WO2016109943A1 - 抗vegf抗体 - Google Patents

抗vegf抗体 Download PDF

Info

Publication number
WO2016109943A1
WO2016109943A1 PCT/CN2015/070209 CN2015070209W WO2016109943A1 WO 2016109943 A1 WO2016109943 A1 WO 2016109943A1 CN 2015070209 W CN2015070209 W CN 2015070209W WO 2016109943 A1 WO2016109943 A1 WO 2016109943A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
vegf
cdr1
cdr3
Prior art date
Application number
PCT/CN2015/070209
Other languages
English (en)
French (fr)
Inventor
方海洲
曲伟
郑赞顺
庄兰芳
王新志
Original Assignee
珠海亿胜生物制药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 珠海亿胜生物制药有限公司 filed Critical 珠海亿胜生物制药有限公司
Priority to EP15864295.9A priority Critical patent/EP3078674B1/en
Priority to PCT/CN2015/070209 priority patent/WO2016109943A1/zh
Priority to AU2015376558A priority patent/AU2015376558B9/en
Priority to US15/101,472 priority patent/US10456466B2/en
Priority to ES15864295T priority patent/ES2833530T3/es
Publication of WO2016109943A1 publication Critical patent/WO2016109943A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • 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
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • 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
    • C07K2317/50—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
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to antibodies and their use, in particular, the present invention relates to antibodies, particularly heavy chain antibodies, more particularly single domain antibodies, which specifically bind to Vascular Endothelial Growth Factor (VEGF); Preparation methods and therapeutic uses.
  • VEGF Vascular Endothelial Growth Factor
  • Angiogenesis refers to the development of new blood vessels from existing capillaries or posterior veins of the capillaries. It is a complex process involving multiple molecules of multiple cells. Angiogenesis is a complex process in which the angiogenic factors and inhibitors coordinate. Under normal conditions, the two are in equilibrium. Once this balance is broken, the vascular system is activated, causing excessive angiogenesis or inhibiting the vascular system to degenerate blood vessels.
  • diseases are known to be associated with uncontrolled angiogenesis and undesired angiogenesis.
  • diseases include, but are not limited to, tumors such as so-called solid tumors and liquid (or blood) tumors (such as leukemias and lymphomas), inflammation such as rheumatoid or rheumatic inflammation, especially arthritis (including rheumatoid arthritis), or Other chronic inflammations such as chronic asthma, arteriosclerosis or post-transplant atherosclerosis, endometriosis, ocular neovascular diseases such as retinopathy (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis.
  • Other diseases associated with uncontrolled angiogenesis and undesired angiogenesis will be apparent to those skilled in the art.
  • Vascular endothelial growth factor a heparin-binding growth factor specific for vascular endothelial cells, induces angiogenesis in vivo.
  • VEGF-A vascular endothelial growth factor
  • VEGF-B vascular endothelial growth factor
  • VEGF-C vascular endothelial growth factor
  • VEGF-D vascular endothelial growth factor
  • VEGF-E vascular endothelial growth factor
  • placental growth factors include VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factors.
  • VEGF-A The main role of VEGF-A is to promote vascular endothelial cell proliferation, migration and lumen formation, as well as increase vascular leakage, promote monocyte chemotaxis and B cell production.
  • the biological effect of VEGF-A is mediated by binding to its specific receptor, and its The main receptors are the specific receptors vascular endothelial growth factor receptor 1 (VEGFR-1) and VEGFR-2.
  • VEGFR-2 is considered to be the main VEGFR, which has an important influence on the proliferation of vascular endothelial cells.
  • VEGFR-2 induces VEGF-binding dimers and receptors that require autophosphorylation by intracellular kinases, thereby enhancing mitosis (Klettner A, Roider J.
  • VEGF-A includes 8 exons and 7 introns, which are transcribed into multiple subtypes, mainly: VEGF121, VEGF145, VEGF206, VEGF165, VEGF189. These subtypes have different molecular mass, solubility and heparin. Binding ability, wherein VEGF165 is the most important subtype of VEGF-A (Ferrara N, Gerber HP, Le Couter J. The biology of VEGF and its receptors. Nat Med, 2003, 9(6): 669-676).
  • VEGF165 is a secreted soluble protein that directly acts on vascular endothelial cells to promote vascular endothelial cell proliferation, accelerate the repair of vascular endothelial cell injury, increase vascular permeability, reduce intravascular thrombosis and thrombotic occlusion, and inhibit intimal hyperplasia.
  • vascular endothelial growth factor drugs include pegaptanib sodium (trade name Macugen), Ranibizumab (trade name Lucentis), bevacizumab (trade name Avastin), VEGF Trap, and the like.
  • the current focus of controversy over anti-VEGF formulations is that it may aggravate the formation of tissue fiber membranes.
  • Anti-VEGF drugs currently used clinically for the treatment of various diseases require frequent intraocular injections, leading to the potential risk of endophthalmitis, which is a significant problem in the treatment of anti-VEGF.
  • VEGF-165 and VEGF-121 mainly affect blood vessel growth
  • VEGF-189 mainly affects fibers.
  • Both vaccinizumab and ranibizumab inhibit all active VEGF-A isoforms (Van Bergen T, Vandewalle E, Van de Veire S, et al. The role of Different VEGF isoforms in scar formation after glaucoma filtration surgery.
  • anti-VEGF drugs need repeated treatment every 4 to 6 weeks.
  • the average annual injection volume of ranibizumab treatment in the first year is about 6.9 times, and that they are about 7.7 times (Li X, Hu Y, Sun X, Zhang J).
  • Zhang M. Bevacizumab for neovascular age-related macular degeneration in China. Ophthalmology. 2012 Oct., 119 (10): 2087-93) such frequent intraocular injection treatment has the potential risk of developing endophthalmitis, urgently needed to develop drugs Effectiveness, retinal permeability absorbs better new antibody drugs to prolong the dosing cycle and reduce the discomfort and risk of injection administration.
  • a heavy chain antibody is an antibody isolated from the serum of a camelid. It consists of only a heavy chain, and its antigen-binding region is only a single domain joined to the Fc region through a hinge region, and this antigen-binding region is self-antibody. Since it still has the function of binding antigen after separation, it is called a single-domain antibody (sdAb) or a nanobody. Unlike traditional antibodies, a single domain antibody is a peptide chain of about 110 amino acids with a molecular weight of about 1/10 that of a conventional antibody, which provides a new method for molecular construction of antibodies (Muyldermans. Single domain camel) Antibodies: current status. J Biotechnol 2001, 74: 277-302).
  • Single-domain antibodies have small molecular weight, good thermal stability, stability in detergents and high concentrations of uric acid, good tissue permeability and good solubility in vivo (Stanfield R, Dooley H, Flajnik M, Wilson I. Crystal structure of a Shark single-domain antibody V region in complex with lysozyme.Science.2004, 305 (5691)), easy to express, facilitate expression of prokaryotic system, low production cost, unique antigen recognition epitope, and can recognize hidden antigenic sites, etc. Characteristics, in the immune experiment, diagnosis and treatment, gradually play a huge function beyond imagination (Dirk Saerens, Gholamreza Hassanzadeh Ghassabeh, Serge Muyldermans. Single-domain antibodies as building blocks for novel therapeutics. Current Opinion in Pharmacology 2008, 8: 600-608).
  • the invention provides an anti-VEGF antibody, variant or derivative thereof, wherein the antibody comprises a heavy chain variable region comprising: (i) SEQ ID NO: 1, SEQ ID NO: CDR1, CDR2 and CDR3 of SEQ ID NO: 3 or a functionally active variant thereof; or (ii) CDR1, CDR2 and CDR3 of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a functionally active variant thereof; or (iii) CDR1, CDR2 and CDR3 of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a functionally active variant thereof; or (iv) SEQ ID NO: 10.
  • the invention provides a heavy chain antibody consisting of a heavy chain, the variable region of the heavy chain comprising: (i) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 CDR1, CDR2 and CDR3; or (ii) CDR1, CDR2 and CDR3 of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or a functionally active variant thereof; Iii) CDR1, CDR2 and CDR3 of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a functionally active variant thereof; or (iv) SEQ ID NO: 10, SEQ ID NO: 11 and CDR1, CDR2 and CDR3 of SEQ ID NO: 12 or a functionally active variant thereof; or (v) CDR1, CDR2 and CDR3 of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15 or a functionally active variant thereof; or (vi) CDR1, CDR2 and CDR3 of SEQ ID NO: 1,
  • the heavy chain variable region of an antibody of the invention can comprise at least one amino acid addition, insertion, deletion, and/or substitution.
  • the antibodies of the invention can be monoclonal antibodies, chimeric or humanized antibodies, multispecific antibodies and/or bispecific antibodies, and fragments thereof.
  • the antibody of the invention is a humanized antibody.
  • the heavy chain of an antibody of the invention may also contain a constant region.
  • the heavy chain of an antibody of the invention further comprises an Fc fragment.
  • the antibodies of the invention are heavy chain antibodies, ie, consist only of heavy chains. In some specific embodiments, the antibodies of the invention are single domain antibodies body.
  • the present invention provides an antibody which competes with a reference antibody for binding to VEGF, and the reference antibody is any one of the above antibodies.
  • the invention also relates to nucleic acid sequences encoding the above antibodies; vectors comprising the nucleic acid sequences; and host cells which express and/or comprise such nucleic acid sequences or vectors.
  • the present invention also provides a method of producing an antibody, the method comprising: cultivating the above host cell under conditions permitting expression of the antibody; and purifying the antibody from the obtained culture product.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may further comprise one or more therapeutically active compounds, such as known anti-VEGF drugs or anti-tumor drugs.
  • the invention relates to an antibody-drug conjugate (ADC) comprising an antibody of the invention conjugated to another agent, such as a chemotherapeutic agent, a growth inhibitor, a toxin (eg, a bacterium, An enzymatically active toxin of fungal, plant or animal origin, or a fragment thereof) or a radioisotope (ie, a radioactive conjugate).
  • ADC antibody-drug conjugate
  • the antibody-conjugated drug can also comprise a linker unit between the drug unit and the antibody unit.
  • the invention relates to methods of modulating VEGF activity by administering an effective amount of an antibody of the invention.
  • the present invention relates to a method of inhibiting angiogenesis by administering an effective amount of an antibody of the present invention to a patient in need thereof.
  • the invention also provides a method of treating a disease or condition associated with VEGF, the method comprising administering to a patient in need thereof an effective amount of at least one antibody of the invention.
  • the disease or condition includes a tumor or cancer or an ophthalmic disease.
  • the tumor or cancer includes breast cancer, brain tumor, kidney cancer, ovarian cancer, thyroid cancer, lung cancer, colorectal cancer, endometrial cancer, angiosarcoma, bladder cancer, embryonic tissue cancer, neck tumor, malignant glioma , stomach cancer, pancreatic cancer, nasopharyngeal cancer, etc.
  • the ophthalmic diseases include macular edema caused by various causes (including diabetic macular edema, post-cataract surgery, or after uveitis). Disease-induced macular edema, age-related macular degeneration, diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma, and other ophthalmic diseases involving ne
  • the present invention relates to the use of the antibody of the present invention for the preparation of a medicament for regulating VEGF activity; the use of the antibody of the present invention for the preparation of a medicament for inhibiting angiogenesis; the antibody of the present invention is prepared for treatment and Use in drugs for VEGF-related diseases or conditions.
  • the invention also provides a kit comprising a) an antibody of the invention, or the pharmaceutical composition; and b) instructions for use.
  • Figure 1 is a graph showing the results of SDS-PAGE detection of purified hVEGF165 protein.
  • the first lane is the standard protein Marker (Invitrogen, Cat. No.: LC5677); the second lane is 2 ⁇ g of non-reduced hVEGF165; the third lane is 5 ⁇ g of non-reduced hVEGF165; the fourth lane is 2 ⁇ g of reduced hVEGF165; the fifth lane is 5 ⁇ g of reduced hVEGF165.
  • Figure 2 is a result of an immunoreaction test showing that the animal produced a better immune response after injection of the antigen, and the serum titer was about 1:100k.
  • Figure 3 shows the results of agarose gel electrophoresis of total RNA, indicating that the resulting RNA quality is consistent with the library construction requirements.
  • Fig. 4 is a result of agarose gel electrophoresis purification of the amplified V H H fragment obtained by reverse transcription of the total RNA of Fig. 3 into cDNA.
  • FIG 5 is a map of phagemid vector connecting V H H fragments.
  • Figure 6 is a graph showing the detection of the phage display library fragment insertion rate.
  • Fig. 7 is a diagram showing the diversity of the library of the single domain antibody library obtained by sequencing the positive clone having the inserted fragment in Fig. 6, showing that the library diversity is good.
  • Figure 8 is a FASEBA screening specific vector map.
  • the carrier is ampicillin resistant Sex, containing SASA and 6 ⁇ His tag, can be used for secretory expression of antibodies.
  • Figure 9 is an affinity ranking of antibodies after FASEBA screening; 9A, 9B, 9C are the results of affinity ranking for 3 different batches.
  • the upper left picture the sensory map of the binding and dissociation of different clones; the upper right picture: the matrix of the combination and dissociation rate of different clones; the lower left picture: the sensor map of different clones normalized; the lower right picture: Sensing map of antibodies with higher affinity.
  • Figure 10 is a graph of receptor competitive screening results in which 15 single domain antibodies, preferably subjected to expression level screening and affinity sequencing, were used for the screening. Based on this competitive result, 7 of the better clones were selected for heavy chain antibody preparation for cell proliferation inhibition experiments compared to controls.
  • Figure 11 is a graph of heavy chain antibody against HUVEC cell proliferation inhibition assay. Judging by the degree of inhibition of cell proliferation by different concentrations of antibodies, 13 heavy chain antibodies have inhibitory functions, among which A80887, A80723 and A69458 have the strongest inhibitory function at the cellular level.
  • Figure 12 is a variable region sequence of 13 heavy chain antibodies in Example 11.
  • Figure 13 is a schematic diagram of the intestines of the zebrafish. After a certain period of administration, 15 zebrafish were randomly taken from each group and photographed under a fluorescence microscope to quantitatively analyze the area of the inferior vascular plexus (SIVs). T-test was used to compare the two groups. One-way analysis of variance and Dunnett's T-test were used for statistical analysis. P ⁇ 0.05 showed statistical difference.
  • the formula for calculating the effect of inhibiting angiogenesis was as follows:
  • Figure 14 is a graph of the area of the intestine vessel. As can be seen from the figure, A80887, like Avastin, has significant neovascular inhibition.
  • Fig. 15 is a graph showing the area inhibition rate of the intestinal plexus.
  • Avastin was a positive control drug.
  • A80887 inhibited neovascularization efficiency by 23.2%, which was significantly better than Avastin (9%) (p ⁇ 0.001).
  • the present invention relates to antibodies, variants or derivatives thereof that specifically bind to VEGF; and to methods of preparation and therapeutic use of such antibodies.
  • the invention relates to heavy chain antibodies that specifically bind to VEGF, more particularly single domain antibodies.
  • the antibodies of the invention show superior effects over inhibition of cell proliferation and angiogenesis over prior art anti-VEGF monoclonal antibodies (e.g., Avastin), as further described in the Examples below.
  • the single domain antibody of the present invention has a smaller molecular weight than the Fab fragment and the full-length IgG antibody, generally 12-15 kD, and can be used for constructing multivalent antibodies, and is genetically engineered to increase affinity and prolong half-life, and to extend the interval of administration. And other characteristics.
  • the binding ability of single-domain antibody drugs to antigens is more stable under extreme conditions such as high temperature, gastric acid and protease, and has high conformational stability.
