF165 by ELISA.
  • Figure 4 shows the species cross-binding between VEGF-H988 Fab and mVEGF164 detected by ELISA.
  • Figure 5 shows that VEGF-H988 Fab blocked the binding of VEGF165 to VEGFR2 protein detected by ELISA.
  • Figure 6 shows the effect of VEGF-H988 Fab versus Lucentis in neutralizing different concentrations of VEGF165.
  • Figure 7 shows the effect of VEGF-H988 Fab versus EYLEA in neutralizing different concentrations of VEGF165.
  • Figure 8 shows the effect of VEGF-H988 Fab versus Avastin in neutralizing VEGF165 at different concentrations.
  • Figure 9 shows the effect of VEGF-H988 Fab in contrast to Conbercept in neutralizing different concentrations of VEGF165.
  • Figure 10 shows the effect of VEGF-H988 Fab versus Brolucizumab in neutralizing different concentrations of VEGF165.
  • Various aspects of the present invention relate to isolated anti-VEGF Fab antibody fragments, antibody-drug conjugates comprising the antibody fragments or antigen-binding fragments thereof, nucleic acids and expression vectors encoding the Fab antibody fragments, and hosts containing the nucleic acids or expression vectors Cells, methods for producing the anti-VEGF Fab antibody fragments, pharmaceutical compositions containing the anti-VEGF Fab antibody fragments, and methods for using the anti-VEGF Fab antibody fragments to treat diseases related to angiogenesis.
  • 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.
  • Fab fragments typically comprise a heavy chain variable region (V H) and a heavy chain constant region 1 (C H 1) and light chain variable region (V L) and a light chain constant region (C L).
  • 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 such as an antibody or antigen-binding fragment thereof chemically linked to one or more chemical drugs (also referred to herein as agents), which may optionally Ground is a therapeutic or cytotoxic agent.
  • 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 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 an antigen of interest, such as a tumor-associated polypeptide antigen target (herein, VEGF), 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.
  • an antigen of interest such as a tumor-associated polypeptide antigen target (herein, VEGF)
  • VEGF 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).
  • K D 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.
  • K D 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, the binding of a ligand to a specific protein Tightness. 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 to produce 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, such as 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.
  • Human Umbilical Vein Endothelial Cells are isolated from umbilical cord veins and are generally used in physiology and pharmacological research, such as macromolecular transport, blood coagulation, angiogenesis and fibrinolysis. In particular, it can be used as a model for angiogenesis research and other VEGF-dependent signal pathway research (related endothelial growth factor) research.
  • the present invention firstly uses recombinant human VEGF165 protein to immunize rabbits, and then obtains the antibody clone VEGF165-R988 which specifically binds to recombinant human VEGF165 protein through phage display library screening. Then, the nucleotide sequences encoding the heavy chain and light chain variable regions of the VEGF165-R988scFv antibody were inserted into the pSTEP2 vector with the nucleotide sequence of the rabbit heavy chain IgG1 constant region or the rabbit light chain kappa constant region, and Perform culture expression. Use protein A purification column for purification to obtain high-purity antibody. ELISA test showed that the rabbit antibody VEGF165-R988 can effectively inhibit the binding of VEGF165 protein to VEGFR2 protein, and VEGF165-R988 can effectively neutralize the ability of VEGF165 to promote HUVEC cell proliferation.
  • the light chain or heavy chain variable region of a human antibody that is closer to the rabbit light chain or heavy chain variable region is selected as the template, and each of the rabbit antibody light chain or heavy chain is selected as the template.
  • Three CDRs (Table 1) were inserted into the variable region of the human antibody to obtain humanized light chain variable region (VL) and heavy chain variable region (VH) sequences. Since the rabbit-derived framework region has key points that are essential for supporting the activity of CDR, the key points are backmutated to the corresponding sequence of the rabbit antibody.
  • the VEGF-H988-10 light chain/heavy chain expression vector was obtained by the method of full gene synthesis, transfected into HEK-293 cells and cultured and expressed, and the culture supernatant was purified by a protein A purification column to obtain high-purity VEGF-H988-10 antibody .
  • an SDM library of the CDR regions of the heavy and light chain variable domains (including LCDR1, LCDR3, HCDR2 and HCDR3) was constructed, and the 4 mutant libraries were constructed into scFv sequences to be fused with gene 3.
  • the protein form was cloned into a phagemid vector, and the best clones were screened for each CDR through the binding ability of the soluble antigen VEGF, and finally the VEGF-H988 Fab antibody fragment with optimized CDR affinity and stability was obtained and tested.
  • the antibody of the Fab fragment Compared with the full-length structure, the antibody of the Fab fragment has stronger penetrability, lower gastrointestinal perforation, high blood pressure and bleeding and other side effects and cannot stimulate the complement cascade reaction, thereby reducing the occurrence of endophthalmitis and The risk of autoimmune inflammation.
  • 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: 2-3, 4-7, 16-17, 20-21, 41-49, and 52-53.
  • 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, and examples of eukaryotic host cells are yeast, insect or mammalian cells. It should be understood that the design of an expression vector including a selection regulatory sequence is affected by many factors, such as the choice of host cell, the desired protein expression level, 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 (ATCC37017). 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.
  • the antibodies of the present invention can be used to treat diseases related to angiogenesis, including ocular diseases characterized by choroidal neovascularization, including but not limited to age-related age-related macular degeneration (AMD), diabetic macular edema (DME), retinal edema, and degeneration The occurrence of sexual myopia, choroidal neovascularization (CNV).
  • AMD age-related age-related macular degeneration
  • DME diabetic macular edema
  • CNV choroidal neovascularization
  • 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 rabbit antibodies that block the binding of VEGF165 and VEGFR2 using phage antibody display library
  • Recombinant human VEGF165 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.11066-HNAH) was used to immunize rabbits.
  • the amino acid sequence of the extracellular region Met1-Arg191 of the human VEGF165 protein (UniProtKB P15692-4) is SEQ ID NO:1.
  • the specific method is: mixing recombinant human VEGF165 protein with Freund's adjuvant, and using the mixture for four immunizations, each with a dose of 500 ⁇ g, subcutaneous injection, and the immunization interval is 3 weeks, 2 weeks, and 2 weeks.
  • blood was collected from the orbital intracanthal venous plexus four days after immunization.
  • the ELISA method was used to coat recombinant human VEGF165 protein to detect the titer of rabbit anti-VEGF165 serum.
  • the serum titer of the fifth immunization reached 25000 times dilution.
  • 25 ⁇ g of recombinant human VEGF165 protein was injected intravenously for boost. Seven days later, the rabbits were sacrificed and the spleen tissues of the rabbits were frozen and stored in liquid nitrogen.
  • TriPure Isolation Reagent reagent (source: Roche, Cat. No. 11 667 165 001) to extract RNA from rabbit spleen tissue, and use reverse transcription kit (source: Invitrogen, Cat. No. 18080-051) for reverse transcription.
  • cDNA Design 10 pairs of primers to amplify the light chain variable region sequence of the rabbit antibody, and 4 pairs of primers to amplify the heavy chain variable region sequence (Barbas C F et al., CSHL Press. 2004).
  • the overlapping extension splicing PCR method was used to splice the sequences encoding the light chain and heavy chain variable regions of the rabbit antibody into a nucleotide sequence encoding scFv, and the light and heavy chain variable regions were connected through a linker:
  • phage vector pComb3x source: Beijing Yiyi
  • restriction endonuclease Sfi I source: Fermentas
  • X-Blue competent cells were electrotransformed to construct a phage display scFv antibody library for immunizing rabbits.
  • the recombinant human VEGF165 protein was coated on the ELISA plate, and the phage library enriched with anti-VEGF165 positive antibodies was screened according to the phage antibody panning process (O'Brien, PM, & Aitken, R.
  • Monoclonal phages were selected from the enriched library for expression, and the binding to recombinant human VEGF165 protein was detected by ELISA method.
  • the antibody clones that specifically bind to recombinant human VEGF165 were screened and the clones obtained were sent to the sequencing company for sequencing to obtain VEGF165.
  • the nucleotide sequence of the R988scFv antibody SEQ ID NO: 3).
