CN108101985A

CN108101985A - Anti-VEGF antibody

Info

Publication number: CN108101985A
Application number: CN201810103433.8A
Authority: CN
Inventors: 方海洲; 曲伟; 郑赞顺; 庄兰芳; 王新志
Original assignee: YISHENG BIOLOGICAL PHARMACEUTICAL CO Ltd SHUHAI
Current assignee: YISHENG BIOLOGICAL PHARMACEUTICAL CO Ltd SHUHAI
Priority date: 2015-01-06
Filing date: 2015-01-06
Publication date: 2018-06-01
Anticipated expiration: 2035-01-06
Also published as: CN108148136B; CN105820244B; CN108101986B; CN108101986A; CN105820244A; CN108285484A; CN108285484B; CN108148136A; CN108101985B

Abstract

The invention discloses the antibody of specific binding vascular endothelial growth factor (VEGF), particularly heavy chain antibody, more particularly single domain antibody.Also disclose the preparation method and therapeutical uses of the antibody.

Description

Anti-VEGF antibody

The application for Application No. 201510004038.0 application for a patent for invention divisional application, original application day 2015 06 day 01 month year, publication date are on 08 03rd, 2016, entitled " anti-VEGF antibody ".

Technical field

The present invention relates to antibody and its applications, and specifically, the present invention relates to specific binding vascular endothelial growth factor The antibody of (Vascular Endothelial Growth Factor, VEGF), particularly heavy chain antibody, more particularly single domain resist Body；And the preparation method and therapeutical uses of the antibody.

Background technology

Angiogenesis refers to from vein after existing capillary or capillary develop and form new blood vessel, is one It is related to the complex process of the different kinds of molecules of various kinds of cell.Angiogenesis is to promote angiogenesis factor and inhibiting factor coordinative role Complex process both under normal circumstances in equilibrium state, once this balance, which is broken, will activate vascular system, makes angiogenesis Excessively or inhibition vascular system makes vascular deterioration.

Known many diseases and angiogenesis associated angiogenesis out of control and undesirable.These diseases include but unlimited In for example so-called solid tumor of tumour and liquid (or blood) knurl (such as leukaemia and lymthoma), inflammation such as rheumatoid or rhematic inflammation After disease, especially arthritis (including rheumatoid arthritis) or other chronic inflammations such as chronic asthma, artery sclerosis or transplanting Artery sclerosis, mullerianosis, neovascular diseases of the eye, such as retinopathy (including diabetic retinopathy), age-related macular Denaturation, psoriasis, hemangioblastoma, hemangioma, artery sclerosis.It is other to be given birth to angiogenesis blood vessel out of control and undesirable It is obvious to those skilled in the art into related disease.

Vascular endothelial growth factor is the heparin binding growth factor for having specificity to vascular endothelial cell, can be in body Interior induction of vascular is newborn.Including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placenta growth factor.

The main function of VEGF-A can also increase to promote vascular endothelial cell proliferation, migration and the formation of tube chamber Vascular leakage promotes monocyte chemotactic and B cell generation.The biological effect of VEGF-A is to rely on and its specific receptor knot It closes and mediates, mainly specific receptor Vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in connection. Wherein, VEGFR-2 is considered as main VEGFR, and the hyperplasia of vascular endothelial cell is had a major impact.VEGFR-2 passes through Intracellular kinases induction VEGF binding dimers and the receptor for needing autophosphorylation, so as to strengthen the mitosis of cell (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 includes 8 extrons and 7 intrones, and transcription montage is mainly into multiple hypotypes：VEGF121、VEGF145、 VEGF206, VEGF165, VEGF189, these hypotypes have different molecular masses, solubility and heparin affinity, wherein VEGF165 is the most important hypotypes 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 secreting type soluble protein, Vascular endothelial cell can be directly acted on and promote vascular endothelial cell proliferation, accelerate the reparation of vascular endothelial cell damage, increased Vasopermeability reduces Intravascular Thrombus formation and thrombus occlusion, and inhibits endometrial hyperplasia (yellow stars at dawn, Shen ancestral's light blood vessels The research of endothelial growth factors and the application J. China Reconstructive surgery magazine .2002,160 in tissue repair:64–68).

Existing vascular endothelial growth factor drug includes Macugen (Pegaptanib sodium, trade name Macugen), Ranibizumab (trade name Lucentis), bevacizumab (Bevacizumab, trade name Avastin), VEGF Trap etc..Focus currently for the dispute of anti-vegf preparation is possible to aggravate the formation of tissue fibers film.It is clinical at present to use In the anti vegf agents for treating a variety of diseases (such as age-related macular degeneration), it is necessary to frequently carry out intraocular injection, cause to send out The potential risk of raw entophthamia, this is apparent problem existing for anti-vegf treatment.Have researcher using huve cell and The fibrocyte of Tenon capsules, observation cut down the effect of monoclonal antibody and Macugen to VEGF different subtypes again, as a result confirm VEGF-165, VEGF-121 mainly influences angiogenic growth, and VEGF-189 mainly influences fibrosis forming process.Monoclonal antibody and Lucentis energy are cut down again Inhibit all active VEGF-A hypotypes (Van Bergen T, Vandewalle E, Van de Veire S, et a1.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), this may be and cuts down monoclonal antibody again to cause some patient's glass In glass body cavity the reason for fibrosis.

The drug of anti-vegf needs repetitive treatment for every 4~6 weeks at present, and Lucentis treats the average year injection volume of the 1st year About 6.9 times, (Li X, Hu Y, Sun X, Zhang J, the Zhang M.Bevacizumab for that cut down monoclonal antibody again about 7.7 times neovascular age-related macular degeneration in China.Ophthalmology.2012Oct., 119(10):2087-93), so frequently there is the potential risk that entophthamia occurs in intraocular drug-injection in treatment, be badly in need of exploitation drug effect Long, retina is penetrating to absorb better novel antibodies drug, to extend dosage period, reduces drug administration by injection and is brought not to patient Suitable and risk.