  • single-domain antibody drugs lack an Fc fragment and do not cause a complement effect.
  • the antibody can have better permeability in ocular tissue and tumor tissue administration. Stability in protease, extreme temperature and pH environments, high affinity, and oral and other routes of administration provide feasibility.
  • Single-domain antibodies can be expressed in large scale in prokaryotic or eukaryotic cells, such as E. coli or yeast cells, and the expression amount is large, which greatly facilitates mass production, is conducive to controlling production costs, and is also beneficial for later drug development. Market prospects.
  • antibody is well understood in the biological and biomedical fields and generally refers to intact antibodies and any antibody fragments or single chains thereof.
  • An antibody is a glycoprotein secreted by specialized B lymphocytes called plasma cells. It is also known as immunoglobulin (Ig) because it contains a consensus domain found in many proteins.
  • the antibody most likely comprises two heavy (H) chains and two light (L) chains or antigen binding portions thereof, usually joined by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region ( VH ) and a heavy chain constant region.
  • VH heavy chain variable region
  • L heavy chain constant region
  • the light chain constant region consists of one domain CL.
  • V I and V H regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs of), interspersed with regions that are more conserved, termed framework regions (FR) of.
  • CDRs of complementarity determining regions
  • FR framework regions
  • the antibodies of the invention consist only of heavy chains.
  • the antibodies of the invention are single domain antibodies.
  • the complementarity determination of a given antibody can be determined using the method described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. Region (CDR) and framework region (FR).
  • the invention includes "variants" of antibodies, for example, the heavy chain variable region of an antibody of the invention may comprise at least one amino acid addition, insertion, deletion and/or substitution, for example 10, 20, 30, 40, 50, preferably for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid additions, insertions, deletions, and/or substitutions.
  • the invention also includes “derivatives” of antibodies.
  • a “derivative” of an antibody is a chemically modified antibody, for example by binding to other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • albumin e.g., human serum albumin
  • phosphorylation e.g., phosphorylation
  • glycosylation e.g., glycosylation
  • antibody includes fragments, derivatives, variants thereof.
  • the invention provides an anti-VEGF antibody, variant or derivative thereof, wherein the antibody comprises a heavy chain variable region comprising: (i) SEQ ID NO: 1, SEQ ID NO : 2 and CDR1, CDR2 and CDR3 of SEQ ID NO: 3 or a functionally active variant thereof; or (ii) CDR1, CDR2 of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: And CDR3 or a functionally active variant thereof; or (iii) CDR1, CDR2 and CDR3 of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a functionally active variant thereof; or (iv) SEQ ID NO: 10, CDR1, CDR2 and CDR3 of SEQ ID NO: 11 and SEQ ID NO: 12 or a functionally active variant thereof; or (v) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO : CDR1, CDR2 and CDR3 or functionally active variants thereof; or (vi) CDR
  • the functionally active variant is at least 70%, such as at least 75%, at least 80%, at least 85%, at least 90%, such as 91%, 92%, with the amino acid sequence of any one of SEQ ID NOs: 1-33, Functionally active variants of 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity.
  • the invention provides a heavy chain antibody consisting of a heavy chain, the variable region of the heavy chain comprising: (i) SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, CDR1, CDR2 and CDR3 or a functionally active variant thereof; or (ii) CDR1, CDR2 and CDR3 of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 or functions thereof An active variant; or (iii) CDR1, CDR2 and CDR3 of SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9 or a functionally active variant thereof; or (iv) SEQ ID NO: CDR1, CDR2 and CDR3 set forth in SEQ ID NO: 11 and SEQ ID NO: 12, or a functionally active variant thereof; or (v) SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: CDR1, CDR2 and CDR3 or functionally active variants thereof; or (vi) CDR1, CDR2
  • the heavy chain of an antibody of the invention may also contain a constant region.
  • the heavy chain of an antibody of the invention further comprises an Fc fragment.
  • the antibodies of the invention are heavy chain antibodies, ie, consist only of heavy chains. In some specific embodiments, the antibodies of the invention are single domain antibodies.
  • the heavy chain variable region sequence of an antibody of the invention is SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45 or SEQ ID NO: 46.
  • the present invention provides a competitive binding of a VEGF antibody to a reference antibody, which is any one of the above antibodies.
  • the invention also relates to nucleic acid sequences encoding the above antibodies; vectors comprising the nucleic acid sequences; and host cells which express and/or comprise such nucleic acid sequences or vectors.
  • a "host cell” is a cell used to express a nucleic acid, such as a nucleic acid of the invention.
  • the host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a single cell eukaryote (eg, yeast).
  • the nucleic acid sequence is SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58 or SEQ ID NO: 59, as detailed in the Examples below .
  • the invention also provides a method of making an antibody, the steps comprising: cultivating the host cell under conditions permitting expression of the antibody; and purifying the antibody from the resulting culture product, as detailed in the Examples below.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an antibody of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition may further comprise one or more therapeutically active compounds, such as known anti-VEGF drugs or anti-tumor drugs.
  • the therapeutically active compound can be administered simultaneously or sequentially with the antibody of the invention.
  • compositions can be prepared according to techniques known in the art.
  • excipient generally refers to any component other than one or more active therapeutic ingredients.
  • the excipient can be an inert substance, an inactive substance, and/or a pharmaceutically inactive substance.
  • Excipients can be used for a variety of purposes, for example, as carriers, vehicles, diluents, tablet adjuvants, and/or to improve administration and/or absorption of the active substance.
  • Formulation of pharmaceutically active ingredients with various excipients is known in the art, see, for example, Remington: The Science and Practice of Pharmacy (e.g., 19th Edition (1995), and any subsequent editions).
  • Non-limiting examples of excipients are: solvents, diluents, buffers, preservatives, tonicity adjusters, chelating agents, and stabilizers.
  • the antibody of the present invention can be administered in the form of a pharmaceutical composition. It can be prepared not only as a liquid preparation such as an injection solution, a lyophilized preparation, or a spray, but also as a solid preparation such as a capsule.
  • the route of administration may be, for example, intravenous injection, oral or topical administration, such as transdermal, transconjunctival, and/or transocular. In a specific embodiment, the route of administration is oral. In another specific embodiment, the route of administration is via the eye.
  • the invention relates to an antibody-conjugated drug comprising an antibody of the invention conjugated to another agent, such as a chemotherapeutic agent, a growth inhibitor, a toxin (eg, a bacterial, fungal, plant or animal derived enzyme) An active toxin, or a fragment thereof) or a radioisotope (ie, a radioactive conjugate).
  • a chemotherapeutic agent such as a chemotherapeutic agent, a growth inhibitor, a toxin (eg, a bacterial, fungal, plant or animal derived enzyme) An active toxin, or a fragment thereof) or a radioisotope (ie, a radioactive conjugate).
  • a chemotherapeutic agent such as a chemotherapeutic agent, a growth inhibitor, a toxin (eg, a bacterial, fungal, plant or animal derived enzyme) An active toxin, or a fragment thereof) or a radioisotope (ie, a
  • Antibody-conjugated drugs typically comprise a linker between the drug unit and the antibody unit unit.
  • the linker can be cleaved under intracellular conditions such that cleavage of the linker results in release of the drug unit from the antibody in the intracellular environment.
  • the linker can be, for example, a peptidyl linker that can be cleaved by an intracellular peptidase or protease, including but not limited to: lysosomal or endosomal proteases.
  • the peptidyl linker is at least two amino acids in length or at least 3 amino acids in length.
  • the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker.
  • the linker unit is not cleavable and the drug is released by, for example, degradation of the antibody.
  • the present invention relates to a method of modulating (preferably inhibiting) VEGF activity and inhibiting angiogenesis in a mammal by administering an effective amount of the antibody of the present invention.
  • the invention relates to methods of modulating VEGF activity by administering an effective amount of an antibody of the invention.
  • the present invention relates to a method of inhibiting angiogenesis by administering an effective amount of an antibody of the present invention to a patient in need thereof.
  • the invention also provides a method of treating a disease or condition associated with VEGF, the method comprising administering to a patient in need thereof an effective amount of at least one antibody of the invention.
  • the disease or condition includes a tumor or cancer or an ophthalmic disease.
  • the tumor or cancer includes breast cancer, brain tumor, kidney cancer, ovarian cancer, thyroid cancer, lung cancer, colorectal cancer, endometrial cancer, angiosarcoma, bladder cancer, embryonic tissue cancer, neck tumor, malignant glioma , stomach cancer, pancreatic cancer, nasopharyngeal cancer, etc.
  • the ophthalmic diseases include macular edema caused by various causes (including diabetic macular edema, macular edema caused by various diseases such as cataract surgery or uveitis), age-related macular degeneration, diabetic retinopathy, and central retina Venous obstruction, neovascular glaucoma, and other ophthalmic diseases involving neovascularization.
  • the "patient in need” means any mammal such as, but not limited to, human, horse, cow, cat, mouse, rabbit, rat, goat, and the like.
  • the mammal is a human.
  • a plurality of antibodies of the invention are administered in combination.
  • the present invention also relates to the preparation of the antibody of the present invention for regulating VEGF activity Use in a medicament; use of an antibody of the invention in the manufacture of a medicament for inhibiting angiogenesis; use of an antibody of the invention in the manufacture of a medicament for the treatment of a disease or condition associated with VEGF.
  • the invention also provides a kit comprising a) an antibody of the invention, or the pharmaceutical composition; and b) instructions for use.
  • the antigen targeted by the present embodiment is human VEGF165 (Human vascular endothelial growth factor 165, hVEGF 165) molecule (Park JE, Keller GA, Ferrara N.
  • the optimized DNA is obtained by total synthesis, and cloned into the eukaryotic expression vector pTT5 (authorized by the inventor NRC) for preparation of transfection level.
  • Alpaca (Lama pacos) was selected as an experimental animal, and six points of immunization were performed on the shoulder and back at four different time points.
  • PBS is a dilution of the antigen
  • each immunization volume is 1 ml
  • the antigen amount and adjuvant information are shown in Table 2.
  • the immunological reagent contains BSA at a final concentration of 1 mg/ml, and the antigen and the adjuvant are freshly prepared and mixed before injection, and then immunized.
  • the immune process design (Table 3) was collected in the jugular vein at four different times, and anticoagulant was added when blood was collected. The first blood collection was 5 ml, and the remaining three were 15 ml each. After centrifugation with Ficoll 1.077 reagent (Sangon, Cat. No.: F760014-100) and anticoagulation, peripheral blood lymphocytes were isolated and cell resuspended counted, and RNAlater (TIANGEN, Cat. No.: DP408-) was added. 02), stored at -20 °C. Serum obtained by gradient centrifugation was also stored at -20 °C.
  • Serum samples from pre-immune, third-immunization and fourth immunization were tested for antigen-specific immunoreactivity by enzyme-linked immunosorbent assay (ELISA).
  • the immunogen was diluted with NaHCO 3 (pH 9.6) solution, coated with a microplate (Coming, Cat. No.: 9018), overnight at 4 °C. After washing the plate four times with a PBS-T solution in a plate washer, 3% BSA blocking solution was used and blocked at 37 ° C for 2 h. After four washes of PBS-T, the gradient-diluted serum was incubated overnight at 37 °C.
  • RNA is morphologically intact on the agarose gel electrophoresis map ( Figure 3) and is qualitatively compatible with library construction requirements.
  • specific forward primers and reverse primers were selected for VHH amplification (A. Bell et al., Differential tumor-targeting abilities of three single-domain antibody formats, Cancer Lett. 2010 Mar 1). ; 289(1): 81-90; and Honda Toshio, Akahori, Yasushi, Kurosawa Yoshikazu. Methods of constructing camel antibody libraries.
  • United States Patent 2005/0037421 A1 the specific sequence of the primers is shown in Table 4.
  • a fragment of VHH of about 600 bp is isolated and purified according to the molecular weight of the PCR product, and then a fragment of VHH is obtained by a second round of PCR amplification, and two recognition sequences are simultaneously introduced at both ends of the DNA fragment.
  • a total of 101 ⁇ g of gel-purified VHH fragment was obtained by different Sfi I restriction sites (Fig. 4).
  • the cells with different batches and different primers derived V H H fragments were mixed, and then using the restriction enzyme Sfi I digested.
  • Sfi I restriction enzyme
  • the gel electrophoresis was carried out to separate, purify and obtain the digested vector.
  • the carrier/fragment molar ratio was 1:3, 1:5, 1:10, respectively, and T4 ligase (NEB, Cat.) was added.
  • connection reaction system After No.: M0202L), the same volume connection reaction system was prepared, and the ligation was carried out overnight at 16 °C.
  • the ligating system was subjected to phenol/chloroform extraction, chloroform extraction, and ethanol precipitation, and the purified ligation product was subjected to concentration measurement by a light absorption method.
  • the products obtained from the three different ligation systems were subjected to electroporation of equal amounts of DNA and TG1 electrotransformation competent, and the transformation efficiency of the three ligation systems was calculated by coating plate and gradient dilution method, and positive clones were randomly picked.
  • Send test to detect library diversity Choose the system with the highest conversion efficiency for a large number of connections and conversions, and count the storage capacity. According to the plate count results, the library storage capacity was approximately 1.8 ⁇ 10 8 (Table 5).
  • the hVEGF165 protein was biotinylated according to the procedure described in the EZ-Link Sulfo-NHS-LC-Biotinylation kit (Pierce, Cat. No.: 21335). The extent of protein biotinylation labeling was determined by HABA assay. Biotinylated labeled hVEGF165 was mixed with 0.5 ml of M-280 streptavidin magnetic beads (Invitrogen, Cat. No.: 112.06D), incubated overnight at 4 ° C, then magnetic beads were separated by magnetic stand, and PBS solution was used. A biotinylated protein that failed to bind to the magnetic beads was eluted to prepare an antigen magnetic bead coupled complex. After biotinylation and purification, 0.52 mg of hVEGF165 protein was obtained, and HABA experiments showed that the biotin coupling level was 6 moles of biotin molecule per mole of protein.
  • Approximately 100 ⁇ l (MOI approximately 20) of the phage library stock was inoculated into 2YT medium, cultured at 225 rpm, 30 ° C, and M13KO7 helper phage (NEB, Cat) was added during incubation to logarithmic growth phase (OD 600 0.5). .No.: N0315S), 225 rpm, 30 ° C overnight culture.
  • the phage were collected by centrifugation, the culture supernatant was mixed with a PEG/NaCl solution, and the phage was pelleted by centrifugation, and the phage particles obtained by resuscitation were suspended in 1-2 ml of PBS by multiple centrifugation and resuspension.
  • the titer of the phage library obtained by the resuscitation was calculated by the finite gradient dilution method, and a library of 3.15 ⁇ 10 13 pfu/ml was obtained.