  • the nucleotide sequence of the heavy chain variable region of the VEGF165-R988scFv antibody was amplified by PCR, and inserted into the heavy chain signal peptide (SEQ ID NO: 43) and the rabbit heavy chain IgG1 constant region (SEQ ID NO: 6)
  • the heavy chain (SEQ ID NO: 52) expression vector is obtained from the pSTEP2 vector digested with Sca I+Kpn I (Fermentas).
  • the nucleotide sequence of the light chain variable region of the VEGF-R988scFv antibody was amplified by PCR, and inserted into the light chain signal peptide (SEQ ID NO: 44) and the rabbit light chain kappa constant region (SEQ ID NO: 7) by in-fusion method.
  • the light chain (SEQ ID NO: 53) expression vector was obtained from the pSTEP2 vector digested with Sca I+BamH I (Fermentas).
  • the recombinant plasmid was extracted, transfected into HEK-293 cells and cultured and expressed for 7 days, and the culture supernatant was purified by a protein A purification column to obtain high-purity antibodies.
  • the VEGF165 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) at a concentration of 1 ⁇ 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, sealed at room temperature for 1 hour, and then 100 ⁇ L 5 ⁇ g/mL VEGFR2-biotin protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) and different concentrations of VEGF-R988 antibody were added and incubated together. Wash the plate to remove the unbound antibody, add streptavidin/HRP (source: Beijing Zhongshan Jinqiao) to incubate the plate repeatedly, and finally add the substrate color solution for color development.
  • streptavidin/HRP source: Beijing Zhongshan Jinqiao
  • Inhibition rate PI% (OD blank-OD sample) / OD blank ⁇ 100%, where OD blank represents the OD value of the group with only VEGFR2-biotin and no antibody added, and OD sample represents the waiting for VEGFR2-biotin and antibody Test the OD value of the group.
  • the VEGF-R988 antibody can effectively bind to the coated VEGF165 protein, and can effectively inhibit the binding of VEGFR165 protein to VEGFR2 protein.
  • WST-8 method was used to detect the proliferation of umbilical vein endothelial cell (HUVEC) cells by neutralizing VEGF165 by chimeric antibody.
  • a 96-well plate was seeded with HUVEC cells 4 ⁇ 10 3 cells/well, cultured in M199 medium containing 10% FBS and 5% L-Gln for 4 hours, and then added with different concentrations of VEGF-R988 antibody, 50 ⁇ L/well. Subsequently, 10 ⁇ L/well of VEGF165 with a final concentration of 10 ng/mL was added. Place the 96-well plate in a CO 2 incubator at 37° C. and 5% CO 2 for 3 days.
  • HUVEC umbilical vein endothelial cell
  • test blank well B no cells
  • negative control group M seeding cells, no sample, plus VEGF165
  • M' seeding cells, no sample and VEGF165
  • 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 concentration of the sample, the ordinate is the neutralization rate, the regression equation is a four-parameter equation, and the "S" type is obtained Curve, calculate the half effective concentration (EC 50 ) of the sample.
  • the VEGF-R988 antibody can effectively reduce the ability of VEGF165 to promote HUVEC proliferation.
  • Example 1.2 According to the nucleotide sequence of the VEGF-R988scFv antibody determined in Example 1.2, the amino acid sequences of the heavy chain and light chain variable regions of the VEGF-R988scFv were deduced, see SEQ ID NO: 8/9.
  • Rabbit antibody humanization adopts the classic humanization method CDR transplantation method, selects the human antibody light chain or heavy chain variable region that is closer to the rabbit light chain or heavy chain variable region as the template, and combines the rabbit antibody light chain or heavy chain variable region as the template.
  • Each of the three CDRs of the 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) sequences.
  • the human template of the light chain variable region of VEGF-R988 used was IGKV1-27*01, which had 65.30% homology with the light chain of VEGF-R988, and the human template of the heavy chain variable region was IGHV4-4 *08, the template has 53.20% homology with the heavy chain of VEGF-R988.
  • the key points are backmutated to the corresponding sequence of the rabbit antibody, where the first back mutation of the light chain is E and the second back mutation is L ,
  • the back mutation at position 4 is L
  • the back mutation at position 63 is K
  • the back mutation at position 3 of the heavy chain is V
  • the back mutation at position 37 is V
  • the back mutation at position 47 is Y
  • the back mutation at position 78 is V
  • the 79th back mutation is D
  • the 91th back mutation is F
  • the reversion sites are all Kabat numbering.
  • Humanized antibody VEGF-H988-10 was obtained by CDR humanization transplantation and framework region back mutation.
  • the VEGF-H988-10 heavy chain variable region (SEQ ID NO: 20) was obtained by the method of full gene synthesis, and inserted into the heavy chain signal peptide (SEQ ID NO: 43) and human IgG1 constant region (
  • the VEGF-H988-10 heavy chain (SEQ ID NO: 16) expression vector was obtained from the pSTEP2 vector digested with Sca I+Nhe I (Fermentas) of SEQ ID NO: 47).
  • the VEGF-H988-10 light chain variable region (SEQ ID NO: 21) was obtained by the method of full gene synthesis, and inserted into the light chain signal peptide (SEQ ID NO: 44) and the human kappa constant region (
  • the VEGF-H988-10 light chain (SEQ ID NO: 17) expression vector was obtained from the pSTEP2 vector digested with Sca I+BsiW I (Fermentas) of SEQ ID NO: 48). After the plasmid was extracted, it was transfected into HEK-293 cells and cultured and expressed for 7 days. The culture supernatant was purified by a protein A purification column to obtain high-purity antibodies.
  • the heavy chain and light chain variable domain CDR regions construct an SDM library, including three saturation mutation libraries of LCDR1, LCDR3, and HCDR2; at the same time, in order to improve the chemical stability of the antibody, it is necessary to remove the The amide or isomerization site is changed to another amino acid residue. Deamidation of asparagine can lead to the production of isoaspartic acid residues on NG, NS, NA, NT and other sequences, which affects the stability or biological function of the antibody.
  • VEGF-H988 variable domain HCDR3 has easy deamidation sites.
  • an SDM library was constructed at the same time.
  • VEGF-H988 light chain and heavy chain CDR sequences are shown in Table 2.
  • VEGF-H988 Fab light chain nucleotide sequence (SEQ ID NO: 42) that encodes the signal peptide, it contains the nucleotide sequence of the light chain signal peptide (SEQ ID NO: 44) connected in sequence ,
  • the VEGF-H988 Fab heavy chain Fab nucleotide sequence contains the nucleotide sequence of the heavy chain signal peptide (SEQ ID NO: 43), humanized anti- The nucleotide sequence of the variable region of the heavy chain (SEQ ID NO: 45) and the nucleotide sequence of the CH1 constant region of the human IgG1 heavy chain (SEQ ID NO: 49) were inserted by in-fusion method to construct the correct containing light In the pGS vector (Nhe I+Not I) of the chain, the correct VEGF-H988 Fab light and heavy chain expression vector was verified by sequencing.
  • the expression vector contains eukaryotic expression vector of GS screening gene and antibody light and heavy chain expression elements.
  • the expression vector was transfected into CHO-K1-GS-deficient cells, and VEGF-H988 Fab high-expressing cell line was obtained by MSX screening. ELISA is used to select clones with high antibody expression, and the high-expressing cell lines are screened by combining the cell growth status and the key quality attribute analysis results of antibody drugs.
  • the VEGF-H988 Fab-producing CHO cell line was cultivated in a serum-free feeding suspension culture method to obtain high-purity and high-quality VEGF-H988 Fab.
  • VEGF-H988 Fab specifically binds to VEGF165
  • the plate was washed the next day and blocked at room temperature for 1 hour, then 100 ⁇ L of 1 ⁇ g/mL VEGF165-H988 Fab, Lucentis (source: Novartis) and negative control antibody H7N9-R1 Fab (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) were added and incubated for 1 hour Then wash the plate to remove unbound antibody, add the secondary antibody goat F(ab')2 anti-human IgG F(ab')2/HRP (source: Jackson ImmunoResearch), incubate the plate repeatedly, add the substrate color development solution for visualization After termination, the microplate reader reads OD450.
  • a streptavidin-coated Sensor was used to immobilize the biotin-labeled VEGF165 protein, and the affinity of VEGF-H988 Fab and Lucentis to VEGF165 at multiple concentrations was detected.