In addition, anti-vegf preparation expression and purification complex process at present, generally existing is of high cost, and stability is poor, application The realistic problems such as face is not wide.

Heavy chain antibody is a kind of antibody isolated from the serum of camellid, is only made of heavy chain, antigen knot It is only a single domain being connected by hinge area with Fc areas to close area, and this antigen binding domain separated from antibody after still Have the function of with reference to antigen, therefore referred to as single domain antibody (single-domain antibody, sdAb) or nano antibody (nanobody).Unlike conventional antibodies, single domain antibody is a peptide chain containing about 110 amino acid, molecule Amount is about the 1/10 of conventional antibodies, this just provides a new method (Muyldermans.Single for the molecule construction of antibody domain camel antibodies:current status.J Biotechnol 2001,74:277-302).This kind of single domain Antibody have molecule is small, thermal stability is good, under detergent and high concentration uric acid environment stabilization, in-vivo tissue good penetrability, can Good (Stanfield R, Dooley H, Flajnik M, the Wilson I.Crystal structure of a shark of dissolubility single-domain antibody V region in complex with lysozyme.Science.2004,305 (5691)), easily expression, beneficial to prokaryotic system expression, production cost is low, antigen recognizing epitope is unique, and can identify hiding resist The characteristics such as antigenic sites in immunization experiment, Clinics and Practices, gradually play huge function (Dirk beyond imagination Saerens,Gholamreza Hassanzadeh Ghassabeh,Serge Muyldermans.Single-domain antibodies as building blocks for novel therapeutics.Current Opinion in Pharmacology 2008,8:600–608)。

Therefore, this field needs a kind of antibody for the drawbacks described above that can overcome existing anti-vegf preparation, such as can be special Property combination VEGF simultaneously inhibits its active single domain antibody.

The content of the invention

The present invention provides anti-VEGF antibody, its variant or derivative, wherein the antibody includes heavy chain variable region, it is described Heavy chain variable region includes：(i)SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:CDR1, CDR2 and CDR3 shown in 3 or Its functional activity variant；Or (ii) SEQ ID NO:4、SEQ ID NO:5 and SEQ ID NO:CDR1, CDR2 shown in 6 and CDR3 or its functional activity variant；Or (iii) SEQ ID NO:7、SEQ ID NO:8 and SEQ ID NO:CDR1 shown in 9, CDR2 and CDR3 or its functional activity variant；The functional activity variant is and SEQ ID NO:The amino acid of any of 1-9 Sequence has the functional activity variant of at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity.

In specific embodiments, the present invention provides heavy chain antibody, the antibody is made of heavy chain, the heavy chain Variable region includes：(i)SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:CDR1, CDR2 and CDR3 or its work(shown in 3 It can active variant；Or (ii) SEQ ID NO:4、SEQ ID NO:5 and SEQ ID NO:CDR1, CDR2 and CDR3 shown in 6 or Its functional activity variant；Or (iii) SEQ ID NO:7、SEQ ID NO:8 and SEQ ID NO:CDR1, CDR2 shown in 9 and CDR3 or its functional activity variant.

On the one hand, the heavy chain variable region of antibody of the present invention can include at least one amino acid addition, insertion, missing and/or Displacement.On the other hand, antibody of the invention can be monoclonal antibody, chimeric antibody or humanized antibody, multi-specificity antibody And/or bispecific antibody and their segment.In a specific embodiment, antibody of the invention resists for humanization Body.

In a specific embodiment, the heavy chain of antibody of the present invention can also contain constant region.In another specific reality It applies in scheme, the heavy chain of antibody of the present invention also contains Fc segments.

In some specific embodiments, antibody of the invention is heavy chain antibody, i.e. is only made of heavy chain.At some In specific embodiment, antibody of the invention is single domain antibody.

In addition, the present invention provides the antibody with reference antibody competitive binding VEGF, the reference antibody is above-mentioned antibody Any one.

The invention further relates to the nucleotide sequences of encoding such antibodies；Carrier comprising these nucleotide sequences；And host is thin Born of the same parents, the above-mentioned antibody of the host cell expression and/or include these nucleotide sequences or carrier.

The present invention also provides the method for preparing antibody, step includes：It is cultivated under conditions of allowing to express the antibody Above-mentioned host cell；With the antibody purification from gained cultured products.

The invention further relates to pharmaceutical composition, antibody and pharmaceutically acceptable excipient comprising the present invention.The medicine Compositions can also include one or more therapeutical active compounds, for example known anti-vegf medicine of the therapeutical active compound Object or antitumor drug.

On the other hand, the invention further relates to antibody coupling drug (Antibody-drug conjugate, ADC), it includes It is coupled to the antibody of the present invention of other medicaments, other medicaments such as chemotherapeutics, growth inhibitor, toxin is (such as bacterium, true Bacterium, the enzyme activity toxin of plant or animal origin or its segment) or radio isotope (i.e. radioconjugates).

Antibody coupling drug can also include the connector unit between drug unit and antibody units.

Moreover, it relates to adjust the method for VEGF activity by giving the antibody of a effective amount of present invention.This hair It is bright be related to inhibit the method for angiogenesis by giving the antibody of a effective amount of present invention to patient in need.

The present invention also provides a kind of method treated with VEGF relevant diseases or illness, the described method includes in need Patient give the antibody of a effective amount of at least one present invention.The disease or illness include tumour or cancer or ophthalmology disease Disease.The tumour or cancer include breast cancer, brain tumor, kidney, oophoroma, thyroid cancer, lung cancer, colorectal cancer, intrauterine Film cancer, angiosarcoma, carcinoma of urinary bladder, embryonic tissue cancer, tumor colli, glioblastoma, stomach cancer, cancer of pancreas, nasopharyngeal carcinoma etc..It is described Ophthalmology disease includes macular edema caused by a variety of causes (including diabetic macular edema, postcataract or uveitis The macular edema caused by various diseases such as afterwards), age-related macular degeneration, diabetic retinopathy, retinal centre Vein obstruction, neovascular glaucoma and other ophthalmology diseases for being related to new vessels.