  • the phage was eluted and separated, and rapidly added to Tris-HCl buffer for neutralization; the phage production after the first round of screening was calculated by finite gradient dilution method, and the phage obtained in the first round of elution were cultured and expanded overnight.
  • the specific parameters and procedures are the same as those described for previous library resuscitation.
  • the second round of screening will use ⁇ 10 11 pfu of the first round of phage amplified library as input, and 1ul of antigen magnetic bead coupled complex for incubation and screening, the specific operation process and parameters and the first round of screening the same.
  • the cells were collected by centrifugation, and the supernatant was taken and added to a microplate which was pre-coated with hVEGF165 and blocked, and the HRP/anti-M13 monoclonal antibody (GE Healthcare, Cat.
  • the phage DNA produced by the last round of phage display was extracted, and the fragment encoding VHH was amplified by PCR and cloned into the patented FASEBA vector. All the constructed clone structures were V H H-linker-SASA-6 ⁇ His (Fig. 8). .
  • the ligation product will be transformed into TG1 cells.
  • the cells were collected by centrifugation, and the clarified 100 ⁇ l culture supernatant was removed and added to a microplate prepared in advance with BSA and blocked with HPR-labeled mouse anti-His monoclonal antibody as a second antibody (GenScript, Cat. No.).
  • the BSA protein was immobilized on the surface of a CM5 (GE healthcare, Cat. No.: BR-1006-68) chip by a standard coupling procedure according to the BIAcore T200 device instruction manual.
  • the basic procedure is as follows: at 25 ° C, the HBS-EP solution (0.01M HEPES [pH 7.4], 0.15M NaCl, 3mM EDTA and 0.005% [v / v] surfactant P20) as the equipment running buffer, flow rate It is 10 ml/min.
  • the above-mentioned sdAb-SASA clone expression supernatant was centrifuged through a 96-well filter plate (Pall, Cat. No.: PN5045) at 4000 g for 5 minutes at 4000 g to remove bacteria and other particles, using HBS-EP.
  • the sdAb antibody was detected by solution dilution and flowed sequentially through the surface of the BSA coupled chip.
  • the sequencing analysis process consists of the following four steps: a. capture of the SASA-conjugated single domain antibody using a fixed BSA chip; b. injection of hVEGF165 to bind to the surface of the chip with the single domain antibody captured; c. injection of running buffer, The dissociation phase was monitored for 300 s; d.
  • the surface of the BSA-conjugated chip was injected with 10 mM glycine/HCl (pH 2.0), 30 ⁇ l/min, and regenerated for 30 s. Each round of chip capture antibody, antigen binding, antigen dissociation, and BSA chip surface need to be regenerated.
  • Purified SASA protein at a concentration of 200 nM was passed through the surface of the BSA chip as a test for detecting the rejuvenation effect of the chip.
  • 138 clones were sorted into 3 batches, with reference to clone A10981. The consistency of 6 A10981 repeat clones was very good among different batches.
  • the dissociation degree of 400s was about -20%, and 93 clones had a dissociation rate ratio of A10981. Slow ( Figure 9), pick and sort. Of these, 53 single-domain antibodies were selected for prokaryotic expression and tested using cell proliferation inhibition assays, of which 15 single domain antibodies were used for further competitive screening.
  • the 15 single-domain antibodies which have been subjected to expression level screening and affinity sequencing, are used for receptor competitive screening in order to obtain an antibody capable of blocking the antigen hVEGF165 and its receptor hVEGFR.
  • the specific process is as follows:
  • hVEGFR2 protein was immobilized on the surface of the CM5 chip by the method of amino-coupled immobilization (same as 5.2.2); b. The hVEGF165 protein was injected, the binding characteristics were observed, and the injection was stopped when the binding curve was close to saturation; c. The chip bound with hVEGF165 No injection on the surface The same single domain antibody and observation of binding characteristics, while injection of the already marketed anti-VEGF drug Avastin as a control; d.
  • the antibody binds to the VEGF165 epitope is just VEGF and VEGFR2 binding epitope, the antibody will no longer be able to bind to VEGF, Alternatively, if the VEGF that binds VEGFR2 competes, the binding signal will be significantly smaller than VEGF itself; if the antibody binds to the VEGF165 epitope and the VEGF&VEGFR2 binding epitope is different or unrelated, the antibody will bind to the VEGF165 that has bound to the receptor, and the binding signal produced. Will be significantly higher than VEGF itself. Based on the competitive results compared to the control ( Figure 10), seven of the better clones were selected for heavy chain antibody preparation for cell proliferation inhibition experiments.
  • Prokaryotic expression, purification, and endotoxin removal of single domain antibodies are as follows.
  • IPTG mother liquor 1M, 0.22 ⁇ m filter filtration treatment, 1-2ml packaging, frozen at -20 °C (valid for 3 months);
  • MgCl 2 mother liquor 1M, moist heat sterilization at 121 ° C for 30 min, 1-2 ml dispensing, storage at 4 ° C (valid for 6 months);
  • CaCl 2 mother liquor 1M, 0.22 ⁇ m filter filtration treatment, 1-2ml packaging, storage at 4 ° C (valid for 6 months);
  • VB1 mother liquor 50mg/ml, 0.22 ⁇ m filter filtration treatment, 1-2ml packaging, storage at 4 °C (valid for 6 months);
  • Glucose mother liquor 20% (W / V), 0.22 ⁇ m filter filtration treatment, storage at 4 ° C (valid for 3 months);
  • Glycerol mother liquor 50% (V / V), 121 ° C wet heat sterilization for 30 min, 4 ° C storage (valid for 6 months);
  • Casein acid hydrolysate mother liquor 4%, sterilized by damp heat at 121 °C for 30 min, stored at room temperature (valid for 3 months);
  • 2YT medium 1.6% (W / V) Tryptone, 1.0% (W / V) yeast extract, 0.5% (W / V) NaCl, 121 ° C wet heat sterilization for 30 min;
  • 10X TB medium 12% (W/V) Tryptone, 24% (W/V) yeast extract, 4% (V/V) glycerol, and heat-sterilized at 121 °C for 30 min.
  • DNase I 10mg/ml nuclease: usually 1 ⁇ l per gram of wet weight bacteria (Life Science Product and Service, DD0099-1);
  • Lysozyme usually 100 ⁇ l per gram of wet weight bacteria (Xingxing Biological, L0005-10);
  • Lysis buffer 20 mM HEPES, 150 mM NaCl, 10% (v/v) glycerol, 40 mM imidazole, pH 8.0;
  • Binding buffer 20 mM Na 2 HPO 4 , 0.5 M NaCl, 20 mM imidazole, pH 7.4;
  • Washing buffer 20 mM Na 2 HPO 4 , 0.5 M NaCl, 40 mM imidazole, pH 7.4;
  • Elution buffer 20 mM Na 2 HPO 4 , 0.5 M NaCl, 300 mM imidazole, pH 7.4;
  • 1X PBS 137 mM NaCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4.
  • ToxinEraser TM Endotoxin Removal Resin 1.5ml resin (GenScript, L00402);
  • TAL sensitivity 0.25EU/ml Xiamen City ⁇ reagent Experimental Factory Co., Ltd.
  • Millex-GP Filter Unit 0.22 ⁇ m (Millipore, Lot: R4AA43868).
  • Loading Buffer (5X, non-reducing): 0.25 M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 7.8% DTT;
  • Loading Buffer (5X, reduction): 0.25 M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol.
  • a) transformation the prokaryotic expression plasmid constructed into the single domain antibody gene was transferred into the strain TG1 by chemical transformation or electrotransformation, coated on a 2YT ampicillin resistant plate, and cultured at 37 ° C overnight;
  • M9 medium Preparation of M9 medium: adding a final concentration of 0.2% (w/v) glucose, 1 mM MgCl 2 , 0.1 mM CaCl 2 , 0.4% (W/V) Casein acid hydrolysate, 5 mg/L VBl, 200 ⁇ g to the 1X M9 salt solution. /ml ampicillin, preheated at 37 ° C shaker;
  • lysis buffer 10X BugBuster Protein Extraction Reagent diluted to 1X with binding buffer and added to a final concentration of 100 ⁇ g/ml lysozyme, 2 ⁇ g/ Ml nuclease and 1 mM PMSF), incubated for 1 h at room temperature with moderate shaking;
  • washing The heteroprotein is eluted with at least 50 column volumes of the wash buffer. (Bradford dyeing solution is used as indicator in the washing process: 5 ⁇ l washing solution is added to 200 ⁇ l Bradford dyeing solution to see if it turns blue. If it turns blue, continue to carry out impurity protein elution until the dyeing solution is basically not discolored, then it can be carried out. step.);
  • the target protein is eluted with at least 10 column volumes of elution buffer. (The elution process uses Bradford dye solution as an indicator, and the method is the same as step iii to determine whether the elution is complete).
  • Loading Add appropriate amount (0.5ml) of nickel column purified protein solution to HiTrapTM Desalting column at appropriate flow rate (0.5ml/min);
  • Sample treatment before the purification, use 1M sodium chloride to adjust the ionic strength to 0.2 ⁇ 0.5M, using 0.1M sodium hydroxide or 0.1M hydrochloric acid to adjust the pH value of 7.4 ⁇ 0.2;
  • Activated resin Fix PD-10Columns vertically, remove the cover at the top of pre-packed column, add ToxinEraser TM Endotoxin Removal resin, open the flow controller, let the protective solution flow dry under gravity, add 5ml regeneration buffer, adjust the flow rate The controller keeps the flow rate at 0.25 ml/min (about 10 drops/min), the reaction buffer is drained, and then 5 ml of regeneration buffer is added, and the operation is repeated twice to ensure that the system remains pyrogen-free (ie, no endotoxin). ;
  • Iii. Balanced resin After activation of PD-10Columns, add 6ml of equilibration buffer, adjust the flow rate controller, keep the flow rate at 0.5ml/min, drain the equilibration buffer, and repeat this operation twice;
  • Iv. Endotoxin removal Turn off the flow controller, add the sample with a non-heat source gun head, open the controller, control the flow rate to no more than 0.25ml/min, and the volume of the effluent reaches 1.5ml, then start using the non-heat source receiving tube After the sample is drained, it is rinsed by adding 1.5 ml-3.0 ml of equilibration buffer, and the eluent is collected. Sample concentration and endotoxin levels were measured (Bradford stain was used as an indicator during the elution process to determine if the collection was complete).
  • Dilution sample According to the sensitivity of sputum reagent (0.25 EU / ml), the sample needs to be diluted to a suitable concentration (0.005 ⁇ g / ml; 0.05 ⁇ g / ml; 0.5 ⁇ g / ml; 5 ⁇ g / ml);
  • Iii. Detection Take TAL reagent, add 100 ⁇ l bacterial endotoxin test water gently shake for at least 30s until the reagent is completely dissolved, taking care not to cause air bubbles, add 100 ⁇ l samples: positive control (endotoxin standard 0.5EU/ml), Negative control (no endotoxin) Water), sample to be tested after dilution: (1) Four concentrations of the sample to be tested, close the nozzle, gently shake it, place it vertically in a 37 ° C incubator for 1 hour, and then take it out and observe;
  • the sample was filtered through a 0.22 ⁇ m filter in a sterile operation in a biosafety cabinet and an appropriate amount of sample was taken for subsequent testing.
  • protein concentration UV absorption of the protein solution (A 280 ) / absorption coefficient of the protein
  • a certain amount of protein for example, 2 ⁇ g
  • an equivalent amount of a standard protein for example, 2 ⁇ g of BSA
  • HUVEC HUVEC
  • the cells subjected to trypsin digestion were collected, and the cells were cleared and resuspended twice with M199 buffer, and the cells were resuspended to a cell density of 1 ⁇ 10 5 .
  • 50 ⁇ L of the cell suspension was added to each well of the microplate; the microplate to which the cells were added was placed in the culture, and cultured at 37 ° C, 5% CO 2 for 96 hours;
  • Example 7 Six of the single-domain antibodies that were initially screened for a certain proliferation inhibition function and that were competitively screened by the receptor were firstly screened in Example 7, and fused together with the Fc fragment of human IgG1 (SEQ ID NO: 60) to construct a heavy chain antibody.
  • the pTT5 vector was cloned into HEK293E for expression purification and endotoxin removal. The specific process is as follows:
  • HEK293 suspension cells were taken out from liquid nitrogen or -86 ° C refrigerator, quickly placed in a 37 ° C water bath, and the cells were thawed within 1-2 min;
  • HEK293 suspension cells were passaged at a suitable density.
  • the cell density on the day of transfection should be 1.5-2.0 ⁇ 10 6 cells/ml, and the cell viability needs to be greater than 95%;
  • the culture supernatant was collected by centrifugation on the 6th day after transfection and filtered through a 0.22 ⁇ m filter. To be purified.
  • Balance column balance the Protein A column with 5 times the column volume of ddH 2 O and Binding Buffer;
  • Washing Washing the protein with at least 30 times the volume of Binding Buffer (Bradford dyeing solution is used as an indicator: 5 ⁇ l of the washing solution is added to 200 ⁇ l of Bradford dye solution to see if it turns blue. If it turns blue, then Continue the washing process until the dyeing solution is basically not discolored, and then proceed to the next step);
  • Binding Buffer Branford dyeing solution is used as an indicator: 5 ⁇ l of the washing solution is added to 200 ⁇ l of Bradford dye solution to see if it turns blue. If it turns blue, then Continue the washing process until the dyeing solution is basically not discolored, and then proceed to the next step);
  • the protein was purified by affinity HiTrap TM Desalting column
  • the purifier 10 system was replaced with PBS buffer. Subsequent steps of endotoxin removal, filtration sterilization, concentration, purity determination and the like are the same as in Example 6.
  • Single domain antibody clone Heavy chain antibody clone Amino acid sequence number Nucleotide sequence number A14575 A69451 SEQ ID NO:37 SEQ ID NO: 50 A14942 A69452 SEQ ID NO:38 SEQ ID NO: 51 A15411 A69457 SEQ ID NO:39 SEQ ID NO:52 A14614 A69458 SEQ ID NO:36 SEQ ID NO:49 A14972 A69462 SEQ ID NO:40 SEQ ID NO:53 A10981 A60724 SEQ ID NO:41 SEQ ID NO: 54 A15578 A80723 SEQ ID NO: 35 SEQ ID NO:48 A15922 A80744 SEQ ID NO:42 SEQ ID NO: 55 A15637 A80730 SEQ ID NO:43 SEQ ID NO:56
  • a total of 8 heavy-chain antibodies (Table 8) were set to 8 gradient dilutions, the initial concentration of the antibody was 20 ⁇ g/ml, and the gradient was diluted 1:4.
  • Avastin (A68467) was used as a control.
  • the other experimental conditions were exactly the same as in Example 7. . Judging by the degree of inhibition of cell proliferation by different concentrations of antibodies, 13 heavy chain antibodies have different degrees of inhibitory function (Fig. 11), among which A80887, A80723 and A69458 have the strongest inhibitory function at the cellular level.
  • the sample to be tested (A80887) has a molecular weight of about 75 kDa, a concentration of 5.1 mg/ml, and is stored at -80 ° C in portions, diluted with 1 ⁇ PBS (pH 7.4) before use, and placed on ice for storage.
  • Transgenic vascular fluorescent zebrafish embryos are propagated in a natural paired mating manner. Prepare 4 to 5 pairs of adult zebrafish for each mating, with an average of 200 to 300 embryos per pair. Embryos were cleaned 6 hours (ie 6 hpf) and 24 hpf (removed dead embryos) and appropriate embryos were selected based on the embryo's developmental stage (Kimmel, 1995). Incubate embryos with fish culture water at 28 °C (fish culture water quality: 200 mg of instant sea salt per 1 L of reverse osmosis water, conductivity 480-510 ⁇ S/cm; pH 6.9-7.2; hardness 53.7-71.6 mg/L CaCO 3 ).
  • the sample was injected into the blood circulation of the transgenic vascular fluorescent zebrafish at the highest concentration and maximum injection volume (equivalent to human intravenous administration), with no death and apparent abnormal phenotype.
  • 1/10 maximum injection dose (maximum concentration ⁇ maximum injection volume), 1/3 maximum injection dose and maximum injection dose 3 doses were selected for detection, and a positive control group (Avastin) and a solvent control group were set ( PBS) and blank control group, each group treated 30 zebrafish.
  • PBS positive control group
  • PBS solvent control group
  • the angiogenesis inhibition rates of Avastin doses of 400 ng (2.68 pmol) and 1 ⁇ g (6.7 pmol) were 6.9% (p>0.05) and 19.5% (p ⁇ 0.01), respectively, and p ⁇ 0.05.
  • the zebrafish angiogenesis model has been widely recognized for its pharmacodynamic evaluation and new drug target validation.
  • anticancer drugs including drugs that have been approved for FDA approval
  • Vatalanib Novartis
  • Celgene Thalidomide
  • Compound 6 Tumorogene
  • Rosuvastatin Wang2010
  • Solenopsin Eli Lilly
  • the effect of the compound angiogenic zebrafish inferior vascular plexus or interstitial vascular assessment compound on neovascularization is usually selected. This project selects the method of quantifying the size of the intestine vascular plexus to evaluate the effect of sample A80887 on neovascularization.
  • Avastin is a recombinant human monoclonal IgG1 antibody that binds to vascular endothelial growth factor (VEGF) and prevents it from binding to receptors on the surface of endothelial cells (Flt-1 and KDR).
  • VEGF vascular endothelial growth factor
  • Flt-1 and KDR endothelial cells
  • both 1A80887 and Avastin have significant inhibitory effects on zebrafish neovascularization.
  • A80887 was significantly better than Avastin in inhibiting neovascularization.
  • the reference doses for human administration were about 81.6, 163.2, 272, 544, 816, and 1632 ⁇ g/kg body weight, respectively.