  • the method is as follows: Use a 5000MW ultrafiltration tube to pass through the buffer solution of the recombinant VEGF165 protein (20mM Tris, 150mM NaCl, pH8.0) Replaced with PBS. After UV quantification, 567.57 ⁇ g protein was obtained. The obtained protein was mixed with 20mM biotin solution at a molar ratio of 1:2, and reacted for 30 minutes in the dark at room temperature. Filtered again through a 5000MW ultrafiltration tube to remove unlabeled biotin. UV After quantification, an equal volume of glycerol and a final concentration of 0.1% BSA were added to obtain a biotin-labeled protein. Its concentration was 2.08mg/mL by UV detection.
  • VEGF-H988 Fab to recombinant human VEGF165 protein binding affinity KD value of 1.54E-10 (M), the binding constants k on value of 2.74E + 05 (1 / Ms) , the value of the dissociation constant k dis Lucentis VEGF165 protein binding affinity KD value of 5.78E-11 (M), the binding constants k on value of 5.36E + 04 (1 / Ms) , dissociation constant value from k dis; is 4.21E-05 (1 / s) It is 3.10E-05 (1/s).
  • the results show that the affinity of VEGF-H988 Fab is stronger than that of Lucentis, which is about 3.75 times that of Lucentis. Therefore, VEGF-H988 Fab has a stronger ability to bind VEGF165 protein than Lucentis.
  • Dilute recombinant human VEGF165 protein and recombinant mouse mVEGF164 protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) to 0.1 ⁇ g/mL, 1 ⁇ g/mL and 10 ⁇ g/mL and coat them on 96-well plates with 100 ⁇ L per well. Coated overnight at 4°C. Wash the plate the next day, block at room temperature for 1 hour, add 100 ⁇ L of VEGF165-H988 Fab, Lucentis and the negative control antibody H7N9-R-Fab at a concentration of 2 ⁇ g/mL, and incubate for 1 hour.
  • VEGF165-H988 Fab has specific binding to recombinant human VEGF165 protein, and cross-binding with recombinant mouse mVEGF164 protein. Lucentis does not cross-binding with recombinant mouse mVEGF164 protein.
  • the VEGF165 protein at a concentration of 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 VEGFR2-his protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.), and then add different concentrations of humanized VEGF-H988 Fab, Lucentis and negative control antibodies H7N9-R1-Fab (source: Beijing Yiqiao Shenzhou Technology Co., Ltd.) was incubated together. Wash the plate to remove unbound antibody, add C-his-R023/HRP to incubate and repeat the washing. Finally, add the substrate color developing solution for color development. After termination, the microplate reader reads OD450, and each group is twice in parallel.
  • Inhibition rate PI% (OD blank- OD sample )/OD blank ⁇ 100, where OD blank represents the OD value of the group with only VEGFR2-his added without antibody, and OD sample represents the OD of the test group with both VEGFR2-his and antibody added value.
  • VEGFR2 protein can effectively bind to the coated VEGF165 protein
  • VEGF-H988 Fab can effectively inhibit the binding of VEGFR2 protein to VEGF165 protein, and the blocking ability of VEGF-H988 Fab is significantly better than Lucentis, and the negative control antibody has no inhibition. effect.
  • VEGF-H988 Fab blocks the growth inhibitory activity of VEGF165 at different concentrations
  • the WST-8 method was used to detect the proliferation effect of humanized antibody neutralizing VEGF165 on HUVEC cells.
  • a 96-well plate was seeded with HUVEC cells 4 ⁇ 10 3 /well, cultured in M199 medium containing 10% FBS and 5% L-Gln for 4 hours, and then antibodies of different concentrations were added at 50 ⁇ L/well. Subsequently, 10 ⁇ L/well of VEGF165 with final concentrations of 1000 ng/mL, 100 ng/mL, and 10 ng/mL were added. Place the 96-well plate in a CO 2 incubator at 37° C. and 5% CO 2 for 3 days.
  • test blank well B no cells
  • negative control group M seeding cells, no sample, plus VEGF165
  • M' seeding cells, no sample and VEGF165
  • 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 concentration of the sample, the ordinate is the neutralization rate, the regression equation is a four-parameter equation, and the "S" type is obtained Curve, calculate the half effective concentration (EC 50 ) of the sample.
  • the neutralizing effect of VEGF-H988Fab is stronger than Lucentis under the conditions of different concentrations of recombinant human VEGF165.
  • concentration of VEGF165 is 1000ng/mL, 100ng/mL
  • the neutralizing activity of VEGF-H988 Fab is stronger than EYLEA
  • concentration of VEGF165 is 10ng/mL
  • VEGF-H988 Fab is slightly weaker than EYLEA.
  • the activity of VEGF-H988 Fab is stronger than that of Bevacizumab and Brolucizumab, but is equivalent to the activity of Conbercept.
  • concentration of VEGF165 increased, VEGF-H988 Fab maintained a high maximum neutralization rate, while EYLEA and Avastin decreased.