In addition, the purposes the invention further relates to antibody of the invention in the drug for adjusting VEGF activity is prepared；This Purposes of the antibody of invention in the drug for inhibiting angiogenesis is prepared；The present invention antibody prepare for treat with Purposes in VEGF relevant diseases or the drug of illness.

The present invention also provides medicine boxs, and it includes antibody or described pharmaceutical composition a) of the invention；And it b) uses and says It is bright.

Description of the drawings

Fig. 1 is the figure of the SDS-PAGE testing results of the hVEGF165 albumen of purifying.Wherein, the 1st swimming lane is standard protein Marker(Invitrogen,Cat.No.:LC5677)；2nd swimming lane is the 2 non-reduced hVEGF165 of μ g；3rd swimming lane is 5 μ g non-also Former hVEGF165；4th swimming lane is 2 μ g reduction hVEGF165；5th swimming lane is 5 μ g reduction hVEGF165.

Fig. 2 is immune response test result, shows that animal generates preferable immune response, serum after antigen has been injected Potency is about 1:100k.

Fig. 3 is the agarose gel electrophoresis testing result of total serum IgE, shows that gained RNA mass meets the demand of library construction.

Fig. 4 is into the amplification V after cDNA, obtained through PCR by the total serum IgE reverse transcription of Fig. 3_HThe Ago-Gel electricity of H segments Swimming purification result.

Fig. 5 is for connecting V_HThe phagemid vector collection of illustrative plates of H segments.

Fig. 6 is the figure of phage display library segment insertion rate detection.It is detected by the PCR to 72 Random clones, In there are 69 to clone to have the insertion of single domain antibody genetic fragment, insertion rate 69/72=95.8%.

Fig. 7 is and the obtained single domain antibody library sequence by the way that the positive colony for having Insert Fragment in Fig. 6 is sequenced Diversity detection figure, it is seen that library diversity is good.

Fig. 8 is FASEBA screening special carrier collection of illustrative plates.The carrier is amicillin resistance, contains SASA and 6 × His Tag can be used for the secreting, expressing of antibody.

Fig. 9 is the affinity sequence of antibody after FASEBA screenings；9A, 9B, 9C are the affinity sequence knot of 3 different batches Fruit.Wherein the picture left above：The combination of difference clone, the sensing figure of dissociation；Top right plot：The combination of difference clone, the square of dissociation rate The system of battle formations；Lower-left figure：Difference clone schemes by normalized sensing；Bottom-right graph：The sensing of the higher antibody of selected part affinity Figure.

Figure 10 is Receptor Competition the selection result figure, wherein the horizontal screening of expression quantity and affinity sequence will be passed through and it is preferred that 15 single domain antibodies be used for the screening.It is compared according to the competitiveness result with control, wherein 4 preferable clones is selected to be used for Prepared by heavy chain antibody, for cell growth inhibition assay.

Figure 11 is the graph that heavy chain antibody tests HUVEC cell inhibitory effects.Pass through various concentration antibody on cell The degree of Proliferation Ability judges that 5 heavy chain antibodies are respectively provided with inhibition function.

Figure 12 is the variable region sequences of 5 heavy chain antibodies in embodiment 11.

Specific embodiment

The present invention relates to antibody, its variant or the derivatives of specific binding VEGF；And the preparation method of the antibody And therapeutical uses.For example, the present invention relates to the heavy chain antibodies of specific binding VEGF, more particularly single domain antibody.Meanwhile this Invention antibody exhibited improvements in terms of cell Proliferation and angiogenesis is inhibited prior art Anti-X activity (such as Avastin excellent effect), as following embodiment is further described.

The single domain antibody of the present invention has molecular weight more smaller than Fab segment and overall length IgG antibody, and general 12-15kD can For building multivalent antibody, and pass through genetic engineering and transform to improve affinity and extend half-life period, extend doses at intervals week The characteristics such as phase.Compared with common antibody drug, the binding ability of single domain antibody drug and antigen, in high temperature, hydrochloric acid in gastric juice, protease etc. More stablize under extreme condition, and the conformational stability with height.It is anti-that complement effector cell poison is easily induced with whole antibody drug Should be different, single domain antibody drug lacks Fc segments, will not cause complement effect.Meanwhile because single domain antibody molecular weight is small, this is anti- Body can have more preferable permeability in ocular tissue and tumor tissues administration.It is steady in protease, extreme temperature and pH environment Qualitative, high-affinity makes its oral and other administration route provide feasibility.

Single domain antibody can be in protokaryon or eukaryotic, such as scale table is carried out in Escherichia coli or yeast cells It reaches, expression quantity is very big, thus greatly facilitates batch production, is conducive to control production cost, is also beneficial to later stage drug The market prospects of exploitation.

Unless it is defined otherwise herein, there should be those of ordinary skill in the art institute with relevant scientific and technical terms herein The meaning of understanding.

Term " antibody " is well understood in biology and biomedical sector, typically refers to complete antibody and any anti- Body segment or its is single-stranded.Antibody is the glycoprotein secreted by being known as the specialization bone-marrow-derived lymphocyte of thick liquid cell.Its also referred to as immune ball Albumen (Ig), because it contains the apokoinou construction domain being present in many protein.Antibody most probable is included usually by disulfide bond 2 weight (H) chains and 2 light (L) chains of connection or its antigen-binding portion thereof.Each heavy chain is by heavy chain variable region (V_H) and heavy chain perseverance Determine district's groups into.Every light chain is equally by variable region (V_L) and constant region composition.Constant region of light chain is made of a domain C L.V_H And V_LArea can be further subdivided into hypervariable region, be known as complementary determining region (CDR), be scattered with the more conservative of referred to as framework region (FR) Region.In some specific embodiments, antibody of the invention is only made of heavy chain.In some specific embodiments In, antibody of the invention is single domain antibody.

Kabat etc. can be used in Sequences of Proteins of Immunological Interest, the 5th Version, US Dept.of Health and Human Services, PHS, NIH, NIH Publication no.91-3242, Method described in 1991 determines the given complementary determining region (CDR) of antibody and framework region (FR).