Abstract

本发明公开了特异性结合血管内皮生长因子(VEGF)的抗体，特别是重链抗体，更特别是单域抗体。还公开了所述抗体的制备方法和治疗用途。

Description

抗VEGF抗体 技术领域
本发明涉及抗体及其应用，具体而言，本发明涉及特异性结合血管内皮生长因子(Vascular Endothelial Growth Factor，VEGF)的抗体，特别是重链抗体，更特别是单域抗体；以及所述抗体的制备方法和治疗用途。
背景技术
血管生成是指从已有的毛细血管或毛细血管后静脉发展而形成新的血管，是一个涉及多种细胞的多种分子的复杂过程。血管生成是促血管形成因子和抑制因子协调作用的复杂过程，正常情况下二者处于平衡状态，一旦此平衡打破就会激活血管系统，使血管生成过度或抑制血管系统使血管退化。
已知许多疾病与血管生成失控和不希望有的血管生成有关。这些疾病包括但不限于肿瘤如所谓的实体瘤和液体(或血液)瘤(如白血病和淋巴瘤)，炎症如类风湿或风湿性炎症，尤其是关节炎(包括类风湿性关节炎)，或其它慢性炎症如慢性哮喘，动脉硬化或移植后动脉硬化、子宫内膜异位，眼新生血管疾病，如视网膜病(包括糖尿病视网膜病)、老年黄斑变性、牛皮癣、成血管细胞瘤、血管瘤、动脉硬化。其它与血管生成失控和不希望有的血管生成有关的疾病对本领域技术人员是显然的。
血管内皮生长因子，是对血管内皮细胞具有特异性的肝素结合生长因子，可在体内诱导血管新生。包括VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGF-E、VEGF-F和胎盘生长因子。
VEGF-A的主要作用为促进血管内皮细胞增殖、迁移和管腔的形成，同时也会增加血管渗漏、促进单核细胞趋化和B细胞生成。VEGF-A的生物学效应是依靠与其特异性受体结合而介导的，与其结 合的主要是特异性受体血管内皮生长因子受体1(VEGFR-1)和VEGFR-2。其中，VEGFR-2被认为是主要的VEGFR，其对血管内皮细胞的增生有重要影响。VEGFR-2通过细胞内激酶诱导VEGF绑定二聚体和需要自身磷酸化的受体，从而加强细胞的有丝分裂(Klettner A，Roider J.Treating age-related macular degeneration interaction of VEGF-antagonists with their target.Mini Rev Med Chem，2009，9(9)：1127-1135)。VEGF-A包括8个外显子和7个内含子，其转录剪接成多个亚型，主要为：VEGF121、VEGF145、VEGF206、VEGF165、VEGF189，这些亚型有不同的分子质量、溶解度和肝素结合能力，其中VEGF165是VEGF-A最主要的亚型(Ferrara N，Gerber HP，Le Couter J.The biology of VEGF and its receptors.Nat Med，2003，9(6)：669-676)。VEGF165是分泌型可溶性蛋白，能直接作用于血管内皮细胞促进血管内皮细胞增殖，加速血管内皮细胞损伤的修复，增加血管通透性，使血管内血栓形成及血栓性闭塞减少，并抑制内膜增生(黄晨星，沈祖光.血管内皮生长因子的研究及在组织修复中的应用J.中国修复重建外科杂志.2002，160：64-68)。
现有的血管内皮生长因子药物包括哌加他尼钠(Pegaptanib sodium，商品名Macugen)、Ranibizumab(商品名为Lucentis)、贝伐单抗(Bevacizumab，商品名为Avastin)、VEGF Trap等。目前针对抗VEGF制剂争议的焦点是可能加重组织纤维膜的形成。目前临床用于治疗多种疾病(如年龄相关性黄斑变性)的抗VEGF药物，需要频繁进行眼内注药，导致发生眼内炎的潜在风险，这是抗VEGF治疗存在的明显问题。有研究者使用脐静脉内皮细胞和Tenon囊的纤维细胞，观察倍伐单抗和Macugen对VEGF不同亚型的作用，结果证实VEGF-165、VEGF-121主要影响血管生长，VEGF-189主要影响纤维化形成过程。倍伐单抗和雷珠单抗能抑制全部有活性的VEGF-A亚型(Van Bergen T，Vandewalle E，Van de Veire S，et al.The role of  different VEGF isoforms in scar formation after glaucoma filtration surgery.Exp Eye Res，2011，93：689-699；and CATT research group，Martin DF，Maguire MG，et al.Ranibizumab and Bevacizumab for neovascular age-related macular degeneration.N Engl J Med，2011.364：1897-1908)，这可能是倍伐单抗导致某些患者玻璃体腔内纤维化的原因。
目前抗VEGF的药物每4～6周需要重复治疗，雷珠单抗治疗第1年的平均年注射量约6.9次，倍伐单抗约7.7次(Li X，Hu Y，Sun X，Zhang J，Zhang M.Bevacizumab for neovascular age-related macular degeneration in China.Ophthalmology.2012Oct.，119(10)：2087-93)，如此频繁的眼内注药治疗存在发生眼内炎的潜在风险，急需开发药效长，视网膜通透吸收更好的新型抗体药物，以延长给药周期，降低注射给药给患者带来的不适与风险。
除此之外，目前抗VEGF制剂表达纯化工艺复杂，普遍存在成本高，稳定性差、应用面不广等现实问题。
重链抗体是从骆驼科动物的血清中分离出的一种抗体，仅由重链构成，其抗原结合区仅是一个通过铰链区与Fc区连接的单结构域，而且这个抗原结合区自抗体上分离后仍具有结合抗原的功能，故称为单域抗体(single-domain antibody，sdAb)或纳米抗体(nanobody)。与传统抗体不同的是，单域抗体是一个含有大约110个氨基酸的肽链，其分子量约为传统抗体的1/10，这就为抗体的分子构建提供了一个新方法(Muyldermans.Single domain camel antibodies：current status.J Biotechnol 2001，74：277-302)。这类单域抗体具有分子小、热稳定性好、在清洁剂和高浓度尿酸环境下稳定、体内组织渗透性好、可溶性好(Stanfield R，Dooley H，Flajnik M，Wilson I.Crystal structure of a shark single-domain antibody V region in complex with lysozyme.Science.2004，305(5691))、易表达、利于原核系统表达、生产成本低、抗原识别表位独特，且能识别隐藏的抗原性位点等特性，在免疫实验、诊断与治疗中，逐渐发挥着超乎想象的巨大功能 (Dirk Saerens，Gholamreza Hassanzadeh Ghassabeh，Serge Muyldermans.Single-domain antibodies as building blocks for novel therapeutics.Current Opinion in Pharmacology 2008，8：600-608)。
因此，本领域需要一种能够克服现有抗VEGF制剂的上述缺陷的抗体，例如可特异性结合VEGF并抑制其活性的单域抗体。
发明内容
本发明提供了抗VEGF抗体、其变体或衍生物，其中所述抗体包含重链可变区，所述重链可变区包含：(i)SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的CDR1、CDR2和CDR3或其功能活性变体；或(ii)SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的CDR1、CDR2和CDR3或其功能活性变体；或(iii)SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的CDR1、CDR2和CDR3或其功能活性变体；或(iv)SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的CDR1、CDR2和CDR3或其功能活性变体；或(v)SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的CDR1、CDR2和CDR3或其功能活性变体；或(vi)SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的CDR1、CDR2和CDR3或其功能活性变体；或(vii)SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的CDR1、CDR2和CDR3或其功能活性变体；或(viii)SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24所示的CDR1、CDR2和CDR3或其功能活性变体；或(ix)SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27所示的CDR1、CDR2和CDR3或其功能活性变体；或(x)SEQ ID NO：28、SEQ ID NO：29和SEQ ID NO：30所示的CDR1、CDR2和CDR3或其功能活性变体；或(xi)SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：33所示的CDR1、CDR2和CDR3或其功能活性变体；所述功能活性变体是与SEQ ID NO：1-33中任一个的氨基酸序列具有至少70％、75％、80％、85％、90％、95％、98％或99％序列同一性的功能活性变体。
在具体的实施方案中，本发明提供了重链抗体，所述抗体由重链组成，所述重链的可变区包含：(i)SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的CDR1、CDR2和CDR3；或(ii)SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的CDR1、CDR2和CDR3或其功能活性变体；或(iii)SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的CDR1、CDR2和CDR3或其功能活性变体；或(iv)SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的CDR1、CDR2和CDR3或其功能活性变体；或(v)SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的CDR1、CDR2和CDR3或其功能活性变体；或(vi)SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的CDR1、CDR2和CDR3或其功能活性变体；或(vii)SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的CDR1、CDR2和CDR3或其功能活性变体；或(viii)SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24所示的CDR1、CDR2和CDR3或其功能活性变体；或(ix)SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27所示的CDR1、CDR2和CDR3或其功能活性变体；或(x)SEQ ID NO：28、SEQ ID NO：29和SEQ ID NO：30所示的CDR1、CDR2和CDR3或其功能活性变体；或(xi)SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：33所示的CDR1、CDR2和CDR3或其功能活性变体。
一方面，本发明抗体的重链可变区可包含至少一个氨基酸添加、插入、缺失和/或置换。另一方面，本发明的抗体可以是单克隆抗体、嵌合抗体或人源化抗体、多特异性抗体和/或双特异性抗体以及它们的片段。在一个具体实施方案中，本发明的抗体为人源化抗体。
在一个具体的实施方案中，本发明抗体的重链还可含有恒定区。在另一具体的实施方案中，本发明抗体的重链还含有Fc片段。
在一些具体的实施方案中，本发明的抗体是重链抗体，即，仅由重链组成。在一些具体的实施方案中，本发明的抗体是单域抗 体。
另外，本发明提供了与参比抗体竞争结合VEGF的抗体，所述参比抗体为上述抗体的任一个。
本发明还涉及编码上述抗体的核酸序列；包含这些核酸序列的载体；以及宿主细胞，所述宿主细胞表达上述抗体，和/或包含这些核酸序列或载体。
本发明还提供了制备抗体的方法，步骤包括：在允许表达所述抗体的条件下培养上述宿主细胞；和从所得培养产物中纯化抗体。
本发明还涉及药物组合物，包含本发明的抗体和药学上可接受的赋形剂。所述药物组合物还可包含一种或多种治疗活性化合物，所述治疗活性化合物例如已知的抗VEGF药物或抗肿瘤药物。
另一方面，本发明还涉及抗体偶联药物(Antibody-drug conjugate，ADC)，其包含偶联于其它药剂的本发明抗体，所述其它药剂例如化疗剂、生长抑制剂、毒素(例如细菌、真菌、植物或动物来源的酶活性毒素，或其片段)或放射性同位素(即放射性偶联物)。
抗体偶联药物还可包含位于药物单元和抗体单元之间的接头单元。
另外，本发明涉及通过给予有效量的本发明的抗体来调节VEGF活性的方法。本发明涉及通过向有需要的患者给予有效量的本发明的抗体来抑制血管生成的方法。
本发明还提供了一种治疗与VEGF相关疾病或病症的方法，所述方法包括向有需要的患者给予有效量的至少一种本发明的抗体。所述疾病或病症包括肿瘤或癌症或眼科疾病。所述肿瘤或癌症包括乳腺癌、脑肿瘤、肾癌、卵巢癌、甲状腺癌、肺癌、结直肠癌、子宫内膜癌、血管肉瘤、膀胱癌、胚胎组织癌、颈部肿瘤、恶性胶质瘤、胃癌、胰腺癌、鼻咽癌等。所述眼科疾病包括各种原因引起的黄斑水肿(包括糖尿病性黄斑水肿、白内障术后或葡萄膜炎后等各种 疾病引起的黄斑水肿)、年龄相关性黄斑变性、糖尿病性视网膜病变、视网膜中央静脉阻塞、新生血管性青光眼以及其它涉及新生血管的眼科疾病。
另外，本发明还涉及本发明的抗体在制备用于调节VEGF活性的药物中的用途；本发明的抗体在制备用于抑制血管生成的药物中的用途；本发明的抗体在制备用于治疗与VEGF相关疾病或病症的药物中的用途。
本发明还提供了药盒，其包含a)本发明的抗体，或所述药物组合物；以及b)使用说明。
附图说明
图1是纯化的hVEGF165蛋白的SDS-PAGE检测结果的图。其中，第1泳道是标准蛋白Marker(Invitrogen，Cat.No.：LC5677)；第2泳道是2μg非还原hVEGF165；第3泳道是5μg非还原hVEGF165；第4泳道是2μg还原hVEGF165；第5泳道是5μg还原hVEGF165。
图2是免疫反应测试结果，表明动物在注射了抗原后产生了较好的免疫反应，血清效价约为1∶100k。
图3是总RNA的琼脂糖凝胶电泳检测结果，表明所得RNA质量符合文库构建的需求。
图4是将图3的总RNA反转录成cDNA后，经PCR得到的扩增VHH片段的琼脂糖凝胶电泳纯化结果。
图5是用于连接VHH片段的噬菌粒载体图谱。
图6是噬菌体展示文库片段插入率检测的图。通过对72个随机克隆的PCR检测，其中有69个克隆有单域抗体基因片段的插入，插入率69/72＝95.8％。
图7是通过对图6中有插入片段的阳性克隆进行测序，而得到的单域抗体文库序列多样性检测图，可见文库多样性良好。
图8是FASEBA筛选专用载体图谱。该载体为氨苄青霉素抗 性，含有SASA及6×His tag，可以用于抗体的分泌表达。
图9是FASEBA筛选后抗体的亲和力排序；9A、9B、9C为3个不同批次的亲和力排序结果。其中左上图：不同克隆的结合、解离的传感图；右上图：不同克隆的结合、解离速率的矩阵图；左下图：不同克隆经过归一化的传感图；右下图：选取部分亲和力较高的抗体的传感图。
图10是受体竞争性筛选结果图，其中将经过表达量水平筛选和亲和力排序而优选的15个单域抗体用于该筛选。根据该竞争性结果和对照相比，选择其中7个较好的克隆用于重链抗体制备，用于细胞增殖抑制实验。
图11是重链抗体对HUVEC细胞增殖抑制试验的曲线图。通过不同浓度抗体对细胞增殖抑制的程度来判断，13个重链抗体均具有抑制功能，其中A80887、A80723及A69458在细胞水平的抑制功能最强。
图12是实施例11中13个重链抗体的可变区序列。
图13是斑马鱼肠下血管示意图。在给药一定时间后，每组随机取15尾斑马鱼在荧光显微镜下拍照，对肠下血管丛(SIVs)面积进行定量分析。两组间比较采用T-检验，多组比较采用单因素方差分析和Dunnett′s T-检验进行统计学分析，p＜0.05表明具有统计学差异，抑制血管形成的药效计算公式如下：
Figure PCTCN2015070209-appb-000001
图14是肠下血管面积的图。从图中可以看出，A80887与Avastin一样，都有显著的新生血管抑制作用。
图15是肠下血管丛面积抑制率的图。Avastin为阳性对照药，在等摩尔质量(均约2.7pmol)时，A80887的抑制新生血管形成效率为23.2％，明显优于Avastin 6.9％(两者比较，p＜0.001)。与按临床用量换算的Avastin 1μg(6.7pmol)比较，对新生血管的抑制率未见统计学差异。
发明详述