Abstract

本发明属于肿瘤免疫治疗领域，涉及人源化抗VEGF Fab抗体片段。本发明公开编码所述抗体片段的核酸序列(包括重/轻链可变区)、含有所述核酸序列的载体、药物组合物和试剂盒。本发明公开的抗VEGF Fab抗体片段可以以高亲和力特异性结合VEGF，阻断VEGF与受体VEGFR2结合，还能中和VEGF对HUVEC细胞的增殖作用。相比于全长结构，Fab片段的抗体具有更强的穿透性，和更低的胃肠道穿孔、高血压和出血等毒副作用且不能激发补体级联反应，从而降低引发眼内炎和自身免疫炎症反应的风险，可用于临床治疗多种以脉络膜新生血管为特征的眼病，包括但不局限于年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。

Description

人源化抗VEGF Fab抗体片段及其用途 技术领域
本发明属于肿瘤免疫治疗领域，具体地涉及人源化抗VEGF Fab抗体片段。
背景技术
血管系统的发育是许多生理和病理过程的基础。血管内皮生长因子(Vascular endothelial growth factor，VEGF)是一组具有重要促血管生成活性的生长因子，具有促进内皮细胞有丝分裂和抗细胞凋亡，增加血管通透性，促进细胞迁移等作用。人VEGF基因定位在6p21.3染色体上，属于VEGF/PDGF超基因家族，编码的VEGF由二硫键连接组成二聚体。在人体，VEGF家族包括多个具有不同功能的成员：VEGFA(VEGF，具有多种不同的剪切形式)、VEGFB、VEGFC、VEGFD、VEGFE、VEGFF和胎盘生长因子(Placenta growth factor,PIGF)。最近，将内分泌源性内皮生长因子(Endocrine gland-derived vascular endothelial growth factor,EG-VEGF)也纳入该家族中(Samson M et al.,J Clin Endocrinol Metab.2004；89(8):4078–4088)。VEGF广泛分布于人体组织器官，其中眼部的视网膜色素上皮细胞、血管内皮细胞、神经细胞等均可表达(Goel H L et al.,Nat Rev Cancer.2013；13(12):871)。VEGF受体有三种类型：VEGFR1、VEGFR2和VEGFR3。VEGF与受体胞外结构域结合触发受体二聚化，促进胞内结构域中酪氨酸残基自磷酸化，从而激活下游促进细胞增殖、迁移、抗凋亡和提高血管通透性的信号。VEGFR1和VEGFR2主要表达在血管内皮细胞，而VEGFR3主要表达在淋巴管内皮细胞。
目前已证实VEGF在调节正常和病理性血管生成的过程中起着重要的作用(Melincovici C S et al.,Rom J Morphol Embryol.2018；59(2):455-467)。VEGF在多种可引起恶性腹水的肿瘤种过表达，并且肿瘤内VEGF的表达量同肿瘤细胞的迁移能力具有相关性。胃肠癌、卵巢癌、乳腺癌和肺癌等实体瘤病人体内VEGF的浓度和疾病分期呈正相关，并具有较低的存活率(Sebastian,K et al.,Oncologist.2009；14(12):1242-1251)。眼后段疾病中多种病变例如年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)等的发生的发展也同VEGF表达水平密切相关(Patel J R et al.,Curr opin ophthalmol.2016；27(5):387-392；Tan G S et al,.Lancet Diabetes Endo.2017；5(2):143-155；Mitchell P  et al.,Lancet.2018；392(10153):1147-1159)。
VEGF的单克隆抗体药物通过抑制VEGF与内皮细胞表面受体VEGFR2和VEGFR1的相互作用，阻断下游信号通路转导，抑制内皮细胞扩增和新生血管生成。FDA批准用于治疗眼科疾病的靶向VEGF的抗体药物有Lucentis(雷珠单抗，2006年批准)，EYLEA(阿柏西普，2004年批准)，在中国上市的还有Conbercept(康柏西普)。Lucentis是一种人源性VEGFA抗体Fab片段，能结合所有活性形式的VEGFA和抑制其与VEGFR1及VEGFR2的结合，从而抑制血管内皮细胞的增殖迁移及降低血管的通透性，抑制脉络膜新生血管的形成。相比于全长结构，Fab片段的抗体更易于透过视网膜达视网膜下间隙，到达靶组织与VEGF紧密结合，从而抑制脉络膜新生血管的生成。通过血液循环渗透到全身系统中的Fab片段抗体仅0.09天或约2小时即被清除，可最大程度降低对正常VEGF的生理功能的影响，降低胃肠道穿孔、高血压和出血等毒副作用(Ferrara,N et al.,Retina.2006；26(8):859-870；Van Wijngaarden et al.,Clin Exp Optom.2008；91(5):427-437)。研究表明AMD同补体效应引起的炎性反应具有一定关系，Fab抗体片段不含Fc片段，不能激发补体级联反应，从而可降低引发眼内炎和自身免疫炎症反应的风险(Ferrara,N et al.,Retina.2006；26(8):859-870)。Lucentis已获批用于治疗湿性AMD、CNV、DME和视网膜水肿。贝伐珠单抗(贝伐单抗)是被FDA批准用于治疗转移性结肠癌、非小细胞肺癌等实体瘤的重组人单抗药，目前作为批准范围外用药用于治疗AMD。阿柏西普和康柏西普为人源化重组融合蛋白，包含VEGFR上与配体结合的特定结构域，可与VEGF特异性高亲和力结合，阻断VEGF与受体的结合。相比贝伐珠单抗和Lucentis，阿柏西普对VEGF165具有更高的亲和力，在治疗DME上显现更优的疗效。阿柏西普已获批用于治疗湿性AMD、分支视网膜静脉阻塞、视网膜中央静脉阻塞、CNV、DME和糖尿病性视网膜病变。康柏西普在中国已获批用于治疗湿性AMD。
由于该类药物为局部玻璃体内注射，频繁给药极易引发眼球和眼周感染而带来损伤。因此需要在药物上进行优化，提高药效，降低给药频率，给病人带来更大的治疗获益。
发明内容
本发明提供了新的人源化抗VEGF Fab抗体片段，可用于治疗以脉络膜新生血管为特征的眼病，包括但不限于年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管等的发生(CNV)。
在一个方面，本发明提供了一种分离的抗VEGF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。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段包含具有SEQ ID NO:22所示的氨基酸序列或与SEQ ID NO:22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SEQ ID NO:23所示的氨基酸序列或与SEQ ID NO:23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。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区，优选地所述重链恒定区为氨基酸序列为SEQ ID NO:38的IgG1重链恒定区的氨基酸序列或与SEQ ID NO:38具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链恒定区为氨基酸序列为SEQ ID NO:39的轻链恒定区的氨基酸序列或与SEQ ID NO:39具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段为人源化抗体或嵌合抗体。
在另一个方面，本发明提供了一种分离的抗VEGF抗体或其抗原结合片段，其包含具有SEQ ID NO:27所示的氨基酸序列的重链CDR1域、具有SEQ ID NO:28所示的氨基酸序列的重链CDR2域和具有SEQ ID NO:29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:24所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO:25所示的氨基酸序列的轻链CDR2域和具有SEQ ID NO:26所示的氨基酸序列的轻链CDR3域的轻链可变区。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段包含具有SEQ ID NO:36所示的氨基酸序列或与SEQ ID NO:36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SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37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。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段为Fab片段，所述Fab片段进一步包含重链恒定区CH1和轻链恒定区，优选地所述重链 恒定区CH1为氨基酸序列为SEQ ID NO:40的IgG1重链恒定区的氨基酸序列或与SEQ ID NO:40具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链恒定区为氨基酸序列为SEQ ID NO:39的轻链恒定区的氨基酸序列或与SEQ ID NO:39具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段进一步包含重链信号肽和轻链信号肽，优选地所述重链信号肽为氨基酸序列为SEQ ID NO:34的氨基酸序列或与SEQ ID NO:34具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链信号肽为氨基酸序列为SEQ ID NO:35的氨基酸序列或与SEQ ID NO:35具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段是Fab抗体片段。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段为IgG抗体，优选为IgG1抗体。
在一些实施方式中，所述抗VEGF Fab抗体片段为IgG抗体相关Fab抗体片段，优选为IgG1抗体相关Fab抗体片段。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段为单克隆抗体。
在一些实施方式中，所述抗VEGF Fab抗体片段为单克隆的。
在一些实施方式中，所述抗VEGF抗体或其抗原结合片段与重组人VEGF165蛋白的结合亲和力K D为0.01-8E-10M，优选0.1-5E-10M，更优选0.5-3E-10M，最优选1.54E-10M。
在一些实施方式中，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；其中所述单链抗体分子优选为scFv、di-scFv、tri-scFv、双体抗体或scFab。