The present invention includes " variant " of antibody, for example, the heavy chain variable region of antibody of the present invention can include at least one amino Acid addition, insertion, missing and/or displacement, such as 10,20,30,40,50, preferably such as 1,2,3,4,5,6,7,8,9,10 Amino acid addition, insertion, missing and/or displacement.

The present invention also includes " derivative " of antibody." derivative " of antibody is the antibody through chemical modification, such as is passed through With other chemical parts such as polyethylene glycol, albumin (such as human serum albumins) combination, phosphorylation and glycosylation.It is unless another External declaration, otherwise term " antibody " include its segment, derivative, variant.

On the one hand, the present invention provides anti-VEGF antibody, its variant or derivative, wherein the antibody includes weight chain variable Area, the heavy chain variable region include：(i)SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:CDR1, CDR2 shown in 3 With CDR3 or its functional activity variant；Or (ii) SEQ ID NO:4、SEQ ID NO:5 and SEQ ID NO:CDR1 shown in 6, CDR2 and CDR3 or its functional activity variant；Or (iii) SEQ ID NO:7、SEQ ID NO:8 and SEQ ID NO:Shown in 9 CDR1, CDR2 and CDR3 or its functional activity variant.

The functional activity variant is and SEQ ID NO:The amino acid sequence of any of 1-9 has at least 70%, example Such as at least 75%, at least 80%, at least 85%, at least 90%, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%th, the functional activity variant of 99% sequence identity.

In a more particular embodiment, the weight chain variabl area sequence of antibody of the present invention such as SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:Shown in 14.

In addition, the present invention also provides with reference antibody competitive binding VEGF antibody, the reference antibody be above-mentioned antibody Any one.

The invention further relates to the nucleotide sequences of encoding such antibodies；Carrier comprising these nucleotide sequences；And host is thin Born of the same parents, the above-mentioned antibody of the host cell expression and/or include these nucleotide sequences or carrier." host cell " is for expressing The cell of nucleic acid nucleic acid for example of the present invention.Host cell can be prokaryotes, such as Escherichia coli or its can be eucaryon give birth to Object, such as unicellular eukaryote (for example, yeast).

In specific embodiments, the nucleotide sequence such as SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO: 17、SEQ ID NO:18、SEQ ID NO:Shown in 19, as following embodiment is described in detail.

The present invention also provides the method for preparing antibody, step includes：It is cultivated under conditions of allowing to express the antibody Above-mentioned host cell；With the antibody purification from gained cultured products, as following embodiment is described in detail.

The invention further relates to pharmaceutical composition, antibody and pharmaceutically acceptable excipient it includes the present invention.It is described Pharmaceutical composition can also include one or more therapeutical active compounds, for example known anti-vegf of the therapeutical active compound Drug or antitumor drug.

The therapeutical active compound can be administered simultaneously with the antibody of the present invention or sequential administration.

Pharmaceutical composition can be prepared according to techniques known in the art.Term " excipient " refers broadly to one or more work Any component outside property therapeutic component.Excipient can be inert substance, inactive substance and/or the substance without pharmaceutical activity.It assigns Shape agent can be used as a variety of purposes, for example, as carrier, solvent, diluent, tablet auxiliaries and/or improving giving for active material Medicine and/or absorption.The preparation of pharmacy activity component and various excipient is known in the art, see, for example, Remington:The Science and Practice of Pharmacy (such as the 19th edition (1995), and it is any after version).Excipient Non-limiting examples are：Solvent, diluent, buffer, preservative, tonicity contributor, chelating agent and stabilizer.

The antibody of the present invention can be given in the form of pharmaceutical composition.It can not only prepare as parenteral solution, freeze The liquid preparations such as preparation, spray can also be prepared as solid pharmaceutical preparations such as capsules.Administration route can be, for example, intravenously Injection, oral or topical administration, such as percutaneously, through conjunctiva and/or through eyes etc..In specific embodiments, administration route For by oral administration.In another specific embodiment, administration route is through eyes.

On the other hand, the invention further relates to antibody coupling drug, it includes the antibody of the present invention for being coupled to other medicaments, institutes State other medicaments such as chemotherapeutics, growth inhibitor, toxin (such as bacterium, fungi, the enzymatic activity poison of plant or animal origin Element or its segment) or radio isotope (i.e. radioconjugates).

The local delivery that other medicaments are carried out using antibody coupling drug can be by medicament targeted delivery to tumour, and in tumour Middle intracellular accumulation, and systemic these medicaments not being coupled of giving may result in normal cell and need the tumour removed The unacceptable levels of cytotoxicity of cell.

Antibody coupling drug generally comprises the connector unit between drug unit and antibody units.In some embodiment party In case, which can cut under the conditions of intracellular, so as to the cutting of connector drug unit be caused to be released in intracellular environment from antibody It puts.Connector can be that (can for example be included but not limited to by intracellular peptase or protease：Lysosome or inclusion body protease) cutting Peptidyl linkers.In some embodiments, at least two amino acid of length of peptidyl linkers or at least 3 amino acid. Can be Val-Cit connectors or Phe-Lys connectors by the peptidyl linkers of intracellular protein cleavage in one specific embodiment.

In other embodiments, connector unit is not cleavable, and drug is discharged by, for example, the degradation of antibody.

In addition, the invention further relates to lived by giving the antibody of a effective amount of present invention to adjust and (preferably inhibit) VEGF Property, inhibit mammal angiogenesis method.

" patient in need " means any mammal, the mammal be such as, but not limited to people, horse, ox, Cat, mouse, rabbit, rat, goat etc..Preferably, the mammal is people.

In some treatment uses, the Antibody Combination of a variety of present invention is given.

The present invention is further described with reference to following non-limiting example.

It is prepared by 1. antigen of embodiment

Antigen behaviour VEGF165 (the Human vascular endothelial growth that the present embodiment is directed to Factor 165, hVEGF165) molecule (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 VEGF121and VEGF165using 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), the wherein nucleotide sequence of people VEGF165 antigens such as SEQ ID NO: Shown in 21；The amino acid sequence of people's VEGF165 antigens such as SEQ ID NO:Shown in 22.