本发明涉及特异性结合VEGF的抗体、其变体或衍生物；以及所述抗体的制备方法和治疗用途。例如，本发明涉及特异性结合VEGF的重链抗体，更特别地为单域抗体。同时，本发明抗体在抑制细胞增殖以及血管生成方面显示出优于现有技术抗VEGF单克隆抗体(例如Avastin)的优异效果，如以下实施例所进一步描述的。
本发明的单域抗体具有比Fab片段和全长IgG抗体更小的分子量，一般12-15kD，可以用于构建多价抗体，并通过基因工程改造以提高亲和力和延长半衰期，延长间隔给药周期等特性。与普通抗体药物相比，单域抗体药物与抗原的结合能力，在高温、胃酸、蛋白酶等极端条件下更加稳定，并具有高度的构象稳定性。与全抗体药物易诱发补体效应细胞毒反应不同，单域抗体药物缺少Fc片段，不会引起补体效应。同时，因为单域抗体分子量小，该抗体在眼组织和肿瘤组织给药中可具有更好渗透性。处于蛋白酶、极端温度和pH环境中的稳定性，高亲和力、使其口服和其它给药途径提供了可行性。
单域抗体可以在原核或真核细胞中，例如大肠杆菌或酵母细胞中进行规模化表达，表达量很大，这样就极大地便利了批量生产，有利于控制生产成本，也有利于后期药物开发的市场前景。
除非本文另外定义，与本文相关的科学和技术术语应具有本领域普通技术人员所理解的含义。
术语“抗体”在生物学和生物医学领域中被充分理解，通常是指完整抗体及任何抗体片段或其单链。抗体是由称为浆细胞的特化B淋巴细胞分泌的糖蛋白。其亦被称为免疫球蛋白(Ig)，因为其含有存在于许多蛋白质中的共有结构域。抗体最可能包含通常由二硫键连接的2条重(H)链和2条轻(L)链或其抗原结合部分。每条重链由重链可变区(VH)和重链恒定区组成。每条轻链同样由可变区(VL)和恒定区组成。轻链恒定区由一个结构域CL组成。VH和VI区可进一步再分 成超变区，称为互补决定区(CDR)，其散布有称为构架区(FR)的更保守的区域。在一些具体的实施方案中，本发明的抗体仅由重链组成。在一些具体的实施方案中，本发明的抗体是单域抗体。
可使用Kabat等在Sequences of Proteins of Immunological Interest，第5版.，US Dept.of Health and Human Services，PHS，NIH，NIH Publication no.91-3242，1991中描述的方法确定给定抗体的互补决定区(CDR)和构架区(FR)。
本发明包括抗体的“变体”，例如，本发明抗体的重链可变区可包含至少一个氨基酸添加、插入、缺失和/或置换，例如10、20、30、40、50个，优选例如1、2、3、4、5、6、7、8、9、10个氨基酸添加、插入、缺失和/或置换。
本发明亦包括抗体的“衍生物”。抗体的“衍生物”为经化学修饰的抗体，例如通过与其它化学部分例如聚乙二醇、白蛋白(例如人血清白蛋白)结合、磷酸化和糖基化。除非另外说明，否则术语“抗体”包括其片段、衍生物、变体。
一方面，本发明提供了抗VEGF抗体、其变体或衍生物，其中所述抗体包含重链可变区，所述重链可变区包含：(i)SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的CDR1、CDR2和CDR3或其功能活性变体；或(ii)SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的CDR1、CDR2和CDR3或其功能活性变体；或(iii)SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的CDR1、CDR2和CDR3或其功能活性变体；或(iv)SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的CDR1、CDR2和CDR3或其功能活性变体；或(v)SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的CDR1、CDR2和CDR3或其功能活性变体；或(vi)SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的CDR1、CDR2和CDR3或其功能活性变体；或(vii)SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的CDR1、CDR2和CDR3或其功能活性变体；或(viii) SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24所示的CDR1、CDR2和CDR3或其功能活性变体；或(ix)SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27所示的CDR1、CDR2和CDR3或其功能活性变体；或(x)SEQ ID NO：28、SEQ ID NO：29和SEQ ID NO：30所示的CDR1、CDR2和CDR3或其功能活性变体；或(xi)SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：33所示的CDR1、CDR2和CDR3或其功能活性变体。
所述功能活性变体是与SEQ ID NO：1-33中任一个的氨基酸序列具有至少70％，例如至少75％、至少80％、至少85％、至少90％、例如91％、92％、93％、94％、95％、96％、97％、98％、99％序列同一性的功能活性变体。
在具体的实施方案中，本发明提供了重链抗体，所述抗体由重链组成，所述重链的可变区包含：(i)SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的CDR1、CDR2和CDR3或其功能活性变体；或(ii)SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的CDR1、CDR2和CDR3或其功能活性变体；或(iii)SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的CDR1、CDR2和CDR3或其功能活性变体；或(iv)SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的CDR1、CDR2和CDR3或其功能活性变体；或(v)SEQ ID NO：13、SEQ ID NO：14和SEQ ID NO：15所示的CDR1、CDR2和CDR3或其功能活性变体；或(vi)SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的CDR1、CDR2和CDR3或其功能活性变体；或(vii)SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的CDR1、CDR2和CDR3或其功能活性变体；或(viii)SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24所示的CDR1、CDR2和CDR3或其功能活性变体；或(ix)SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27所示的CDR1、CDR2和CDR3或其功能活性变体；或(x)SEQ ID NO：28、SEQ ID NO：29和SEQ ID NO：30所示的 CDR1、CDR2和CDR3或其功能活性变体；或(xi)SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：33所示的CDR1、CDR2和CDR3或其功能活性变体。
在一个具体的实施方案中，本发明抗体的重链还可含有恒定区。在另一具体的实施方案中，本发明抗体的重链还含有Fc片段。
在一些具体的实施方案中，本发明的抗体是重链抗体，即，仅由重链组成。在一些具体的实施方案中，本发明的抗体是单域抗体。
在更具体的实施方案中，本发明抗体的重链可变区序列如SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45或SEQ ID NO：46所示。
另外，本发明还提供了与参比抗体竞争结合VEGF抗体，所述参比抗体为上述抗体的任一个。
本发明还涉及编码上述抗体的核酸序列；包含这些核酸序列的载体；以及宿主细胞，所述宿主细胞表达上述抗体，和/或包含这些核酸序列或载体。“宿主细胞”为用于表达核酸例如本发明核酸的细胞。宿主细胞可为原核生物，例如大肠杆菌，或者其可为真核生物，例如单细胞真核生物(例如，酵母)。
在具体的实施方案中，所述核酸序列如SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：51、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56、SEQ ID NO：57、SEQ ID NO：58或SEQ ID NO：59所示，如以下实施例所详述的。
本发明还提供了制备抗体的方法，步骤包括：在允许表达所述抗体的条件下培养上述宿主细胞；和从所得培养产物中纯化抗体，如以下实施例所详述的。
本发明还涉及药物组合物，其包含本发明的抗体和药学上可接受的赋形剂。所述药物组合物还可包含一种或多种治疗活性化合物，所述治疗活性化合物例如已知的抗VEGF药物或抗肿瘤药物。
所述治疗活性化合物可与本发明的抗体同时给药或序贯给药。
药物组合物可按照本领域已知的技术制备。术语“赋形剂”概括地指一种或多种活性治疗成分外的任何组分。赋形剂可为惰性物质、无活性物质和/或无药物活性的物质。赋形剂可用作多种目的，例如，用作载体、溶媒、稀释剂、片剂辅助剂，和/或改善活性物质的给药和/或吸收。药学活性成分与各种赋形剂的配制为本领域已知，参见例如Remington：The Science and Practice of Pharmacy(例如第19版(1995)，和任何之后的版本)。赋形剂的非限制性实例为：溶剂、稀释剂、缓冲剂、防腐剂、张度调节剂、螯合剂和稳定剂。
本发明的抗体可以以药物组合物的形式给予。其不仅可以制备成为注射液、冻干制剂、喷雾剂等液体制剂，还可以制备成为胶囊剂等固体制剂。给药途径可为，例如，静脉内注射、口服或局部给药，例如经皮，经结膜，和/或经眼睛等。在具体的实施方案中，给药途径为经口服。在另一具体的实施方案中，给药途径为经眼睛。
另一方面，本发明还涉及抗体偶联药物，其包含偶联于其它药剂的本发明抗体，所述其它药剂例如化疗剂、生长抑制剂、毒素(例如细菌、真菌、植物或动物来源的酶活性毒素，或其片段)或放射性同位素(即放射性偶联物)。
采用抗体偶联药物进行其它药剂的局部递送能将药剂靶向递送到肿瘤，并在肿瘤中胞内积累，而全身性给予这些未偶联的药剂可能会导致对正常细胞以及需要去除的肿瘤细胞不可接受程度的细胞毒性。
抗体偶联药物通常包含位于药物单元和抗体单元之间的接头 单元。在一些实施方案中，该接头可在胞内条件下切割，从而接头的切割导致药物单元在胞内环境中从抗体释放。接头可为例如可被胞内肽酶或蛋白酶(包括但不限于：溶酶体或内含体蛋白酶)切割的肽基接头。在一些实施方案中，肽基接头的长度至少为两个氨基酸或至少为3个氨基酸。在一个具体的实施方式中，可被胞内蛋白酶切割的肽基接头是Val-Cit接头或Phe-Lys接头。
在其它实施方案中，接头单元不可切割，药物通过，例如抗体的降解而释放。
另外，本发明还涉及通过给予有效量的本发明的抗体来调节(优选抑制)VEGF活性、抑制哺乳动物的血管生成的方法。
另外，本发明涉及通过给予有效量的本发明的抗体来调节VEGF活性的方法。本发明涉及通过向有需要的患者给予有效量的本发明的抗体来抑制血管生成的方法。
本发明还提供了一种治疗与VEGF相关疾病或病症的方法，所述方法包括向有需要的患者给予有效量的至少一种本发明的抗体。所述疾病或病症包括肿瘤或癌症或眼科疾病。所述肿瘤或癌症包括乳腺癌、脑肿瘤、肾癌、卵巢癌、甲状腺癌、肺癌、结直肠癌、子宫内膜癌、血管肉瘤、膀胱癌、胚胎组织癌、颈部肿瘤、恶性胶质瘤、胃癌、胰腺癌、鼻咽癌等。所述眼科疾病包括各种原因引起的黄斑水肿(包括糖尿病性黄斑水肿、白内障术后或葡萄膜炎后等各种疾病引起的黄斑水肿)、年龄相关性黄斑变性、糖尿病性视网膜病变、视网膜中央静脉阻塞、新生血管性青光眼以及其它涉及新生血管的眼科疾病。
所述“有需要的患者”意指任何哺乳动物，所述哺乳动物例如但不限于人、马、牛、猫、小鼠、兔、大鼠、山羊等。优选地，所述哺乳动物是人。
在一些治疗应用中，多种本发明的抗体组合给予。
另外，本发明还涉及本发明的抗体在制备用于调节VEGF活性 的药物中的用途；本发明的抗体在制备用于抑制血管生成的药物中的用途；本发明的抗体在制备用于治疗与VEGF相关疾病或病症的药物中的用途。
本发明还提供了药盒，其包含a)本发明的抗体，或所述药物组合物；以及b)使用说明。
参考以下的非限制性实施例进一步描述本发明。
实施例1.抗原制备
本实施例针对的抗原为人VEGF165(Human vascular endothelial growth factor 165，hVEGF 165)分子(Park JE，Keller GA，Ferrara N.The vascular endothelial growth factor(VEGF)isoforms：differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.Mol Biol Cell.1993Dec.，4(12)：1317-26；Gengrinovitch S，Greenberg SM，Cohen T，Gitay-Goren H，Rockwell P，Maione TE，Levi BZ，Neufeld G.Platelet factor-4inhibits the mitogenic activity of VEGF 121and VEGF 165using several concurrent mechanisms.J Biol Chem.1995Jun 23；270(25)：15059-65；and Keyt BA，Berleau LT，Nguyen HV，Chen H，Heinsohn H，Vandlen R，Ferrara N.The carboxyl-terminal domain(111-165)of vascular endothelial growth factor is critical for its mitogenic potency.J Biol Chem.1996Mar 29；271(13)：7788-95)，其中人VEGF165抗原的核酸序列如SEQ ID NO：61所示；人VEGF165抗原的氨基酸序列如SEQ ID NO：62所示。
根据该氨基酸序列，对其进行哺乳动物表达的密码子优化后，通过全合成的方式得到优化后的DNA，克隆入真核表达载体pTT5(由发明机构NRC授权)后，用于制备转染级质粒。转染HEK293E细胞后培养7天，通过离心收集细胞并培养上清液用于手动装配的Capto柱和HiTrapTM Q HP的两步离子交换纯化，纯化并进行去内毒素处理。蛋白浓度检测采用UV280nm吸光值检测，蛋白内毒素水平采用LAL方法检测，抗原的活性通过HUVEC细胞增殖实验测定。共得浓度为1.25mg/ml，体积为22ml，总量为27.5mg的hVEGF165 蛋白，内毒素水平为0.537EU/ml(表1)，SDS-PAGE检测结果见图1，蛋白于-80℃保存。
表1 hVEGF165纯化蛋白信息
Figure PCTCN2015070209-appb-000002
实施例2.动物免疫和免疫反应测定
1.动物免疫
选择羊驼(Lama pacos)作为实验动物，在4个不同时间点，分别在肩胛及背部进行六点注射免疫。PBS为抗原的稀释液，每次免疫体积为1ml，抗原量及佐剂信息见表2。免疫试剂含终浓度1mg/ml的BSA，抗原与佐剂均在注射前新鲜配制混匀后再免疫。
表2羊驼免疫抗原信息
Figure PCTCN2015070209-appb-000003
免疫过程设计(表3)分四次不同时间分别于颈静脉采血，采集血液时加入抗凝剂。首次采血5ml，其余三次各15ml。利用Ficoll1.077试剂(Sangon，Cat.No.：F760014-100)和抗凝血进行梯度离心后，分离外周血淋巴细胞并进行细胞重悬计数，添加RNAlater(TIANGEN，Cat.No.：DP408-02)，于-20℃保存。梯度离心获得的血清也于-20℃保存。
表3羊驼免疫时间安排表
Figure PCTCN2015070209-appb-000004
Figure PCTCN2015070209-appb-000005
2.免疫反应测试
采用酶联免疫吸附试验(ELISA)分别对免疫前、第三次免疫和第四次免疫后的血清样品进行抗原特异性免疫反应测试。以NaHCO3(pH 9.6)溶液稀释免疫原，包被微孔板(Coming，Cat.No.：9018)，4℃过夜。用PBS-T溶液在洗板机中洗板四次后，使用3％BSA封闭液，37℃封闭2h。PBS-T四次洗板后，37℃过夜孵育梯度稀释的血清。PBS-T四次洗板后，孵育辣根过氧化物酶标记的羊抗美洲驼第二抗体(Novus Biologicals，Cat.No.：NB7242)。使用TMB显色10min，加入1M HCl终止显色。反应终止后的体系使用MK3(Thermo)酶标仪检测450nm处的吸光值。通过ELISA的反应结果可以判断，动物在注射了蛋白抗原后产生了较好的免疫反应，血清效价约为1∶100k(图2)。
实施例3.抗体噬菌体文库构建
3.1.RNA提取
根据细胞数目将对应体积的TRIzol试剂加入分离的外周血淋巴细胞中，细胞裂解完成后，按照
Figure PCTCN2015070209-appb-000006