在又一个方面，本发明提供了一种抗体-药物缀合物，其包含本发明的抗VEGF抗体或其抗原结合片段和另外的治疗剂，优选地所述抗VEGF抗体或其抗原结合片段和所述另外的治疗剂通过接头连接。
在又一个方面，本发明提供了一种核酸，其编码本发明的抗VEGF抗体或其抗原结合片段。
在一些实施方式中，所述核酸包含SEQ ID NO:4所示的核苷酸序列和/或SEQ ID NO:5所示的核苷酸序列；或者其包含SEQ ID NO:20所示的核苷酸序列和/或SEQ ID NO:21所示的核苷酸序列；或者其包含SEQ ID NO:45所示的核苷酸序列和/或SEQ ID NO:46所示的核苷酸序列。优选地，所 述核酸进一步包含SEQ ID NO:49所示的核苷酸序列和/或SEQ ID NO:48所示的核苷酸序列。更优选地，所述核酸包含SEQ ID NO:41所示的核苷酸序列和/或SEQ ID NO:42所示的核苷酸序列。
在又一个方面，本发明提供了一种表达载体，其包含本发明的核酸。
在又一个方面，本发明提供了一种宿主细胞，其包含本发明的核酸或本发明的表达载体。
在又一个方面，本发明提供了一种用于产生本发明的抗VEGF抗体或其抗原结合片段的方法，其包括在适合于抗体表达的条件下培养如本发明的宿主细胞，和从培养基中回收表达的抗体。
在又一个方面，本发明提供了一种用于产生本发明的抗VEGF抗体或其抗原结合片段的方法，其包括在适合于Fab抗体片段表达的条件下培养如本发明的宿主细胞，和从培养基中回收表达的Fab抗体片段。
在又一个方面，本发明提供了一种药物组合物，其包含本发明的抗VEGF抗体或其抗原结合片段，或本发明的抗体-药物缀合物，或本发明的核酸，或本发明的表达载体，及药学上可接受的载体。
在又一个方面，本发明提供本发明的抗VEGF抗体或其抗原结合片段或本发明的抗体-药物缀合物或本发明的药物组合物，其用于治疗与血管生成相关的病症。
在一些实施方式中，所述与血管生成相关的病症是眼病。
在一些实施方式中，所述眼病是以脉络膜新生血管为特征的眼病，包括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
在又一个方面，本发明提供了一种用于治疗与血管生成相关的病症的方法，其包括向需要的受试者施用治疗有效量的本发明的抗VEGF抗体或其抗原结合片段或本发明的抗体-药物缀合物或本发明的药物组合物，从而治疗所述与血管生成相关的病症。
在一些实施方式中，所述与血管生成相关的病症是眼病。
在一些实施方式中，所述眼病是以脉络膜新生血管为特征的眼病，包括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
在又一个方面，本发明提供了本发明的抗VEGF抗体或其抗原结合片段或本发明的抗体-药物缀合物或本发明所述的药物组合物在制备用于治疗与血管生成相关的病症的药物中的用途。
在一些实施方式中，所述与血管生成相关的病症是眼病。
在一些实施方式中，所述眼病是以脉络膜新生血管为特征的眼病，包 括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
在又一个方面，本发明提供了一种药物组合，其包含本发明的抗VEGF抗体或其抗原结合片段或本发明的抗体-药物缀合物或本发明的药物组合物与一种或多种另外的治疗剂。
在又一个方面，本发明提供了一种试剂盒，其包含本发明的抗VEGF抗体或其抗原结合片段或本发明的抗体-药物缀合物或本发明的药物组合物，优选地，还进一步包含给药装置。
附图说明
本发明结合附图进行说明，附图中：
图1显示VEGF165兔源抗体VEGF-R988封闭VEGF165与VEGFR2蛋白结合。
图2显示VEGF165兔源抗体VEGF-R988中和VEGF165对HUVEC细胞的增殖。
图3显示ELISA检测人源化抗体VEGF-H988 Fab与VEGF165结合。
图4显示ELISA检测VEGF-H988 Fab与mVEGF164的种属交叉结合。
图5显示ELISA检测VEGF-H988 Fab阻断VEGF165与VEGFR2蛋白结合。
图6显示VEGF-H988 Fab对比Lucentis中和不同浓度VEGF165的作用。
图7显示VEGF-H988 Fab对比EYLEA中和不同浓度VEGF165的作用。
图8显示VEGF-H988 Fab对比Avastin中和不同浓度VEGF165的作用。
图9显示VEGF-H988 Fab对比Conbercept中和不同浓度VEGF165的作用。
图10显示VEGF-H988 Fab对比Brolucizumab中和不同浓度VEGF165的作用。
具体实施方式
本发明的各个方面涉及分离的抗VEGF Fab抗体片段、包含该抗体片段或其抗原结合片段的抗体-药物缀合物、编码该Fab抗体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VEGF Fab抗体片段的方法、包含该抗VEGF Fab抗体片段的药物组合物以及使用该抗VEGF Fab抗体片段治疗与血管生成相关的病症的方法。
定义
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。
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。
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。
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))。
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))。
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。
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。
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)。
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。
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。
“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。Fab片段通常包含重链可变区(V H)和重链恒定区1(C H1)和轻链可变区(V L)和轻链恒定区(C L)。
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。
术语“抗体-药物缀合物”或“ADC”是指与一种或多种化学药物(在本文中也称为药剂)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。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分裂抑制剂、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。
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。
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，携带异源免疫系统的转基因小鼠)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。
“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。
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，VEGF)，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。
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“K D”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。“K D”、“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。
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。
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。
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。
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))。
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。
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。
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)。
"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。
“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。
“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系统的第一组分(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。
“人类脐静脉内皮细胞(HUVEC)”分离自脐带静脉，一般用于生理学和药理学研究，例如例如大分子转运、血液凝固、血管发生和纤维蛋白溶解作用。特别是可以用作血管生成研究和其他依赖VEGF的信号通路研究(相关内皮生长因子)研究的模型。
本发明的抗体的氨基酸序列
本发明首先采用重组人VEGF165蛋白来免疫兔，然后通过噬菌体展示文库筛选获得与重组人VEGF165蛋白特异性结合的抗体克隆VEGF165-R988。之后采用PCR方法将编码VEGF165-R988scFv抗体的重链和轻链可变区的核苷酸序列分别插入带兔重链IgG1恒定区或兔轻链kappa恒定区核苷酸序列的pSTEP2载体中，并进行培养表达。采用蛋白A纯化柱进行纯化获得高纯度抗体。ELISA测试表明，兔源抗体VEGF165-R988可有效抑制VEGF165蛋白与VEGFR2蛋白结合，且VEGF165-R988可有效中和VEGF165促进HUVEC细胞增殖的能力。