According to the amino acid sequence, after carrying out the codon optimization of mammal expression to it, pass through fully synthetic mode DNA after being optimized after being cloned into carrier for expression of eukaryon pTT5 (being authorized by invention mechanism NRC), is used to prepare transfection grade matter Grain.Cultivate 7 days after transfection HEK293E cells, by the way that the Capto columns of cell and culture supernatant for hand assembled are collected by centrifugation And HiTrap^TMThe two-step solution purifying of Q HP, purifies and carries out endotoxin and handle.Protein concentration detection uses UV_280nmLight absorption value detects, and protein endotoxins are horizontal to be detected using LAL methods, and the activity of antigen passes through HUVEC cell proliferation experiments It measures.Concentration be there are as 1.25mg/ml, volume 22ml, total amount is the hVEGF165 albumen of 27.5mg, and level of endotoxin is 0.537EU/ml (table 1), SDS-PAGE testing results are shown in Fig. 1, and albumen is in -80 DEG C of preservations.

1 hVEGF165 purifying protein information of table

2. animal immune of embodiment and immunoreaction measurement

1. animal immune

Alpaca (Lama pacos) is selected, in 4 different time points, to be carried out respectively in omoplate and back as experimental animal 6 injecting immunes.PBS is the dilution of antigen, and immune volume is 1ml every time, and amount of antigen and adjuvant information are shown in Table 2.Immune examination The BSA of agent 1mg/ml containing final concentration, antigen and adjuvant are immunized after Fresh mixing again before the injection.

2 alpaca immunizing antigen information of table

Respectively at jugular vein blood collection, anti-coagulants is added in when gathering blood for different time in four times for immunologic process design (table 3). Take a blood sample 5ml for the first time, remaining each 15ml three times.Utilize Ficoll 1.077 reagent (Sangon, Cat.No.:F760014-100) and After anticoagulation carries out gradient centrifugation, separate peripheral blood lymphocytes and carry out cell resuspension counting, add RNAlater (TIANGEN,Cat.No.:DP408-02), in -20 DEG C of preservations.The serum that gradient centrifugation obtains is also in -20 DEG C of preservations.

3 alpaca immunization time calendar of table

2. immune response is tested

Using enzyme-linked immunosorbent assay (ELISA) respectively to before immune, third time it is immune and the 4th time it is immune after blood Final proof product carry out antigen specific immune reaction test.With NaHCO₃(pH 9.6) solution dilutes immunogene, is coated with microwell plate (Corning, Cat.No.:9018), stay overnight for 4 DEG C.It after board-washing four times, is closed with PBS-T solution in board-washing machine using 3%BSA Liquid, 37 DEG C of closing 2h.After tetra- board-washings of PBS-T, 37 DEG C of serum for being incubated overnight gradient dilution.After tetra- board-washings of PBS-T, it is incubated Goat-anti yamma secondary antibody (the Novus Biologicals, Cat.No. of horseradish peroxidase-labeled:NB7242).It uses TMB colour developing 10min, add in 1M HCl color development stoppings.System after reaction terminating is detected using MK3 (Thermo) microplate reader Light absorption value at 450nm.It may determine that by the reaction result of ELISA, animal generates preferably after proteantigen has been injected Immune response, serum titer is about 1:100k (Fig. 2).

3. antibody phage libraries of embodiment are built

3.1.RNA extraction

The TRIzol reagents of corresponding volume are added in separated peripheral blood lymphocytes according to cell number, cell cracking After finishing, according toOperating instruction (Invitrogen, the Cat.No. of Plus RNA purification systems:It is 12183-555) complete Extraction into total serum IgE separates.The quality of total serum IgE is detected by agarose gel electrophoresis, and the dense of RNA is measured using light absorption method Degree.According to measurement result, 105.6 μ g total serum IgEs are obtained altogether.RNA forms in agarose gel electrophoresis figure are complete (Fig. 3), quality On meet the demand of library construction.

3.2. reverse transcription PCR

Technical specification is used according to SuperScriptTM III First-Strand Synthesis System (Invitrogen, Cat.No.:18080-051), using 20 primers of Oligo (dT) by total serum IgE reverse transcription into cDNA.According to white horse with a black mane The sequence signature of hunchbacked antibody, select specificity forward primer and reverse primer for VHH amplification (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 2005/0037421 A1 of States Patent), the particular sequence of primer is shown in Table 4.By the first round to the PCR of cDNA, according to The molecular weight of PCR product isolates and purifies the segment containing VHH of about 600bp, is hereafter obtained again by the second wheel PCR amplification The segment of VHH simultaneously introduces two different Sfi I restriction enzyme sites of identification sequence at DNA fragmentation both ends simultaneously, obtains altogether The VHH segments (Fig. 4) of 101 μ g gel-purifieds.

4. primer sequence information of table and PCR amplification effect

3.3 library construction

The V that will be expanded with different batches cell and different primers_HH segments mix, and then utilize restriction enzyme Sfi I carry out digestion.The V after digestion is separated, purified and is obtained by 2% agarose gel electrophoresis_HH；Meanwhile utilize limit Property restriction endonuclease Sfi I processed carry out digestion to phagemid vector (Fig. 5), are separated, purified by 1.5% agarose gel electrophoresis And obtain the carrier after digestion.V after digestion is measured by light absorption method_HAfter the concentration of H and phagemid vector, according to carrier/piece Section molar ratio is respectively 1:3,1:5,1:10 mixing, add in T4 ligases (NEB, Cat.No.:M0202L same volume company) is carried out afterwards The preparation of reaction system is connect, is attached overnight in 16 DEG C.Linked system is carried out in order phenol/chloroform, chloroform, Ethanol precipitation carries out the connection product that purifying obtains by light absorption method the measure of concentration.It is obtained for 3 different linked systems Product carry out the electricity conversion of equivalent DNA and TG1 electricity transformed competence colibacillus, pass through spread plate and gradient dilution method and calculate 3 The size of library, that is, transformation efficiency of linked system, random picking positive colony send survey detection library diversity.Selection conversion effect The highest system of rate largely connect and convert, and counts storage capacity.According to plate count the results show that library storage capacity is about 1.8×10⁸(table 5).