Plus RNA纯化系统的操作说明(Invitrogen，Cat.No.：12183-555)完成总RNA的提取分离。通过琼脂糖凝胶电泳检测总RNA的质量，并利用吸光法测定RNA的浓度。根据测定结果，共获得105.6μg总RNA。RNA在琼脂糖凝胶电泳图上形态完整(图3)，质量上符合文库构建的需求。
3.2.反转录PCR
根据SuperScriptTM III First-Strand Synthesis System使用技术说明书(Invitrogen，Cat.No.：18080-051)，使用Oligo(dT)20引物将总RNA反转录成cDNA。根据骆驼抗体的序列特征，选择特异性的正向引物和反向引物用于VHH的扩增(A.Bell et al.，Differential tumor-targeting abilities of three single-domain antibody formats，Cancer Lett.2010Mar 1；289(1)：81-90；and Honda Toshio，Akahori，Yasushi，Kurosawa Yoshikazu.Methods of constructing camel antibody libraries.United States Patent 2005/0037421 A1)，引物的具体序列见表4。通过第一轮对cDNA的PCR，根据PCR产物的分子量分离纯化约为600bp的含有VHH的片段，此后再通过第二轮PCR扩增获得VHH的片段并同时在DNA片段两端引入两个识别序列不同的Sfi I限制性酶切位点，共获得101μg凝胶纯化的VHH片段(图4)。
表4.引物序列信息及PCR扩增作用
Figure PCTCN2015070209-appb-000007
Figure PCTCN2015070209-appb-000008
3.3文库构建
将用不同批次细胞和不同引物扩增得到的VHH片段混合，然后利用限制性内切酶Sfi I进行酶切。通过2％琼脂糖凝胶电泳进行分离、纯化并获得酶切后的VHH；同时，利用限制性内切酶Sfi I对噬菌粒载体(图5)进行酶切，通过1.5％琼脂糖凝胶电泳进行分离、纯化并获得酶切后的载体。通过吸光法测定酶切后的VHH和噬菌粒载体的浓度后，按照载体/片段摩尔比分别为1∶3，1∶5，1∶10混合，加入T4连接酶(NEB，Cat.No.：M0202L)后进行同体积连接反应体系的制备，于16℃过夜进行连接。对连接体系按顺序进行酚/氯仿抽提、氯仿抽提、乙醇沉淀，对纯化得到的连接产物通过吸光法进行浓度的测定。对于3个不同连接体系获得的产物均进行等量DNA和TG1电转化感受态的电转化，通过涂布平板和梯度稀释法计算3个连接体系的文库即转化效率的大小，随机挑取阳性克隆送测检测文库多样性。选择转化效率最高的体系进行大量连接和转化，并统计库容。根据平板计数结果显示，文库库容约为1.8×108(表5)。
表5单域抗体噬菌体展示文库库容计算
Figure PCTCN2015070209-appb-000009
对文库菌落进行随机克隆PCR，可见文库的片段插入率为95.8％(图6)，对其中有插入片段的阳性克隆进行测序，通过对单域抗体CDR区域的氨基酸序列进行比对，可见文库多样性良好(图7)。对涂布的过夜平板使用含100μg/ml氨苄青霉素和2％葡萄糖的2YT培养基进行菌体收集，5,000g离心去除细胞代谢产物，并用相同培养基重悬细胞，分为两份进行文库储存。
实施例4.噬菌体展示及筛选
4.1.抗原生物素酰化复合物制备
根据EZ-Link Sulfo-NHS-LC-Biotinylation试剂盒(Pierce，Cat.No.：21335)的操作过程说明，将hVEGF165蛋白进行生物素化标记。通过HABA实验检测蛋白生物素化标记的程度。将生物素化标记的hVEGF165与0.5ml M-280链霉亲和素的磁珠混合(Invitrogen，Cat.No.：112.06D)，4℃过夜孵育，然后通过磁力架分离磁珠，并用PBS溶液洗脱未能和磁珠结合的生物素化蛋白，用来制备抗原磁珠偶联复合物。生物素酰化反应和纯化后获得0.52mg hVEGF165蛋白，HABA实验显示生物素偶联水平为每摩尔蛋白偶联6摩尔生物素分子。
4.2.噬菌体文库复苏
取大约100μl(MOI约为20)的噬菌体文库储液接种到2YT培养基中，225rpm，30℃进行培养，在培养至对数生长期(OD600＝0.5)时加入M13KO7辅助噬菌体(NEB，Cat.No.：N0315S)，225rpm，30℃过夜培养。离心收集噬菌体，将培养上清液和PEG/NaCl溶液混合并离心沉淀噬菌体，通过多次离心及重悬，最后将复苏得到的噬菌体颗粒悬浮在1-2ml的PBS中。通过有限梯度稀释法计算复苏得到的噬菌体文库的效价，获得3.15×1013pfu/ml的文库。
4.3.针对靶蛋白噬菌体筛选
取～2×1011pfu的噬菌体作为第一轮的投入和10ul的抗原磁珠偶 联复合物进行常温孵育，并在旋转仪上温和混匀2小时；通过磁力架将磁珠分离，将没有与磁珠结合的噬菌体洗去，对磁珠的重悬和非特异性结合的洗脱需要进行7次；将最后一次重悬的磁珠加入TEA溶液，将与抗原磁珠偶联复合物结合的噬菌体洗脱并分离，迅速加入Tris-HCl缓冲液进行中和；通过有限梯度稀释法计算第一轮筛选后噬菌体的产出，同时对第一轮洗脱得到的噬菌体进行过夜培养和扩增，具体参数和过程和此前文库复苏描述相同。第二轮筛选将以～1011pfu的第一轮产出噬菌体扩增后的文库作为投入，和1ul的抗原磁珠偶联复合物进行孵育和筛选，具体操作过程和参数与第一轮筛选相同。
4.4.噬菌体ELISA鉴定
挑取用于第二轮筛选噬菌体产出计算的过夜平板上的单克隆噬菌斑，放入每孔含有500μl 2YT培养基的96孔深孔板，225rpm，30℃进行培养，在培养至对数生长期(OD600＝0.5)时加入M13KO7辅助噬菌体(NEB，Cat.No.：N0315S)，225rpm，30℃过夜培养。离心收集菌体，取上清液，加入预先包被hVEGF165并封闭好的微孔板中，以HRP/抗-M13单克隆抗体(GE Healthcare，Cat.No.：27-9421-01)作为第二抗体检测，其它ELISA操作参数与免疫反应检测相同，根据吸光值评估产出的阳性率。将随机挑取的部分与抗原识别的阳性噬菌体克隆进行VHH片段的测序，通过序列比对和分析，推断通过噬菌体展示产出的克隆多样性。通过阳性率和序列的多样性来确定是否需要进行更多轮次的噬菌体展示筛选。噬菌体克隆阳性大于50％，且满足多样性需求。因此选用第二轮产出噬菌体克隆sdAb基因构建FASEBA文库，用于进一步克隆筛选。
表6噬菌体淘筛及ELISA检测
Figure PCTCN2015070209-appb-000010
Figure PCTCN2015070209-appb-000011
实施例5.FASEBA筛选
5.1.FASEBA文库构建
提取最后一轮噬菌体展示产出的噬菌体DNA，通过PCR扩增编码VHH的片段通过连接克隆到专利的FASEBA载体中，所有构建克隆结构为VHH-linker-SASA-6×His(图8)。连接产物将转化入TG1菌体。
5.2.FASEBA筛选
5.2.1.样品制备及表达水平评估
从构建的FASEBA文库中随机挑取单克隆，放入每孔含有500μl 2YT培养基的96孔深孔板，在培养至OD600＝0.6-0.8时，加入IPTG过夜诱导表达。离心收集菌体后去除澄清的100μl培养上清液，加入到预先包被BSA并封闭的微孔板中，使用HPR标记的鼠抗His单克隆抗体作为第二抗体检测(GenScript，Cat.No.：A00186)；同时，取等量培养上清液加入到预先包被hVEGF165蛋白并封闭的微孔板中，使用HPR标记的鼠抗His单克隆抗体作为第二抗体检测(GenScript，Cat.No.：A00186)，以OD450吸光值来评估不同克隆的表达水平。不同批次总计超过5000个单克隆被用于表达量和抗原结合力的筛选，将表达量较高且与抗原有高结合力的138个阳性克隆用于亲和力排序和后续筛选。
5.2.2.芯片准备
根据BIAcore T200设备使用说明书，将BSA蛋白通过标准偶联操作流程固定在CM5(GE healthcare，Cat.No.：BR-1006-68)芯片表面上。基本过程如下：在25℃条件下，以HBS-EP溶液(0.01M HEPES[pH 7.4]，0.15M NaCl，3mM EDTA和0.005％[v/v]表面活性剂P20)为设备运行缓冲液，流速为10ml/min。首先注射大于7分钟的0.4M 1-乙基-3-(二甲基氨基丙基)碳二亚胺盐酸盐(EDC)/0.1M N-羟 基琥珀酰亚胺(NHS)(1∶1)，用于活化羧甲基葡聚糖表面，然后通过注射7分钟稀释于10mM醋酸钠(pH4.5)的20μg/ml BSA蛋白溶液，最后再注射7分钟的1M氨基乙醇(pH8.5)对未结合的活化位点进行封闭。上述操作获得BSA偶联反应水平在327反应单位(resonance units，RU)。
5.2.3.抗hVEGF165单域抗体的亲和力排序
将上述提到的sdAb-SASA克隆表达上清液，经96孔过滤板(Pall，Cat.No.：PN5045)在4℃，以4000g离心过滤5分钟以除去细菌和其它颗粒，用HBS-EP溶液稀释检测sdAb抗体并顺序流过BSA偶联芯片表面。排序分析过程包括以下四个步骤：a.使用固定BSA的芯片捕获SASA偶联的单域抗体；b.注射hVEGF165，使其与捕获有单域抗体的芯片表面结合；c.注射运行缓冲液，并监测解离阶段300s；d.对偶联BSA的芯片表面注射10mM甘氨酸/HCl(pH2.0)，30μl/min，30s进行重生。每一轮的芯片捕获抗体、抗原结合、抗原解离、BSA芯片表面均需要再生。以浓度200nM的纯化SASA蛋白流过BSA芯片表面作为对照检测芯片重生效果的检验。138个克隆分3批次进行排序，以克隆A10981为参照，6个A10981重复克隆在不同批次间的一致性很好，400s解离程度约～20％，其中93个克隆解离速率比A10981慢(图9)，挑取进行测序。选取其中53个单域抗体用于原核表达并用细胞增殖抑制实验进行测试，其中15个单域抗体用于进一步的竞争性筛选。
5.2.4.hVEGFR2竞争性筛选
将经过表达量水平筛选和亲和力排序优选的15个单域抗体用于受体竞争性筛选，以期获得能够封闭抗原hVEGF165和其受体hVEGFR结合抗体。具体过程如下：
a.通过氨基偶联固定的方法(同5.2.2)将hVEGFR2蛋白固定在CM5芯片表面；b.注射hVEGF165蛋白，观察结合特征，在结合曲线接近饱和时停止注射；c.在hVEGF165结合的芯片表面上注射不 同的单域抗体并观察结合特征，同时注射已经上市的抗VEGF的药物Avastin作为对照；d.如果抗体结合VEGF165的表位恰是VEGF和VEGFR2结合表位的话，抗体将不能再与VEGF结合，或者将已结合VEGFR2的VEGF竞争下来，结合信号将明显小于VEGF本身；如果抗体结合VEGF165的表位和VEGF&VEGFR2结合表位不同或者无关的话，抗体将结合已经与受体结合的VEGF165，产生的结合信号将明显高于VEGF本身。根据竞争性结果和对照相比(图10)，选择其中的7个较好的克隆用于重链抗体制备用于细胞增殖抑制实验。
实施例6：单域抗体的制备
单域抗体的原核表达、纯化、内毒素去除过程如下。
6.1.试剂准备
6.1.1.原核表达试剂
Tryptone，OXOID LP0042
Yeast extract，OXOID LP0021
Casein acid hydrolysate，Sigma C9386
KH2PO4，国药AR CAS[7778-77-0]
Na2HPO4.12H2O，国药AR 10020318
NH4Cl，国药AR CAS[12125-02-9]
NaCl，国药AR 10019318
MgCl2，国药AR 7791-18-6
CaCl2，国药AR 10043-52-4
葡萄糖，国药AR 10010518
甘油，Sigma G5516-500ML
IPTG，Amresco 0487-100G
VB1，阿拉丁AR 1099302
氨苄青霉素，100mg/ml，0.22μm滤器过滤处理；
IPTG母液：1M，0.22μm滤器过滤处理，1-2ml分装，-20℃冻存(有效期3个月)；
MgCl2母液：1M，121℃湿热灭菌30min，1-2ml分装，4℃存放(有效期6个月)；
CaCl2母液：1M，0.22μm滤器过滤处理，1-2ml分装，4℃存放(有效期6个月)；
VB1母液：50mg/ml，0.22μm滤器过滤处理，1-2ml分装，4℃存放(有效期6个月)；
葡萄糖母液：20％(W/V)，0.22μm滤器过滤处理，4℃存放(有效期3个月)；
甘油母液：50％(V/V)，121℃湿热灭菌30min，4℃存放(有效期6个月)；
Casein acid hydrolysate母液：4％，121℃湿热灭菌30min，室温存放(有效期3个月)；
10X M9盐溶液：6％Na2HPO4(W/V)、3％KH2PO4(W/V)、1％NH4Cl(W/V)、0.5％；
NaCl(W/V)，121℃湿热灭菌30min，室温存放(有效期3个月)；
2YT培养基：1.6％(W/V)Tryptone、1.0％(W/V)yeast extract、0.5％(W/V)NaCl、121℃湿热灭菌30min；
10X TB培养基：12％(W/V)Tryptone、24％(W/V)yeast extract、4％(V/V)甘油、121℃湿热灭菌30min。
6.1.2.蛋白纯化试剂及设备
10X BugBuster Protein Extraction Reagent(Novagen，70921-4)；
100mM PMSF：1.74g PMSF in 100ml异丙醇溶液(碧云天，ST506)；
10mg/ml核酸酶(DNase I)：通常每克湿重菌体用1μl(Life Science Product and Service，DD0099-1)；
5mg/ml溶菌酶(Lysozyme)：通常每克湿重菌体用100μl(生兴生物，L0005-10)；
Quick StartTM Bradford试剂(Bio-Rad，500-0204)；
High Affinity Ni-NTA Resin(GenScript，L00250)；
HiTrap TM Desalting，5ml，(GE Healthcare，17-1408-01)；
Figure PCTCN2015070209-appb-000012
purifier 10(GE Healthcare)；
裂解缓冲液：20mM HEPES，150mM NaCl，10％(V/V)甘油，40mM咪唑，pH 8.0；
结合缓冲液：20mM Na2HPO4，0.5M NaCl，20mM咪唑，pH7.4；
洗杂缓冲液：20mM Na2HPO4，0.5M NaCl，40mM咪唑，pH 7.4；
洗脱缓冲液：20mM Na2HPO4，0.5M NaCl，300mM咪唑，pH7.4；
1X PBS：137mM NaCl，10mM Na2HPO4，2mM KH2PO4，pH7.4。
6.1.3.内毒素去除试剂及设备
ToxinEraser TM Endotoxin Removal Resin 1.5ml树脂(GenScript，L00402)；
PD-10Columns；ToxinEraser TM Regeneration Buffer(GenScript，M01053)；
ToxinEraser TM Equilibration Buffer(GenScript M01054)；
凝胶法内毒素检测试剂盒(GenScript，L00451)；
鲎试剂(Tachypleus Amebocyte Lysate简称TAL灵敏度0.25EU/ml，厦门市鲎试剂实验厂有限公司)；
0.1M NaOH(无热源水配制)；
0.1M盐酸(无热源水配制)；
37℃恒温培养箱。
6.1.4.过滤除菌试剂及设备
Millex-GP Filter Unit，0.22μm(Millipore，Lot：R4AA43868)。
6.1.5.蛋白浓度测定及检测试剂及设备
Nanodrop 2000分光光度计(Thermo)；
ExpressPlus PAGE Gel：4-20％，12wells(GenScript，M42012)；
MOPS Running Buffer Powder(GenScript，M00138)；
Loading Buffer(5X，非还原)：0.25M Tris-HCl(pH 6.8)，10％SDS，0.5％溴酚蓝，50％甘油，7.8％DTT；
Loading Buffer(5X，还原)：0.25M Tris-HCl(pH 6.8)，10％SDS，0.5％溴酚蓝，50％甘油。
6.2.方法与流程
6.2.1.菌株制备
a)转化：将构建入单域抗体基因的原核表达质粒通过化学转化或电转化转入菌株TG1，涂布在2YT氨苄青霉素抗性的平板上，37℃恒温过夜培养；
b)挑取单克隆：在10ml的2YT培养基中加入终浓度200μg/ml氨苄青霉素和2％(W/V)葡萄糖；将镊子在酒精灯上充分烧灼，冷却后夹取10μl灭菌枪头，从转化平板上挑取1个单克隆，放入培养基，225rpm，37℃，恒温培养过夜。
6.2.2.转接诱导
a)配制M9培养基：向1X M9盐溶液中添加终浓度0.2％(W/V)葡萄糖、1mM MgCl2、0.1mM CaCl2、0.4％(W/V)Casein acid hydrolysate，5mg/L VBl，200μg/ml氨苄青霉素，置37℃摇床预热；
b)转接：将过夜培养菌液从摇床取出，按1∶100转接体系，取过夜培养产物4000rpm离心10min，用新鲜1X M9盐溶液重悬，4000rpm离心10min，再次重悬后转接至预热的培养基中，后置37℃摇床，225rpm培养24h；