然后，采用经典的人源化方式CDR移植方法，选择与兔轻链或重链可变区较接近的人抗体轻链或重链可变区为模板，将兔抗体轻链或重链的各3个CDR(表1)插入到该人抗体的可变区中，获得人源化的轻链可变区(VL)和重链可变区(VH)序列。由于兔源框架区有关键点对于支撑CDR的活性至关重要，因此将关键位点回复突变为兔抗体对应的序列。通过全基因合成的方法获得VEGF-H988-10轻链/重链表达载体，转染HEK-293细胞并进行培养表达，培养上清采用蛋白A纯化柱进行纯化获得高纯度VEGF-H988-10抗体。为了改善VEGF-H988-10抗体的亲和力，构建重链和轻链可变结构域CDR区(包括LCDR1、LCDR3、HCDR2和HCDR3)的SDM库，将4个突变库构建成scFv序列以基因3融合蛋白形式克隆至噬菌粒载体，对于各CDR通过可溶性抗原VEGF的结合能力筛选较优克隆，最终得到CDR亲和力和稳定性优化的VEGF-H988 Fab抗体片段并检测。
相比于全长结构，Fab片段的抗体具有更强的穿透性，和更低的胃肠道穿孔、高血压和出血等毒副作用且不能激发补体级联反应，从而降低引发眼内炎和自身免疫炎症反应的风险。
本发明的核酸
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:2-3、4-7、16-17、20-21、41-49和52-53。
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。
重组载体和表达
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插入载体例如质粒、噬粒、噬菌体或病毒载体中构建本发明的重组构建体。
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。
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。
细菌表达
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插入可操作阅读框中，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。
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(ATCC37017)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。
在细菌系统中，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。
哺乳动物表达和纯化
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。
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。
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。
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。
本发明的抗体的特性和功能
对本发明的人源化VEGF-H988 Fab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)与VEGF165蛋白结合的能力与Lucentis相近；
(2)与VEGF165蛋白的结合亲和力强于Lucentis的亲和力，约为Lucentis的亲和力的3.75倍；
(3)与重组人VEGF165蛋白有特异性结合，与重组小鼠mVEGF164蛋白无交叉结合；
(4)有效抑制VEGFR2蛋白与VEGF165蛋白的结合，且阻断能力明显优于Lucentis；和
(5)在不同浓度重组人VEGF165条件下，中和作用均强于Lucentis；高浓度VEGF165下，中和活性强于EYLEA；不同浓度的VEGF165条件下，活性均强于贝伐珠单抗及Brolucizumab，而与Conbercept活性相当。
用途
本发明的抗体可用于治疗与血管生成相关的病症，包括以脉络膜新生血管为特征的眼病，包括但不限于年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管等的发生(CNV)。
药物组合物
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。
试剂盒
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。
制备和储存
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。
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。
药物组合
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。
实施例
以下的实施例便于更好地理解本发明，但并不限定本发明。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。
实施例1：采用噬菌体抗体展示文库筛选阻断VEGF165与VEGFR2结合的兔源抗体
1.1兔子免疫
采用重组人VEGF165蛋白(来源：北京义翘神州科技有限公司，Cat.11066-HNAH)来对兔子进行免疫。该人VEGF165蛋白(UniProtKB P15692-4)的胞外区Met1-Arg191氨基酸序列为SEQ ID NO:1。
具体方法为：将重组人VEGF165蛋白与弗氏佐剂混合，使用混合物进行四次免疫，每次免疫剂量为500μg，皮下注射，免疫间隔依次为3周、2 周、2周。从第四次免疫起，免疫后四天经眼眶内眦静脉丛采血。采用ELISA方法，包被重组人VEGF165蛋白以检测兔抗VEGF165的血清效价。第五次免疫血清滴度达到25000倍稀释，第五次免疫9周后使用25μg重组人VEGF165蛋白进行静脉注射加强，7天之后处死兔子，取兔子的脾脏组织冻存于液氮中。
1.2噬菌体抗体库筛选
用TriPure Isolation Reagent试剂(来源：Roche，Cat.No.11 667 165 001)提取兔脾组织的RNA，用反转录试剂盒(来源：Invitrogen，Cat.No.18080-051)进行反转录获得cDNA。设计10对引物扩增兔抗体的轻链可变区序列，4对引物扩增重链可变区序列(Barbas C F et al.,CSHL Press.2004)。采用重叠延伸拼接PCR法将编码兔抗体轻链和重链可变区的序列拼接成编码scFv的核苷酸序列，轻重链可变区通过接头：
TCTAGTGGTGGCGGTGGTTCGGGCGGTGGTGGAGGTGGTAGTTCTAGATCTTCC(SSGGGGSGGGGGGSSRSS)(SEQ ID NO:2)
进行连接(Jones S T et al.,Bio/technology.1991；9(1):88)，再通过限制性内切酶Sfi I(来源：Fermentas)酶切连接到噬菌体载体pComb3x(来源：北京义翘神州科技有限公司)中，电转化X-Blue感受态细胞以构建免疫兔的噬菌体展示scFv抗体库。将重组人VEGF165蛋白包被在ELISA板上，按照噬菌体抗体淘选的流程，筛选获得抗VEGF165阳性抗体富集的噬菌体文库(O'Brien,P.M.,&Aitken,R.(Eds.),Springer Science&Business Media.2002；ISBN:9780896037113)。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VEGF165蛋白的结合，筛选获得与重组人VEGF165特异性结合的抗体克隆，将筛选得到的克隆送测序公司测序获得VEGF165-R988scFv抗体的核苷酸序列(SEQ ID NO:3)。
1.3靶向VEGF165兔源抗体的生产
以PCR扩增VEGF165-R988scFv抗体的重链可变区核苷酸序列，通过in-fusion方法插入到带重链信号肽(SEQ ID NO:43)和兔重链IgG1恒定区(SEQ ID NO:6)的经Sca I+Kpn I(Fermentas)酶切的pSTEP2载体中获得重链(SEQ ID NO:52)表达载体。PCR扩增VEGF-R988scFv抗体的轻链可变区核苷酸序列，通过in-fusion方法插入到带轻链信号肽(SEQ ID NO:44)和兔轻链kappa恒定区(SEQ ID NO:7)的经Sca I+BamH I(Fermentas)酶切的pSTEP2载体中获得轻链(SEQ ID NO:53)表达载体。提取重组质粒，转染HEK-293细胞进行培养表达7天，培养上清采用蛋白A纯化柱进行纯化获 得高纯度抗体。
扩增重链可变区引物：
F1 ACCAGGGTGCTGAGTCAGTCGGTGGAGGAGTCC
R1 TGTGACCAGGGTACCTGGGCCCCA
扩增轻链可变区引物：
F2 ACAGGAGTGCATAGTGAGCTCGATCTGACCCAGAC
R2 GGTGCAACTGGATCCCCTTTGACGACCACCTCGGT
1.4靶向VEGF165兔源抗体功能检测
1.4.1兔源抗体阻断VEGF165结合VEGFR2-his
将浓度为1μg/mL的VEGF165蛋白(来源：北京义翘神州科技有限公司)包被于96孔板上，每孔100μL，4℃包被过夜。次日洗板，室温封闭1小时后，加入100μL 5μg/mL VEGFR2-生物素蛋白(来源：北京义翘神州科技有限公司)和不同浓度VEGF-R988抗体共同孵育。洗板去除未结合抗体，加入链霉亲和素/HRP(来源：北京中杉金桥)孵育后重复洗板，最后加入底物显色液进行显色。终止显色后酶标仪读取OD450。结果分析以抗体浓度为横坐标，抑制率PI％为纵坐标，利用GraphPad Prism绘图分析。抑制率PI％＝(OD空白–OD样品)/OD空白×100％，其中OD空白表示只加VEGFR2-生物素不加抗体组的OD值，OD样品表示同时加VEGFR2-生物素和抗体的待测组OD值。
如图1所示，VEGF-R988抗体可有效结合包被的VEGF165蛋白，并可有效的抑制VEGFR165蛋白与VEGFR2蛋白的结合。
1.4.2兔源抗体对HUVEC增殖的抑制
采用WST-8法，检测嵌合抗体中和VEGF165对脐静脉内皮细胞(HUVEC)细胞的增殖作用。96孔板接种HUVEC细胞4×10 3个细胞/孔，在含有10％FBS及5％L-Gln的M199培养基中培养4h后加入不同浓度的VEGF-R988抗体，50μL/孔。随后加入终浓度为10ng/mL的VEGF165 10μL/孔。将96孔板置于37℃、5％CO 2条件下的CO 2培养箱内3天。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)对照。培养结束后加入WST-8显色液，10μL/孔，将96孔板置于CO 2培养箱中孵育，显色稳定后置酶标仪上于450nm、630nm处测定吸光度。以吸光度值(OD450–OD630)减去检 测空白孔B的读值来计算抗体的中和率，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，横坐标为样品的浓度，纵坐标为中和率，回归方程为四参数方程，得到“S”型曲线，计算样品半数有效浓度(EC 50)。
如图2所示，VEGF-R988抗体可有效降低VEGF165促进HUVEC增殖的能力。