5 single domain antibody phage display library storage capacity of table calculates

Random clones PCR is carried out to library bacterium colony, it is seen that the segment insertion rate in library is 95.8% (Fig. 6), to wherein having The positive colony of Insert Fragment is sequenced, and is compared by the amino acid sequence to single domain antibody CDR region domain, it is seen that library Diversity is good (Fig. 7).The 2YT containing 100 μ g/ml ampicillins and 2% glucose is used to cultivate the overnight tablet of coating Base carries out microorganism collection, 5,000g centrifugation removal products of cellular metabolism, and cell is resuspended with same medium, is divided into two parts of progress Library stores.

4. phage display of embodiment and screening

4.1. prepared by antigen biotinylation compound

According to EZ-Link Sulfo-NHS-LC-Biotinylation kits (Pierce, Cat.No.:21335) Operating process illustrates, hVEGF165 albumen is carried out biotinylation mark.Detection protein biotinylation is tested by HABA to mark Degree.By biotinylation mark hVEGF165 mixed with the magnetic bead of 0.5ml M-280 Streptavidins (Invitrogen, Cat.No.:112.06D), it is incubated overnight for 4 DEG C, magnetic bead is then separated by magnetic frame, and is failed and magnetic bead with PBS solution elution With reference to biotinylated protein, for preparing antigen magnetic bead coupled complex.Biotinylation is reacted and obtained after purification 0.52mg hVEGF165 albumen, HABA experiments show that biotin coupling is horizontal for every mole of albumen coupling 6 moles of biotins point Son.

4.2. phage library is recovered

The phage library liquid storage of about 100 μ l (MOI is about 20) is taken to be inoculated into 2YT culture mediums, 225rpm, 30 DEG C into Row culture, in culture to exponential phase (OD₆₀₀=0.5) M13KO7 helper phages (NEB, Cat.No. are added in when: N0315S), 225rpm, 30 DEG C are incubated overnight.Bacteriophage is collected by centrifugation, by culture supernatant and PEG/NaCl solution mix and from The phage particle that recovery obtains by repeatedly centrifuging and being resuspended, is finally suspended in the PBS of 1-2ml by heart precipitating phage. The potency for the phage library that recovery obtains is calculated by finite gradient dilution method, obtains 3.15 × 10¹³The library of pfu/ml.

4.3. it is directed to target protein phage selection

Take~2 × 10¹¹The bacteriophage of pfu carries out normal as the input of the first round and the antigen magnetic bead coupled complex of 10ul Temperature is incubated, and when mild mixing 2 is small on gyroscope；By magnetic frame by Beads enrichment, the phagocytosis that will do not combined with magnetic bead Body washes away, and the elution of resuspension and non-specific binding to magnetic bead needs to carry out 7 times；The magnetic bead that last time is resuspended adds in TEA solution by the bacteriophage elution combined with antigen magnetic bead coupled complex and separates, be rapidly added Tris-HCl buffer solutions into Row neutralizes；The output of bacteriophage after first round screening is calculated by finite gradient dilution method, while the first round is afforded Bacteriophage is incubated overnight and is expanded, and design parameter and process are identical with the recovery description of library before this.Second wheel screening will be with ~10¹¹Library after the first round output Phage amplification of pfu is carried out as the antigen magnetic bead coupled complex of input and 1ul It is incubated and screens, specific operation process and parameter are identical with first round screening.

4.4. Phage-ELISA is identified

Picking screens the monoclonal plaque on the overnight tablet of bacteriophage output calculating for the second wheel, is put into every hole and contains There are 96 hole depth orifice plates of 500 μ l 2YT culture mediums, 225rpm, 30 DEG C are cultivated, in culture to exponential phase (OD₆₀₀= 0.5) M13KO7 helper phages (NEB, Cat.No. are added in when:N0315S), 225rpm, 30 DEG C are incubated overnight.Bacterium is collected by centrifugation Body takes supernatant, adds in coating hVEGF165 in advance and the microwell plate closed, with the anti-M13 monoclonal antibodies (GE of HRP/ Healthcare,Cat.No.:It 27-9421-01) is detected as secondary antibody, other ELISA operating parameters are examined with immune response Survey it is identical, according to light absorption value assess output positive rate.By the part of random picking and the positive phage clones of antigen recognizing Carry out V_HThe sequencing of H segments by sequence alignment and analysis, infers the clonal diversity by phage display output.Pass through The diversity of positive rate and sequence is screened to determine the need for carrying out the phage display of more rounds.Phage clone is positive More than 50%, and meet diversity requirement.Therefore the second gene constructed FASEBA libraries of wheel output phage clone sdAb are selected, For further colony screening.

6 bacteriophage of table washes in a pan sieve and ELISA detections

Embodiment 5.FASEBA is screened

5.1.FASEBA library construction

The phage DNA of last wheel phage display output is extracted, V is encoded by PCR amplification_HThe segment of H passes through company It connects in the FASEBA carriers for being cloned into patent, all structure clonal structures are V_H- 6 × His of H-linker-SASA (Fig. 8).Connection Product will be transformed into TG1 thalline.

5.2.FASEBA screening

5.2.1. sample preparation and expression assessment

The random picking monoclonal from the FASEBA libraries of structure is put into 96 holes that 500 μ l 2YT culture mediums are contained in every hole Deep-well plates, in culture to OD₆₀₀During=0.6-0.8, add in IPTG and stay overnight induced expression.Removal is clear after thalline were collected by centrifugation 100 μ l culture supernatants are added in coating BSA in advance and the microwell plate closed, the anti-His monoclonals of mouse marked using HPR Antibody detects (GenScript, Cat.No. as secondary antibody:A00186)；Meanwhile it is pre- that aliquot of culture supernatant is taken to be added to In the microwell plate for being first coated with hVEGF165 albumen and closing, using the anti-His monoclonal antibodies of mouse that HPR is marked as secondary antibody Detect (GenScript, Cat.No.:A00186), with OD₄₅₀Light absorption value assesses the expression of different clones.Different batches The screening that 5000 monoclonals are used for expression quantity and antigen binding power is totalled over, expression quantity is higher and have high knot with antigen 138 positive colonies with joint efforts are for affinity sequence and follow-up screening.