c)诱导：向M9培养基中补加终浓度1X TB培养基及终浓度为1mM的IPTG和200μg/ml的氨苄青霉素，225rpm，25℃培养48h(24h补加一次终浓度为200μg/ml氨苄青霉素)；
d)收样：在诱导结束后，将过夜培养物分装至离心杯，4℃， 11,000rpm，离心15min，收集菌体。
6.2.3.蛋白纯化
a)BugBuster裂解法样品制备
i.裂解：表达沉淀根据每克湿重菌体加5ml裂解缓冲液重悬(裂解缓冲液：用结合缓冲液稀释10X BugBuster Protein Extraction Reagent至1X，并加入终浓度100μg/ml溶菌酶、2μg/ml核酸酶及1mM PMSF)，室温中速振荡孵育1h；
ii.蛋白粗提液制备：将裂解的样品于4℃，12,000g，离心30min，收集上清液并用0.22μm滤膜过滤。
b)镍柱亲和纯化
i.平衡柱料：用5倍柱料体积的ddH2O及平衡缓冲液平衡High Affinity Ni-NTA Resin；
ii.结合：将蛋白粗提液与适量High Affinity Ni-NTA Resin混合，震荡孵育1h。孵育结束后，将粗提液与柱料的混合液加入PD-10空柱收集柱料，并收集流出液待下一步分析用；
iii.洗杂：用至少50个柱体积的洗杂缓冲液洗脱杂蛋白。(洗杂过程用Bradford染液作为指示剂：5μl洗杂液加入200μl Bradford染液观察是否变蓝，若变蓝，则继续进行杂蛋白洗脱至染液基本不变色，此时方可进行下一步。)；
iv.洗脱：用至少10个柱体积的洗脱缓冲液洗脱目标蛋白。(洗脱过程用Bradford染液作为指示剂，方法同步骤iii，判断是否洗脱完全)。
c)脱盐/缓冲液置换
i.平衡：在
Figure PCTCN2015070209-appb-000013
purifier 10系统上以适当流速(0.5ml/min)用5倍柱体积的ddH2O及PBS平衡5ml的HiTrapTM Desalting柱；
ii.上样：用适当流速(0.5ml/min)将适量(0.5ml)镍柱纯化后的蛋白液加入HiTrapTM Desalting柱中；
iii.洗脱：在适当流速下(0.5ml/min)继续用至少10倍柱体积的 PBS进行洗脱，收集蛋白所在的目标紫外峰。
6.2.4.内毒素去除及检测
a)内毒素去除
i.样品处理：纯化之前使用1M氯化钠调节离子强度到0.2±0.5M，使用0.1M氢氧化钠或0.1M盐酸调节pH值在7.4±0.2；
ii.活化树脂：将PD-10Columns垂直固定，去除预装柱顶部的盖子，加入ToxinEraser TM Endotoxin Removal树脂，打开流速控制器，使保护液在重力作用下流干，加入5ml的再生缓冲液，调节流速控制器，保持流速在0.25ml/min(约10滴/min)，待再生缓冲液流干，再加入5ml再生缓冲液，重复操作两次，确保体系保持无热原(即无内毒素)存在；
iii.平衡树脂：PD-10Columns活化完毕，加入6ml的平衡缓冲液，调节流速控制器，保持流速在0.5ml/min，流干平衡缓冲液，再按此操作重复两次；
iv.内毒素去除：将流速控制器关闭，使用无热源枪头将样品加入，打开控制器，控制流速不高于0.25ml/min，流出液体积达到1.5ml后，开始使用无热源接收管接受样品，样品流干后，再加入1.5ml-3.0ml平衡缓冲液淋洗，收集淋洗液。检测样品浓度及内毒素水平(洗脱过程用Bradford染液作为指示剂，判断是否收集完全)。
b)凝胶法测定内毒素
i.稀释样品：根据鲎试剂灵敏度(0.25EU/ml)需要将样品稀释到合适的浓度(0.005μg/ml；0.05μg/ml；0.5μg/ml；5μg/ml)；
ii.稀释内毒素标准品：准备好内毒素标准品，用细菌内毒素检查用水复溶后置涡旋振荡器混匀15min，再稀释至适当浓度(0.5EU/ml)；
iii.检测：取TAL试剂，加入100μl细菌内毒素检查用水轻轻振摇至少30s至试剂完全溶解，注意不要引起气泡，分别加入100μl以下样品：阳性对照(内毒素标准品0.5EU/ml)、阴性对照(无内毒素 水)、稀释后待检测样品：(1)中四个浓度待测样品，封闭管口，轻轻摇匀，垂直放置在37℃培养箱孵育1小时，然后取出观察；
iv.结果记录：将试管从恒温培养箱轻轻取出，缓慢倒置试管180°，管内内容物呈现实凝胶，不变形，不从管壁滑落为阳性；其他情况则为阴性。阳性管为阳性，阴性管为阴性，同一范围内状态相同，实验有效。
6.2.5.过滤除菌
在生物安全柜中无菌操作情况下用0.22μm的滤器过滤样品，并取适量样品留待后续检测。
6.2.6.浓度、纯度检测
a)蛋白浓度测定
i.根据提供的蛋白氨基酸序列，计算出该蛋白的吸光系数
ii.测定蛋白溶液的紫外吸收值(A280)
iii.根据公式算出蛋白浓度：蛋白浓度＝蛋白溶液的紫外吸收值(A280)/蛋白的吸光系数
b)蛋白纯度检测
根据上述测定浓度取一定量的蛋白(如2μg)与等量的标准蛋白(如2μg BSA)在还原及非还原条件下进行SDS-PAGE，检测纯度及确认浓度。
实施例7.单域抗体的HUVEC细胞增殖抑制试验
7.1.细胞制备
将约为3×105HUVEC(ATCC，Cat No.PCS-100-010)细胞复苏接种于10cm的培养皿并每隔3-4天加入新鲜的培养基；当细胞融合程度达到85％-95％时分盘培养，并加入新鲜的培养基，7代之内的细胞会用于增殖抑制试验，一般固定在第6代。
7.2.VEGF诱导的HUVEC增殖抑制试验
分别用M199缓冲液体(Medium-1991X Earle′s Salts(Invitrogen#11150-059)；10％Fetal Bovine Serum(Gibco，Cat#10100139)，heat  inactivated；10mM HEPES(Invitrogen，Cat#15630080)；100units/ml Penicillin 100μg/ml Streptomycin(Invitrogen，Cat#10378016))制备2×VEGF和待测不同浓度的抗体样品；将50μL 2×VEGF和各待测不同浓度的抗体样品预混合，在微孔板上设置复孔，以细胞培养基作为反应的空白对照，以Avastin作为阳性对照；将经过胰蛋白酶消解的细胞收集，用M199缓冲液清晰并重悬细胞两次，将细胞重悬至细胞密度为1×105个细胞/ml后，微孔板的每个孔加入50μL细胞重悬液；将加入细胞的微孔板置于培养中，37℃，5％CO2培养96小时；培养完成后使用
Figure PCTCN2015070209-appb-000014
Luminescent Cell Viability Assay Kit(Promega，Cat No：G7571)检测细胞存活率，同时通过PHERAStar Plus(BMG Labtech)检测检测荧光强度并记录其相对发光单位；通过以下公式计算生产抑制率，生长抑制率％＝100*(1-相对发光单位/最大生产值)，其中相关发光单位为各样样品的测定值，最大生产值为只加入VEGF时的相对发光单位。综合最大抑制率和EC50两方面数据，53个原核表达的单域抗体中有6个有较为明显的抑制效果，被选取用于构建重链抗体及后续的增殖抑制实验。
表7：HUVEC细胞增殖抑制试验的EC50值如下：
Figure PCTCN2015070209-appb-000015
Figure PCTCN2015070209-appb-000016
实施例8.重链抗体的表达
将实施例7中初步筛选有一定增殖抑制功能的6个以及经过受体竞争性筛选的7个单域抗体，一起和人IgG1的Fc片段(SEQ ID NO：60)融合构建为重链抗体，克隆入pTT5载体于HEK293E中表达纯化及内毒素去除，具体过程如下：
8.1.试剂准备
同实施例6。
8.2.方法与流程
8.2.1.细胞培养
a)将HEK293悬浮细胞从液氮或-86℃冰箱中取出后，迅速放入37℃水浴锅中，在1～2min内将细胞融解；
b)将融解的细胞加入10倍体积的预热FreeStyleTM 293Expression Medium中，轻柔颠倒混匀，低速离心收集细胞并用适量新鲜预热的培养基重悬；
c)将重悬的细胞转入培养瓶中并在37℃，5％CO2，110rpm转速条件下培养。
8.2.2.转染
a)转染前一天，以合适密度传代HEK293悬浮细胞，转染当天细胞密度需达到1.5～2.0×106个细胞/ml，细胞活力需要大于95％；
b)将适量的DNA与PEI以最佳比例(如1∶3)于适量预热FreeStyleTM 293Expression Medium中充分混匀，室温下静置10分钟；
c)将上述PEI-DNA混合物加入细胞培养物中，轻柔旋转混匀，继续在37℃，5％CO2，110rpm转速条件下培养；
d)转染后16～24小时内，加入终浓度为0.5％(w/v)的Tryptone N1；
e)转染后第6天离心收集培养上清液并用0.22μm滤膜过滤以 待纯化。
8.2.3.蛋白纯化
a)Protein A亲和纯化
i.平衡柱料：用5倍柱料体积的ddH2O及Binding Buffer平衡Protein A柱料；
ii.结合：过滤后的表达上清液与柱料混合后孵育结合1h，孵育结束后将柱料装于PD-10手动纯化柱中；
iii.洗杂：用至少30倍柱料体积的Binding Buffer洗去杂蛋白(洗杂过程用Bradford染液作为指示剂：5μl洗杂液加入200μl Bradford染液观察是否变蓝，若变蓝，则继续进行洗杂过程至染液基本不变色，此时方可进行下一步)；
iv.洗脱：用至少10倍柱料体积的Elution Buffer洗脱目标蛋白并用Neutralization Buffer调节pH值至7.0左右(洗脱过程用Bradford染液作为指示剂，方法同步骤iii，判断是否洗脱完全)。
b)脱盐/缓冲液更换：
将亲和纯化所得蛋白用HiTrapTM Desalting柱在
Figure PCTCN2015070209-appb-000017
purifier10系统上置换到PBS缓冲液中。后续的内毒素去除、过滤除菌、浓度、纯度测定等步骤同实施例6。
表8重链抗体、单域抗体克隆号对应关系及其可变区氨基酸序列和核苷酸序列编号
单域抗体克隆号 重链抗体克隆号 氨基酸序列编号 核苷酸序列编号
A14575 A69451 SEQ ID NO：37 SEQ ID NO：50
A14942 A69452 SEQ ID NO：38 SEQ ID NO：51
A15411 A69457 SEQ ID NO：39 SEQ ID NO：52
A14614 A69458 SEQ ID NO：36 SEQ ID NO：49
A14972 A69462 SEQ ID NO：40 SEQ ID NO：53
A10981 A60724 SEQ ID NO：41 SEQ ID NO：54
A15578 A80723 SEQ ID NO：35 SEQ ID NO：48
A15922 A80744 SEQ ID NO：42 SEQ ID NO：55
A15637 A80730 SEQ ID NO：43 SEQ ID NO：56
A15908 A80890 SEQ ID NO：44 SEQ ID NO：57
A15612 A80726 SEQ ID NO：45 SEQ ID NO：58
A15775 A80887 SEQ ID NO：34 SEQ ID NO：47
A15872 A80740 SEQ ID NO：46 SEQ ID NO：59
实施例9.重链抗体的HUVEC细胞增殖抑制试验
将13个重链抗体(表8)共设置8个梯度稀释浓度，抗体的起始浓度为20μg/ml，1∶4梯度稀释，以Avastin(A68467)作为对照，其它实验条件完全同实施例7。通过对不同浓度抗体对细胞增殖抑制的程度来判断，13个重链抗体均有不同程度的抑制功能(图11)，其中A80887、A80723及A69458在细胞水平的抑制功能最强。具体而言，对于A69458，在抗体浓度为10-31μg/ml时，其抑制效果明显优于对照；对于A80723，在抗体浓度为10-310-1μg/ml时，其抑制效果明显优于对照；对于A80887，在抗体浓度为10-21μg/ml时，其抑制效果明显优于对照。
表9 重链抗体样品信息
Figure PCTCN2015070209-appb-000018
实施例10.斑马鱼模型评价本发明抗体的抗新生血管形成作用
待测样品(A80887)的分子量约为75kDa，浓度为5.1mg/ml，-80℃分装保存，临用前1×PBS(pH7.4)稀释，注射时将样品置于冰上保存。所用实验仪器与试剂包括：显微注射仪(IM300，Narishige)；解剖显微镜(SMZ645，Nikon公司)；电动聚焦连续变倍荧光显微镜(AZ100，Nikon公司)；6孔板(Nest Biotech)；MESAB(Sigma)；Bevacizumab(商品名Avastin，Roche)。
转基因血管荧光斑马鱼胚胎的繁殖以自然成对交配的方式进行。每次交配准备4～5对成年斑马鱼，平均每对能产200～300个胚胎。在受精后6小时(即6hpf)和24hpf对胚胎进行清理(移除已死亡胚胎)，并根据胚胎的发育阶段挑选合适的胚胎(Kimmel，1995)。在28℃条件下用养鱼用水孵育胚胎(养鱼用水水质：每1L反渗透水中加入200mg速溶海盐，电导率为480～510μS/cm；pH为6.9～7.2；硬度为53.7～71.6mg/L CaCO3)。因为胚胎可以从自身的卵黄囊中获取营养物质，所以在受精后9天内(9dpf)不需要喂食。实验完成后，用三卡因甲磺酸对各个发育阶段的斑马鱼进行过度暴露处理，从而将斑马鱼麻醉处死。所有实验操作均严格按照国际实验动物评估和认可委员会(AAALAC)标准执行。
使用显微注射仪，样品以最高浓度、最大注射体积注射到转基因血管荧光斑马鱼血液循环中(相当于人类静脉给药)，未出现死亡和明显异常表型。根据上述实验结果，选择1/10最大注射剂量(最高浓度×最大注射体积)、1/3最大注射剂量和最大注射剂量3个剂量进行检测，同时设置阳性对照组(Avastin)、溶剂对照组(PBS)和空白对照组，每组均处理30尾斑马鱼。在给药一定时间后，每组随机取15尾斑马鱼在荧光显微镜下拍照，对肠下血管丛(SIVs)面积进行定量分析。两组间比较采用T-检验，多组比较采用单因素方差分析和Dunnett′s T-检验进行统计学分析，p＜0.05表明具有统计学差异，抑制血管形成的药效计算公式如下：
Figure PCTCN2015070209-appb-000019
整个实验过程中所有实验组均未出现斑马鱼死亡或异常表型；空白对照组与溶剂对照组无统计学差异(p＞0.05)；阳性对照组(Avastin)与溶剂对照组比较具有统计学差异(p＜0.05)。
通过剂量摸索实验，A80887注射最高浓度为5.1mg/ml，未见斑马鱼出现死亡和明显的异常表型。因此，选择40.8ng(0.544pmol)、136ng(1.81pmol)、408ng(5.44pmol)为给药总剂量；。
实验结果表明，A80887剂量为20.4ng(0.272pmol)、68ng(0.907pmol)和204ng(2.72pmol)时，新生血管形成抑制率依次为10.9％(p＞0.05)、18.5％(p＜0.05)和23.2％(p＜0.001)。Avastin剂量为400ng(2.68pmol)和1μg(6.7pmol)的血管形成抑制率分别为6.9％(p＞0.05)和19.5％(p＜0.01)，且两者比较，p＜0.05。在等摩尔质量(均约2.7pmol)时，A80887的抑制新生血管形成效率为23.2％，明显优于Avastin 6.9％(两者比较，p＜0.001)。而Avastin 1μg(6.7pmol)与A8088720.4ng(0.272pmol)、68ng(0.907pmol)和204ng(2.72pmol)比较，对新生血管的抑制率未见统计学差异。原始数据见下表。
表10.A80887对血管的抑制作用
Figure PCTCN2015070209-appb-000020
斑马鱼血管形成模型进行药效学评价和药物新靶点验证获得广泛认可。目前，多支进入临床前实验(Pre-clinical Trial)或者临床试验(Clinical Trial)阶段的抗癌药物(包括已获得FDA批准上市的药物)，如Vatalanib(Novartis)(Chan，2002)、Thalidomide(Celgene)(Yabu，2005)、Compound 6(TargeGen)(Murphy，2010)、Rosuvastatin(Wang2010)、Solenopsin(Eli Lilly)(Arbiser，2007)等，均成功地利用斑马鱼血管形成抑制模型进行了有效验证。通常选择转基因血管荧光斑马鱼肠下血管丛或体节间血管评价化合物对新生血管形成的影响，本项目选择定量肠下血管丛面积的方法评价样品A80887对新生血管形成的影响。
Avastin是一种重组的人类单克隆IgG1抗体，通过结合血管内皮生长因子(VEGF)并阻止其与内皮细胞表面的受体(Flt-1和KDR)结合。本研究选用Avastin(分子量149kDa)作为大分子类抑制血管形成的阳性对照药。实验结果显示Avastin具有较好的新生血管抑制效果，因此，用Avastin作为阳性对照药评价该模型是可靠的。
综上所述，①A80887与Avastin均对斑马鱼新生血管形成具有明显的抑制作用。②在等摩尔质量(约2.7pmol)时，A80887抑制新生血管形成的效果明显优于Avastin。③A80887的给药量分别为20.4ng、40.8ng、68ng、136ng、204ng和408ng时，相对于人体给药参考剂量分别约为81.6、163.2、272、544、816和1632μg/kg体重。
虽然本发明某些特征已经在本文中阐释和描述，但本领域技术人员将想到许多修改、替代、变更和等同。因此，应理解的是，所附权利要求书意在涵盖落入本发明真实精神范围之内的所有此类修改和变更。