实施例2：兔抗体VEGF-R988的人源化、突变改造和Fab形式抗体的生产
2.1兔抗体VEGF-R988轻链及重链的CDR确定
根据实施例1.2中测定的VEGF-R988scFv抗体的核苷酸序列，推导出VEGF-R988scFv的重链和轻链可变区氨基酸序列，见SEQ ID NO:8/9。
参考Kabat以及IMGT编号方式确定鼠抗体VEGF-R988-scFv轻链及重链各3个CDR的氨基酸序列，参见表1。上述的轻链及重链各3个CDR在后续步骤中被移植到人源化抗体VEGF-H988-10-scFv中，见实施例2.2。
表1：VEGF-R988轻链及重链CDR序列
名称 序列
LCDR1 QSSQTIYANRRLA(SEQ ID NO:10)
LCDR2 GASTLAS(SEQ ID NO:11)
LCDR3 AGYKSYDGDDVG(SEQ ID NO:12)
HCDR1 GIDLSSYAISWV(SEQ ID NO:13)
HCDR2 YIWNAGNTYYASWAKG(SEQ ID NO:14)
HCDR3 ARGTLGDYNGMDP(SEQ ID NO:15)
2.2通过CDR移植将兔VEGF-R988人源化
兔抗体人源化采用经典的人源化方式CDR移植方法，选择与兔轻链或重链可变区较接近的人抗体轻链或重链可变区为模板，将兔抗体轻链或重链的各3个CDR(表1)插入到该人抗体的可变区中，获得人源化的轻链可变区(VL)和重链可变区(VH)序列。所用VEGF-R988的轻链可变区的人源模板为IGKV1-27*01，该模板与VEGF-R988轻链的同源性为65.30％，重链可变区的人源模板为IGHV4-4*08，该模板与VEGF-R988重链的同源性为53.20％。
2.3将人源化可变区序列的框架区回复突变
由于兔源框架区有关键点对于支持CDR的活性至关重要，因此将关键位点点回复突变为兔抗体对应的序列，其中轻链的第1位回复突变为E，第2位回复突变为L，第4位回复突变为L，第63位回复突变为K；重链的第3位回复突变为V，第37位回复突变为V，第47位回复突变为Y，第78位回复突变为V，第79位回复突变为D，第91位回复突变为F，回复位点均为Kabat编号。经CDR人源化移植和框架区回复突变获得人源化抗体VEGF-H988-10。
2.4人源化抗体VEGF-H988-10的生产及CDR亲和力优化
通过全基因合成的方法获得VEGF-H988-10重链可变区(SEQ ID NO:20)，通过in-fusion方法插入到带重链信号肽(SEQ ID NO:43)和人IgG1恒定区(SEQ ID NO:47)的经Sca I+Nhe I(Fermentas)酶切的pSTEP2载体中获得VEGF-H988-10重链(SEQ ID NO:16)表达载体。通过全基因合成的方法获得VEGF-H988-10轻链可变区(SEQ ID NO:21)，通过in-fusion方法插入到带轻链信号肽(SEQ ID NO:44)和人kappa恒定区(SEQ ID NO:48)的经Sca I+BsiW I(Fermentas)酶切的pSTEP2载体中获得VEGF-H988-10轻链(SEQ ID NO:17)表达载体。提取质粒后转染HEK-293细胞进行培养表达7天，培养上清液采用蛋白A纯化柱进行纯化获得高纯度抗体。
全基因合成重链可变区引物：
Figure PCTCN2020102560-appb-000001
Figure PCTCN2020102560-appb-000002
全基因合成轻链可变区引物：
Figure PCTCN2020102560-appb-000003
为了改善VEGF-H988-10的亲和力，重链和轻链可变结构域CDR区构建SDM库，包括LCDR1、LCDR3、HCDR2三个饱和突变库；同时为了提高抗体的化学稳定性，需要将含有脱酰胺或者异构化位点改变成另一氨基 酸残基。天冬酰胺的脱酰胺可在NG、NS、NA、NT等序列上，导致产生异天冬氨酸残基，影响抗体的稳定性或生物功能。VEGF-H988可变结构域HCDR3存在易脱酰胺位点，为了提高抗体的化学稳定性和生物功能同时构建了SDM库。将4个突变库构建成scFv序列以基因3融合蛋白形式克隆至噬菌粒载体，对于各CDR通过可溶性抗原VEGF的结合能力筛选较优克隆，最终得到CDR亲和力和稳定性优化的抗体VEGF-H988。VEGF-H988轻链及重链CDR序列见表2。
表2：VEGF-H988轻链及重链CDR序列
名称 序列
LCDR1 QSSKFLWQGRRLA(SEQ ID NO:24)
LCDR2 GASTLAS(SEQ ID NO:25)
LCDR3 AGYKSYDGDVVG(SEQ ID NO:26)
HCDR1 GIDLSSYAIS(SEQ ID NO:27)
HCDR2 YIWNDLFTYYASWAKG(SEQ ID NO:28)
HCDR3 ARGTLGDYGGMDP(SEQ ID NO:29)
2.5人源化VEGF-H988 Fab的生产
将编码上述含有信号肽的VEGF-H988 Fab轻链核苷酸序列(SEQ ID NO:42)PCR扩增后，其中包含依次连接的轻链信号肽的核苷酸序列(SEQ ID NO:44)、人源化抗体轻链可变区的核苷酸序列(SEQ ID NO:46)和人kappa轻链恒定区的核苷酸序列(SEQ ID NO:48)，通过in-fusion方法插入自主研发的pGS载体(Kpn I+Xba I)中，通过测序验证获得正确的质粒。将VEGF-H988 Fab重链Fab核苷酸序列(SEQ ID NO:41)PCR扩增后，其中包含依次连接的重链信号肽的核苷酸序列(SEQ ID NO:43)、人源化抗体重链可变区的核苷酸序列(SEQ ID NO:45)和人IgG1重链CH1恒定区的核苷酸序列(SEQ ID NO:49)，通过in-fusion方法插入已经构建正确的包含轻链的pGS载体(Nhe I+Not I)中，通过测序验证获得正确的VEGF-H988 Fab轻重链表达载体。该表达载体包含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VEGF-H988 Fab高表达细胞株。采用ELISA检测选取抗体高表达的克隆，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株。采用无血清加料悬浮培养的方式培养VEGF-H988 Fab生产CHO细胞株，获得高纯度和高质量的VEGF-H988  Fab。
实施例3：人源化VEGF-H988 Fab特性分析
3.1 VEGF-H988 Fab结合VEGF165的特性
3.1.1 VEGF-H988 Fab特异性结合VEGF165
将不同浓度(0.15ng/mL、0.46ng/mL、1.37ng/mL、4.12ng/mL、12.35ng/mL、37.04ng/mL、111.11ng/mL、333.33ng/mL、1000ng/mL和3000ng/mL)的重组人VEGF165蛋白(来源：北京义翘神州科技有限公司)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 1μg/mL的VEGF165-H988 Fab、Lucentis(来源：Novartis)和阴性对照抗体H7N9-R1 Fab(来源：北京义翘神州科技有限公司)孵育1h，之后洗板去除未结合抗体，加入二抗山羊F(ab')2抗人IgG F(ab')2/HRP(来源：Jackson ImmunoResearch公司)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VEGF165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VEGF165蛋白结合的EC 50。
结果如图3所示，人源化抗体VEGF165-H988 Fab与重组人VEGF165的特异性结合EC 50为18.75ng/mL，R 2＝0.993；Lucentis结合的EC 50为12.87ng/mL，R 2＝0.989；人源化抗体VEGF165-H988 Fab与VEGF165蛋白结合的能力与Lucentis相近。阴性对照H7N9-R1 Fab与重组人VEGF165蛋白无结合。
3.1.2 VEGF-H988 Fab与VEGF165蛋白的结合亲和力
使用链霉亲和素包被的Sensor固定生物素标记的VEGF165蛋白，检测多个浓度点的VEGF-H988 Fab、Lucentis与VEGF165的亲和力。
首先将重组人VEGF165蛋白与生物素以摩尔比1:2的比率进行标记，方法如下：利用5000MW超滤管通过超滤形式将重组VEGF165蛋白的缓冲液(20mM Tris,150mM NaCl,pH8.0)更换为PBS，UV定量后得到567.57μg蛋白，所得蛋白与20mM生物素溶液以1:2摩尔比混匀，室温避光反应30分钟，通过5000MW超滤管再次过滤去除未标记的生物素，UV定量后加入等体积的甘油和终浓度0.1％的BSA后获得生物素标记蛋白。UV检测其浓度为2.08mg/mL。
然后测定不同浓度梯度VEGF-H988 Fab、Lucentis与生物素化的重组人VEGF165蛋白的亲和力，获得KD值为最终的亲和力。
如表3所示，VEGF-H988 Fab与重组人VEGF165蛋白结合亲和力KD 值为1.54E-10(M)，结合常数k on值为2.74E+05(1/Ms)，解离常数k dis值为4.21E-05(1/s)；Lucentis与VEGF165蛋白结合亲和力KD值为5.78E-11(M)，结合常数k on值为5.36E+04(1/Ms)，解离常数k dis值为3.10E-05(1/s)。结果表明VEGF-H988 Fab亲和力强于Lucentis的亲和力，约为Lucentis的亲和力的3.75倍。因此VEGF-H988 Fab比Lucentis具有更强的结合VEGF165蛋白的能力。
表3 VEGF-H988-48-IgG1(Fab-N103G)、Lucentis与重组VEG165F蛋白的亲和力
Figure PCTCN2020102560-appb-000004
3.1.3 VEGF-H988 Fab种属交叉
将重组人VEGF165蛋白、重组小鼠mVEGF164蛋白(来源：北京义翘神州科技有限公司)稀释到0.1μg/mL、1μg/mL和10μg/mL并分别包被于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VEGF165-H988 Fab、Lucentis和阴性对照抗体H7N9-R-Fab孵育1h，洗板去除未结合抗体，加入检测二抗山羊F(ab')2抗人IgG F(ab')2/HRP(来源：Jackson ImmunoResearch公司)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4所示，VEGF165-H988 Fab与重组人VEGF165蛋白有特异性结合，与重组小鼠mVEGF164蛋白有交叉结合，Lucentis与重组小鼠mVEGF164蛋白无交叉结合。
3.2 VEGF-H988 Fab阻断受体结合特性