5.2.2. chip prepares

According to BIAcore T200 equipment operating manuals, BSA albumen is fixed on CM5 by standard coupling procedure flow (GE healthcare,Cat.No.:BR-1006-68) on chip surface.Basic process is as follows：Under the conditions of 25 DEG C, with HBS- EP solution (0.01M HEPES [pH 7.4], 0.15M NaCl, 3mM EDTA and 0.005% [v/v] surfactant P20) is Equipment running buffer, flow velocity 10ml/min.0.4M 1- ethyl -3- (dimethylamino third of the injection more than 7 minutes first Base) carbodiimide hydrochloride (EDC)/0.1M n-hydroxysuccinimides (NHS) (1:1), for activating Sensor Chip CM 5 table Face then by injecting 7 minutes 20 μ g/ml BSA protein solutions for being diluted in 10mM sodium acetates (pH4.5), finally injects 7 again The 1M ethylaminoethanols (pH8.5) of minute close unbonded activated sites point.It is horizontal that aforesaid operations obtain BSA coupling reactions At 327 reactons (resonance units, RU).

5.2.3. the affinity sequence of anti-hVEGF165 single domain antibodies

By sdAb-SASA clonal expression supernatants mentioned above, through 96 hole filters (Pall, Cat.No.:PN5045) It at 4 DEG C, is degermed and other particles with removing within 5 minutes with 4000g centrifugal filtrations, dilutes detection sdAb antibody simultaneously with HBS-EP solution Order flows through BSA coupling chip surfaces.Ordination process includes following four step：A. captured using the chip of fixed BSA The single domain antibody of SASA couplings；B. hVEGF165 is injected, it is made to be combined with capturing the chip surface for having single domain antibody；C. injection fortune Row buffering liquid, and monitor dissociation stage 300s；D. 10mM glycine/HCl (pH 2.0) is injected to the chip surface for being coupled BSA, 30 μ l/min, 30s live again.Chip capture antibody, antigen binding, antigen dissociation, the BSA chip surfaces of each round are required to Regeneration.BSA chip surfaces flowed through as control test chip using the purifying SASA albumen of concentration 200nM live again the inspection of effect. 138 clone's point 3 batches are ranked up, to clone A10981 as reference, 6 A10981 repeated clonings between different batches one Cause property is fine, and 400s dissociation degrees about~20%, wherein 93 clone's dissociation rates are slower than A10981 (Fig. 9), surveyed by picking Sequence.Wherein 53 single domain antibodies are chosen for prokaryotic expression and are tested with cell growth inhibition assay, wherein 15 single domains Antibody is for further competitive screening.

5.2.4.hVEGFR2 competitive screening

The horizontal screening of expression quantity will be passed through and preferred 15 single domain antibodies of affinity sequence screen for Receptor Competition, It being capable of blocking antigen hVEGF165 and its receptor hVEGFR binding antibodies to obtain.Detailed process is as follows：

A. by the fixed method of amino coupled (same to 5.2.2) by hVEGFR2 proteopexies in CM5 chip surfaces；B. note HVEGF165 albumen is penetrated, observes binding characteristic, stops injection when binding curve is close to saturation；C. the core combined in hVEGF165 Different single domain antibodies is injected on piece surface and observes binding characteristic, while injects the drug of the anti-vegf listed Avastin is as control；D. if the epitope of antibody combination VEGF165 is just VEGF and VEGFR2 combination epitopes, antibody It cannot be combined with VEGF again or be competed the VEGF for having combined VEGFR2, binding signal will be significantly less than VEGF sheets Body；If epitope and the VEGF＆VEGFR2 combinations epitope of antibody combination VEGF165 are different or unrelated, antibody will combine The VEGF165 combined with receptor, the binding signal of generation will be apparently higher than VEGF in itself.According to competitive result and control Compared to (Figure 10), 4 therein preferable clones is selected to be prepared for heavy chain antibody for cell growth inhibition assay.

Embodiment 6：The preparation of single domain antibody

The prokaryotic expression of single domain antibody, purifying, endotoxin removal process are as follows.

6.1. reagent prepares

6.1.1. prokaryotic expression reagent

Tryptone,OXOID LP0042

Yeast extract,OXOID LP0021

Casein acid hydrolysate,Sigma C9386

KH₂PO₄, traditional Chinese medicines AR CAS [7778-77-0]

Na₂HPO₄.12H₂O, traditional Chinese medicines AR 10020318

NH₄Cl, traditional Chinese medicines AR CAS [12125-02-9]

NaCl, traditional Chinese medicines AR 10019318

MgCl₂, traditional Chinese medicines AR 7791-18-6

CaCl₂, traditional Chinese medicines AR 10043-52-4

Glucose, traditional Chinese medicines AR 10010518

Glycerine, Sigma G5516-500ML

IPTG,Amresco 0487-100G

VB1, Aladdin AR 1099302

Ampicillin, 100mg/ml, 0.22 μm of filter filtration treatment；

IPTG mother liquors：1M, 0.22 μm of filter filtration treatment, 1-2ml packing, 20 DEG C of ﹣ freeze (term of validity 3 months)；

MgCl₂Mother liquor：1M, 121 DEG C of moist heat sterilization 30min, 1-2ml packing, 4 DEG C of storages (term of validity 6 months)；

CaCl₂Mother liquor：1M, 0.22 μm of filter filtration treatment, 1-2ml packing, 4 DEG C of storages (term of validity 6 months)；

VB1 mother liquors：50mg/ml, 0.22 μm of filter filtration treatment, 1-2ml packing, 4 DEG C of storages (term of validity 6 months)；

Glucose mother liquid：20% (W/V), 0.22 μm of filter filtration treatment, 4 DEG C of storages (term of validity 3 months)；