Claims (26)

  1. 一种抗VEGF抗体，其中所述抗体包含重链可变区，所述重链可变区包含：
    (i)SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (ii)SEQ ID NO：4、SEQ ID NO：5和SEQ ID NO：6所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (iii)SEQ ID NO：7、SEQ ID NO：8和SEQ ID NO：9所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (iv)SEQ ID NO：10、SEQ ID NO：11和SEQ ID NO：12所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (v)SEQ ID NO：1 3、SEQ ID NO：14和SEQ ID NO：15所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (vi)SEQ ID NO：16、SEQ ID NO：17和SEQ ID NO：18所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (vii)SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：21所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (viii)SEQ ID NO：22、SEQ ID NO：23和SEQ ID NO：24所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (ix)SEQ ID NO：25、SEQ ID NO：26和SEQ ID NO：27所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (x)SEQ ID NO：28、SEQ ID NO：29和SEQ ID NO：30所示的CDR1、CDR2和CDR3或其功能活性变体；或
    (xi)SEQ ID NO：31、SEQ ID NO：32和SEQ ID NO：33所示的CDR1、CDR2和CDR3或其功能活性变体。
  2. 权利要求1的抗VEGF抗体，其中所述功能活性变体是与SEQ ID NO：1-33中任一个的氨基酸序列具有至少70％、75％、 80％、85％、90％、95％、98％或99％序列同一性的功能活性变体。
  3. 权利要求1的抗VEGF抗体，其中所述重链可变区包含至少一个氨基酸添加、插入、缺失和/或置换。
  4. 权利要求1-3中任一项的抗VEGF抗体，其中所述重链还含有恒定区。
  5. 权利要求1-3中任一项的抗VEGF抗体，其中所述重链还含有Fc片段。
  6. 权利要求1-5中任一项的抗VEGF抗体，其中所述抗体由重链组成。
  7. 前述权利要求任一项的抗VEGF抗体，其中所述抗体是单克隆抗体、嵌合抗体或人源化抗体。
  8. 权利要求1-3中任一项的抗VEGF抗体，其中所述抗体是单域抗体。
  9. 权利要求1-3中任一项的抗VEGF抗体，其中所述抗体的重链可变区序列如SEQ ID NO：34、SEQ ID NO：35、SEQ ID NO：36、SEQ ID NO：37、SEQ ID NO：38、SEQ ID NO：39、SEQ ID NO：40、SEQ ID NO：41、SEQ ID NO：42、SEQ ID NO：43、SEQ ID NO：44、SEQ ID NO：45或SEQ ID NO：46所示。
  10. 一种抗VEGF抗体，其与参比抗体竞争结合VEGF，所述参比抗体为权利要求1中所限定抗体的任一个。
  11. 一种分离的核酸序列，其编码权利要求1-10中任一项的抗VEGF抗体。
  12. 权利要求11的核酸序列，其如SEQ ID NO：47、SEQ ID NO：48、SEQ ID NO：49、SEQ ID NO：50、SEQ ID NO：5 1、SEQ ID NO：52、SEQ ID NO：53、SEQ ID NO：54、SEQ ID NO：55、SEQ ID NO：56、SEQ ID NO：57、SEQ ID NO：58或SEQ ID NO：59所示。
  13. 一种载体，其包含权利要求11或12的核酸序列。
  14. 一种宿主细胞，其表达权利要求1-10中任一项的抗VEGF 抗体，和/或包含权利要求11或12的核酸序列或权利要求13的载体。
  15. 一种生产抗VEGF抗体的方法，其包括在允许表达所述抗体的条件下培养权利要求14的宿主细胞；和从所得培养产物中纯化抗体。
  16. 一种药物组合物，所述组合物包含权利要求1-10中任一项的抗VEGF抗体和药学上可接受的赋形剂。
  17. 权利要求16的药物组合物，其还包含一种或多种治疗活性化合物。
  18. 一种抗体偶联药物，其包含偶联于其它药剂的权利要求1-10中任一项的抗VEGF抗体。
  19. 权利要求18的抗体偶联药物，其还包含接头。
  20. 一种药盒，其包含
    a)权利要求1-10中任一项的抗VEGF抗体，或权利要求16的药物组合物；和
    b)使用说明。
  21. 一种用于调节VEGF活性的方法，所述方法包括给予有效量的至少一种权利要求1-10中任一项的抗VEGF抗体。
  22. 一种抑制血管生成的方法，所述方法包括向有需要的患者给予有效量的至少一种权利要求1-10中任一项的抗VEGF抗体。
  23. 一种治疗与VEGF相关疾病或病症的方法，所述方法包括向有需要的患者给予有效量的至少一种权利要求1-10中任一项的抗VEGF抗体。
  24. 权利要求23的方法，其中所述疾病或病症包括肿瘤或癌症或眼科疾病。
  25. 权利要求24的方法，其中所述肿瘤或癌症包括乳腺癌、脑肿瘤、肾癌、卵巢癌、甲状腺癌、肺癌、结直肠癌、子宫内膜癌、血管肉瘤、膀胱癌、胚胎组织癌、颈部肿瘤、恶性胶质瘤、胃癌、 胰腺癌、鼻咽癌。
  26. 权利要求24的方法，其中所述眼科疾病包括黄斑水肿、年龄相关性黄斑变性、糖尿病性视网膜病变、视网膜中央静脉阻塞、新生血管性青光眼以及其它涉及新生血管的眼科疾病，所述黄斑水肿包括糖尿病性黄斑水肿、白内障术后或葡萄膜炎引起的黄斑水肿。
PCT/CN2015/070209 2015-01-06 2015-01-06 抗vegf抗体 WO2016109943A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
EP15864295.9A EP3078674B1 (en) 2015-01-06 2015-01-06 Anti-vegf antibody
PCT/CN2015/070209 WO2016109943A1 (zh) 2015-01-06 2015-01-06 抗vegf抗体
AU2015376558A AU2015376558B9 (en) 2015-01-06 2015-01-06 Anti-VEGF antibody
US15/101,472 US10456466B2 (en) 2015-01-06 2015-01-06 Anti-VEGF antibody
ES15864295T ES2833530T3 (es) 2015-01-06 2015-01-06 Anticuerpo anti-VEGF

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/070209 WO2016109943A1 (zh) 2015-01-06 2015-01-06 抗vegf抗体

Publications (1)

Publication Number Publication Date
WO2016109943A1 true WO2016109943A1 (zh) 2016-07-14

Family

ID=56355406

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/070209 WO2016109943A1 (zh) 2015-01-06 2015-01-06 抗vegf抗体

Country Status (5)

Country Link
US (1) US10456466B2 (zh)
EP (1) EP3078674B1 (zh)
AU (1) AU2015376558B9 (zh)
ES (1) ES2833530T3 (zh)
WO (1) WO2016109943A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110105450A (zh) * 2019-04-12 2019-08-09 深圳普瑞金生物药业有限公司 Vegf单域抗体、核苷酸序列及试剂盒

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9840553B2 (en) 2014-06-28 2017-12-12 Kodiak Sciences Inc. Dual PDGF/VEGF antagonists
WO2017117464A1 (en) 2015-12-30 2017-07-06 Kodiak Sciences Inc. Antibodies and conjugates thereof
AU2020364071A1 (en) 2019-10-10 2022-05-26 Kodiak Sciences Inc. Methods of treating an eye disorder

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037421A1 (en) 2001-09-13 2005-02-17 Institute For Antibodies Co., Ltd Methods of constructing camel antibody libraries
CN101663319A (zh) * 2007-02-21 2010-03-03 埃博灵克斯股份有限公司 针对血管内皮生长因子的氨基酸序列和包括其的多肽用于治疗特征在于过量和/或病理性血管发生或新血管形成的病症和疾病

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6416758B1 (en) * 1999-04-28 2002-07-09 Board Of Regents, The University Of Texax System Antibody conjugate kits for selectively inhibiting VEGF
CN102002104A (zh) 2009-08-28 2011-04-06 江苏先声药物研究有限公司 一种抗vegf的单克隆抗体及含有该抗体的药物组合物
US20120225081A1 (en) * 2010-09-03 2012-09-06 Boehringer Ingelheim International Gmbh Vegf-binding molecules

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050037421A1 (en) 2001-09-13 2005-02-17 Institute For Antibodies Co., Ltd Methods of constructing camel antibody libraries
CN101663319A (zh) * 2007-02-21 2010-03-03 埃博灵克斯股份有限公司 针对血管内皮生长因子的氨基酸序列和包括其的多肽用于治疗特征在于过量和/或病理性血管发生或新血管形成的病症和疾病

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 1995
A. BELL ET AL.: "Differential tumor-targeting abilities of three single-domain antibody formats", CANCER LETT, vol. 289, no. 1, 1 March 2010 (2010-03-01), pages 81 - 90, XP026897079, DOI: doi:10.1016/j.canlet.2009.08.003
DIRK SAERENS; GHOLAMREZA HASSANZADEH GHASSABEH; SERGE MUYLDERMANS.: "Single-domain antibodies as building blocks for novel therapeutics", CURRENT OPINION IN PHARMACOLOGY, vol. 8, 2008, pages 600 - 608, XP002642572, DOI: doi:10.1016/J.COPH.2008.07.006
FERRARA N; GERBER HP; LE COUTER J.: "The biology of VEGF and its receptors", NAT MED, vol. 9, no. 6, 2003, pages 669 - 676, XP008126354, DOI: doi:10.1038/nm0603-669
GENGRINOVITCH S; GREENBERG SM; COHEN T; GITAY-GOREN H; ROCKWELL P; MAIONE TE; LEVI BZ; NEUFELD G.: "Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms", J BIOL CHEM, vol. 270, no. 25, 23 June 1995 (1995-06-23), pages 15059 - 65, XP002098057, DOI: doi:10.1074/jbc.270.25.15059
HUANG CHEN-XING; SHEN ZU-GUANG: "Vascular endothelial growth factor- fundamental research and experimental study in plastic surgery", CHINESE J REPARATIVE AND RECONSTRUCTIVE SURGERY, vol. 160, 2002, pages 64 - 68
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, NIH PUBLICATION NO. 91-3242
KEYT BA; BERLEAU LT; NGUYEN HV; CHEN H; HEINSOHN H; VANDLEN R; FERRARA N.: "The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency", J BIOL CHEM., vol. 271, no. 13, 29 March 1996 (1996-03-29), pages 7788 - 95
KLETTNER A; ROIDER J .: "Treating age-related macular degeneration interaction of VEGF-antagonists with their target", MINI REV MED CHEM, vol. 9, no. 9, 2009, pages 1127 - 1135
LI X; HU Y; SUN X; ZHANG J; ZHANG M.: "Bevacizumab for neovascular age-related macular degeneration in China", OPHTHALMOLOGY, vol. 119, no. 10, October 2012 (2012-10-01), pages 2087 - 93
MARTIN DF; MAGUIRE MG ET AL.: "Ranibizumab and Bevacizumab for neovascular age-related macular degeneration", N ENGL J MED, vol. 364, 2011, pages 1897 - 1908
MUYLDERMANS: "Single domain camel antibodies: current status", J BIOTECHNOL, vol. 74, 2001, pages 277 - 302, XP008019929
PARK JE; KELLER GA; FERRARA N.: "The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the subepithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF", MOL BIOL CELL, vol. 4, no. 12, December 1993 (1993-12-01), pages 1317 - 26
See also references of EP3078674A4
STANFIELD R; DOOLEY H; FLAJNIK M; WILSON I.: "Crystal structure of a shark single-domain antibody V region in complex with lysozyme", SCIENCE, vol. 305, 2004, pages 5691
VAN BERGEN T; VANDEWALLE E; VAN DE VEIRE S: "The role of different VEGF isoforms in scar formation after glaucoma filtration surgery", EXP EYE RES, vol. 93, 2011, pages 689 - 699, XP028115139, DOI: doi:10.1016/j.exer.2011.08.016

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110105450A (zh) * 2019-04-12 2019-08-09 深圳普瑞金生物药业有限公司 Vegf单域抗体、核苷酸序列及试剂盒
CN110105450B (zh) * 2019-04-12 2023-07-04 深圳普瑞金生物药业股份有限公司 Vegf单域抗体、核苷酸序列及试剂盒

Also Published As

Publication number Publication date
AU2015376558A1 (en) 2017-12-07
US10456466B2 (en) 2019-10-29
AU2015376558B2 (en) 2021-06-24
ES2833530T3 (es) 2021-06-15
AU2015376558B9 (en) 2021-07-01
EP3078674B1 (en) 2020-09-23
EP3078674A4 (en) 2017-10-11
EP3078674A1 (en) 2016-10-12
US20180169228A1 (en) 2018-06-21

Similar Documents

Publication Publication Date Title
CN108148133B (zh) 抗vegf抗体
WO2021042694A1 (zh) 抗vegf单域抗体及其应用
US20130078216A1 (en) Liver targeting molecules
US20220235123A1 (en) Synthetic antibodies against vegf and their uses
CN108148136B (zh) 抗vegf抗体
WO2016109943A1 (zh) 抗vegf抗体
CN103003302B (zh) 人源化和嵌合抗-备解素抗体
JP2016027801A (ja) 改良された抗血清アルブミン結合変異体
ES2665851T3 (es) Nuevo anticuerpo anti-CTGF humano
JP6002354B1 (ja) Ａｎｇ２抗体
WO2023279803A1 (zh) Rbv的蛋白结合分子及其用途
JP6074071B2 (ja) ＶＥＧＦＲ２／Ａｎｇ２化合物
WO2023216981A1 (zh) 松弛素或类似物的融合蛋白及其医药用途
TW202302633A (zh) 結合ｖｅｇｆ和ａｎｇ２的雙特異性結合分子以及其用途
TW202202520A (zh) 自主旋鈕(autonomous knob)結構域肽
CN117858907A (zh) 一种TGF-β/VEGF双功能抗体融合蛋白
EP3810179A1 (en) Complement anaphylatoxin binders and their use in treatment of a subject having an ocular wound and/or fibrosis

Legal Events

Date Code Title Description
REEP Request for entry into the european phase

Ref document number: 2015864295

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 15101472

Country of ref document: US

Ref document number: 2015864295

Country of ref document: EP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15864295

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2015376558

Country of ref document: AU

Date of ref document: 20150106

Kind code of ref document: A