将浓度为1μg/mL的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1小时后，加入100μL 2μg/mL VEGFR2-his蛋白(来源：北京义翘神州科技有限公司)，再加入不同浓度的人源化VEGF-H988 Fab、Lucentis和阴性对照抗体H7N9-R1-Fab(来源：北京义翘神州科技有限公司)共同孵育。洗板去除未结合抗体，加入C-his-R023/HRP孵育后重复洗板，最后加入底物显色液进行显色，终止后酶标仪读取OD450，每组2次平行。
以抗体浓度为横坐标，抑制率PI％为纵坐标，利用GraphPad Prism软件分析并绘图并计算IC50值。抑制率PI％＝(OD 空白–OD 样品)/OD 空白×100，其中OD空白表示只加VEGFR2-his不加抗体组的OD值，OD样品表示同时加VEGFR2-his和抗体的待测组OD值。
如图5所示，VEGFR2蛋白可有效结合包被的VEGF165蛋白，VEGF-H988 Fab可有效抑制VEGFR2蛋白与VEGF165蛋白的结合，且VEGF-H988 Fab阻断能力明显优于Lucentis，阴性对照抗体无抑制作用。
3.3 VEGF-H988 Fab阻滞不同浓度VEGF165生长抑制活性
采用WST-8法，检测人源化抗体中和VEGF165对HUVEC细胞的增殖作用。96孔板接种HUVEC细胞4×10 3/孔，在含有10％FBS及5％L-Gln的M199培养基中培养4h后加入不同浓度的抗体，50μL/孔。随后加入终浓度分别为1000ng/mL、100ng/mL、10ng/mL的VEGF165 10μL/孔。将96孔板置于37℃、5％CO 2条件下的CO 2培养箱内3天。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)对照。培养结束后加入WST-8显色液，10μL/孔，将96孔板置于CO 2培养箱中孵育，显色稳定后置酶标仪上于450nm、630nm处测定吸光度。以吸光度值(OD450–OD630)减去检测空白孔B的读值来计算抗体的中和率，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，横坐标为样品的浓度，纵坐标为中和率，回归方程为四参数方程，得到“S”型曲线，计算样品半数有效浓度(EC 50)。
如图6-10及表4所示，在不同浓度重组人VEGF165条件下，VEGF-H988Fab的中和作用均强于Lucentis。当VEGF165浓度为1000ng/mL、100ng/mL时，VEGF-H988 Fab的中和活性强于EYLEA，但当VEGF165浓度为10ng/mL时，VEGF-H988 Fab稍弱于EYLEA。不同浓度的VEGF165条件下，VEGF-H988 Fab的活性均强于贝伐珠单抗及Brolucizumab，而与Conbercept活性相当。随着VEGF165浓度的升高，VEGF-H988 Fab维持了较高的最大中和率，而EYLEA和Avastin则有所降低。
表4 VEGF-H988 Fab中和VEGF165作用的EC 50及最大中和率
Figure PCTCN2020102560-appb-000005
序列表
Figure PCTCN2020102560-appb-000006
Figure PCTCN2020102560-appb-000007
Figure PCTCN2020102560-appb-000008
Figure PCTCN2020102560-appb-000009
Figure PCTCN2020102560-appb-000010
Figure PCTCN2020102560-appb-000011
Figure PCTCN2020102560-appb-000012
Figure PCTCN2020102560-appb-000013
Figure PCTCN2020102560-appb-000014
Figure PCTCN2020102560-appb-000015
Figure PCTCN2020102560-appb-000016
Figure PCTCN2020102560-appb-000017
Figure PCTCN2020102560-appb-000018
Figure PCTCN2020102560-appb-000019
Figure PCTCN2020102560-appb-000020
Figure PCTCN2020102560-appb-000021

Claims (25)

  1. 一种分离的抗VEGF Fab抗体片段，其包含具有SEQ ID NO:27所示的氨基酸序列的重链CDR1域、具有SEQ ID NO:28所示的氨基酸序列的重链CDR2域和具有SEQ ID NO:29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:24所示的氨基酸序列的轻链CDR1域、具有SEQ ID NO:25所示的氨基酸序列的轻链CDR2域和具有SEQ ID NO:26所示的氨基酸序列的轻链CDR3域的轻链可变区。
  2. 如权利要求1所述的抗VEGF Fab抗体片段，其包含具有SEQ ID NO:36所示的氨基酸序列或与SEQ ID NO:36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SEQ ID NO:37所示的氨基酸序列或与SEQ ID NO:37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。
  3. 如权利要求1或2所述的抗VEGF Fab抗体片段，其为Fab片段，所述Fab片段进一步包含重链恒定区CH1和轻链恒定区，优选地所述重链恒定区CH1为氨基酸序列为SEQ ID NO:40的IgG1重链恒定区的氨基酸序列或与SEQ ID NO:40具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链恒定区为氨基酸序列为SEQ ID NO:39的轻链恒定区的氨基酸序列或与SEQ ID NO:39具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
  4. 如权利要求1-3中任一项所述的抗VEGF Fab抗体片段，其进一步包含重链信号肽和轻链信号肽，优选地所述重链信号肽为氨基酸序列为SEQ ID NO:34的氨基酸序列或与SEQ ID NO:34具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列，和/或所述轻链信号肽为氨基酸序列为SEQ ID NO:35的氨基酸序列或与SEQ ID NO:35具有至少90％、92％、95％、98％或99％序列同一性的氨基酸序列。
  5. 如权利要求1-4中任一项所述的抗VEGF Fab抗体片段，其为IgG抗体相关Fab抗体片段，优选为IgG1抗体相关Fab抗体片段。
  6. 如权利要求1-5中任一项所述的抗VEGF Fab抗体片段，其为单克隆的。
  7. 如权利要求1-6中任一项所述的抗VEGF Fab抗体片段，其与重组人VEGF165蛋白的结合亲和力KD为0.01-8E-10M，优选0.1-5E-10M，更优选0.5-3E-10M，最优选1.54E-10M。
  8. 一种抗体-药物缀合物，其包含如权利要求1-7中任一项所述的抗VEGF Fab抗体片段和另外的治疗剂，优选地所述抗VEGF Fab抗体片段和所述另外的治疗剂通过接头连接。
  9. 一种核酸，其编码根据权利要求1-7中任一项所述的抗VEGF Fab抗体片段。
  10. 如权利要求9所述的核酸，其包含SEQ ID NO:45所示的核苷酸序列和/或SEQ ID NO:46所示的核苷酸序列；优选地，其进一步包含SEQ ID NO:49所示的核苷酸序列和/或SEQ ID NO:48所示的核苷酸序列；更优选地，其包含SEQ ID NO:41所示的核苷酸序列和/或SEQ ID NO:42所示的核苷酸序列。
  11. 一种表达载体，其包含如权利要求9或10所述的核酸。
  12. 一种宿主细胞，其包含如权利要求9或10所述的核酸或如权利要求11所述的表达载体。
  13. 一种用于产生如权利要求1-7中任一项所述的抗VEGF Fab抗体片段的方法，其包括在适合于Fab抗体片段表达的条件下培养如权利要求12所述的宿主细胞，和从培养基中回收表达的Fab抗体片段。
  14. 一种药物组合物，其包含如权利要求1-7中任一项所述的抗VEGF Fab抗体片段，或如权利要求8所述的抗体-药物缀合物，或如权利要求9或10所述的核酸，或如权利要求11所述的表达载体，及药学上可接受的载体。
  15. 如权利要求1-7中任一项所述的抗VEGF Fab抗体片段或如权利要求8所述的抗体-药物缀合物或如权利要求14所述的药物组合物，其用于治疗与血管生成相关的病症。
  16. 如权利要求15所述的抗VEGF Fab抗体片段或者抗体-药物缀合物或者药物组合物，其中所述与血管生成相关的病症是眼病。
  17. 如权利要求16所述的抗VEGF Fab抗体片段或者抗体-药物缀合物或者药物组合物，其中所述眼病是以脉络膜新生血管为特征的眼病，包括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
  18. 一种用于治疗与血管生成相关的病症的方法，其包括向需要的受试者施用治疗有效量的如权利要求1-7中任一项所述的抗VEGF Fab抗体片段或如权利要求8所述的抗体-药物缀合物或如权利要求14所述的药物组合物，从而治疗所述与血管生成相关的病症。
  19. 如权利要求18所述的方法，其中所述与血管生成相关的病症是眼病。
  20. 如权利要求19所述的方法，其中所述眼病是以脉络膜新生血管为特征的眼病，包括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
  21. 如权利要求1-7中任一项所述的抗VEGF Fab抗体片段或如权利要求8所述的抗体-药物缀合物或如权利要求14所述的药物组合物在制备用于治疗与血管生成相关的病症的药物中的用途。
  22. 如权利要求21所述的用途，其中所述与血管生成相关的病症是眼病。
  23. 如权利要求22所述的用途，其中所述眼病是以脉络膜新生血管为特征的眼病，包括年龄相关性老年黄斑变性(AMD)、糖尿病性黄斑水肿(DME)、视网膜水肿、退行性近视、脉络膜新生血管(CNV)。
  24. 一种药物组合，其包含如权利要求1-7中任一项所述的抗VEGF Fab抗体片段或如权利要求8所述的抗体-药物缀合物或如权利要求14所述的药物组合物与一种或多种另外的治疗剂。
  25. 一种试剂盒，其包含如权利要求1-7中任一项所述的抗VEGF Fab抗体片段或如权利要求8所述的抗体-药物缀合物或如权利要求14所述的药物组合物，优选地，还进一步包含给药装置。
PCT/CN2020/102560 2019-07-19 2020-07-17 人源化抗VEGF Fab抗体片段及其用途 WO2021013065A1 (zh)

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