Glycerine mother liquor：50% (V/V), 121 DEG C of moist heat sterilization 30min, 4 DEG C of storages (term of validity 6 months)；

Casein acid hydrolysate mother liquors：4%, 121 DEG C of moist heat sterilization 30min, the room temperature storage (term of validity 3 Month)；

10 Χ M9 salting liquids：6%Na₂HPO₄(W/V), 3%KH₂PO₄(W/V), 1%NH₄Cl (W/V), 0.5%；

NaCl (W/V), 121 DEG C of moist heat sterilization 30min, room temperature storage (term of validity 3 months)；

2YT culture mediums：1.6% (W/V) Tryptone, 1.0% (W/V) yeast extract, 0.5% (W/V) NaCl, 121 DEG C of moist heat sterilization 30min；

10 Χ TB culture mediums：12% (W/V) Tryptone, 24% (W/V) yeast extract, 4% (V/V) Glycerol, 121 DEG C of moist heat sterilization 30min.

6.1.2. protein purification reagent and equipment

10 Χ BugBuster Protein Extraction Reagent (Novagen, 70921-4)；

100mM PMSF：1.74g PMSF in 100ml aqueous isopropanols (the green skies, ST506)；

10mg/ml nucleases (DNase I)：Usual every gram of weight in wet base thalline 1 μ l (Life Science Product and Service, DD0099-1)；

5mg/ml lysozymes (Lysozyme)：Usual every gram of weight in wet base thalline is with 100 μ l (giving birth to emerging biology, L0005-10)；

Quick StartTM Bradford reagents (Bio-Rad, 500-0204)；

High Affinity Ni-NTA Resin (GenScript, L00250)；

HiTrapTM Desalting, 5ml, (GE Healthcare, 17-1408-01)；

purifier 10(GE Healthcare)；

Lysis buffer：20mM HEPES, 150mM NaCl, 10% (V/V) glycerine, 40mM imidazoles, pH 8.0；

Combination buffer：20mM Na₂HPO₄, 0.5M NaCl, 20mM imidazoles, pH 7.4；

Wash miscellaneous buffer solution：20mM Na₂HPO₄, 0.5M NaCl, 40mM imidazoles, pH 7.4；

Elution buffer：20mM Na₂HPO₄, 0.5M NaCl, 300mM imidazoles, pH 7.4；

1ΧPBS：137mM NaCl, 10mM Na₂HPO₄, 2mM KH₂PO₄, pH 7.4.

6.1.3. endotoxin removal reagent and equipment

ToxinEraser TM Endotoxin Removal Resin 1.5ml resins (GenScript, L00402)；

PD-10Columns；ToxinEraser TM Regeneration Buffer (GenScript, M01053)；

ToxinEraser TM Equilibration Buffer(GenScript M01054)；

Gel method endotoxin detection kit (GenScript, L00451)；

Reagents (Tachypleus Amebocyte Lysate abbreviation TAL sensitivity 0.25EU/ml, Xiamen City's reagents Test Co., Ltd., Factory)；

0.1M NaOH (no heat source water is prepared)；

0.1M hydrochloric acid (no heat source water is prepared)；

37 DEG C of constant incubators.

6.1.4. filtration sterilization reagent and equipment

Millex-GP Filter Unit, 0.22 μm of (Millipore, Lot：R4AA43868).

6.1.5. determination of protein concentration and detection reagent and equipment

2000 spectrophotometers of Nanodrop (Thermo)；

ExpressPlus PAGE Gel：4-20%, 12wells (GenScript, M42012)；

MOPS Running Buffer Powder (GenScript, M00138)；

Loading Buffer (5 Χ, non-reduced)：0.25M Tris-HCl (pH 6.8), 10%SDS, 0.5% bromine phenol Indigo plant, 50% glycerine, 7.8%DTT；

Loading Buffer (5 Χ, reduction)：0.25M Tris-HCl (pH 6.8), 10%SDS, 0.5% bromophenol blue, 50% glycerine.

6.2. method and flow

6.2.1. prepared by bacterial strain

A) convert：The prokaryotic expression plasmid for being built into single domain antibody gene is transferred to bacterial strain by chemical conversion or electricity TG1 is coated on the tablet of 2YT amicillin resistances, and 37 DEG C of constant temperature are incubated overnight；

B) picking monoclonal：200 μ g/ml ampicillins of final concentration and 2% (W/ are added in the 2YT culture mediums of 10ml V) glucose；Tweezers on alcolhol burner are fully burnt, 10 μ l sterilizing pipette tips are gripped after cooling, from picking 1 on conversion tablet Monoclonal is put into culture medium, and 225rpm, 37 DEG C, constant temperature incubation is overnight.

6.2.2. switching induction

A) M9 culture mediums are prepared：Final concentration 0.2% (W/V) glucose, 1mM MgCl are added into 1X M9 salting liquids₂、 0.1mM CaCl₂, 0.4% (W/V) Casein acid hydrolysate, 5mg/LVB1,200 μ g/ml ampicillins, put 37 DEG C of shaking table preheatings；

B) transfer：Bacterium solution will be incubated overnight to take out from shaking table, by 1:100 switching systems, take and are incubated overnight product 4000rpm 10min is centrifuged, is resuspended with fresh 1X M9 salting liquids, 4000rpm centrifuges 10min, and the culture medium of preheating is forwarded to after being resuspended again In, 37 DEG C of shaking tables of postposition, 225rpm is cultivated for 24 hours；

C) induce：The IPTG and 200 μ g/ml of final concentration 1X TB culture mediums and final concentration of 1mM are added into M9 culture mediums Ampicillin, 225rpm, 25 DEG C culture 48h (adding once final concentration of 200 μ g/ml ampicillins for 24 hours)；

D) sample is received：After induction, overnight culture is dispensed to Centrifuge Cup, 4 DEG C, 11,000rpm, centrifuges 15min, Collect thalline.

6.2.3. protein purification

A) BugBuster cracking process sample preparation

I. crack：Expression precipitation adds 5ml lysis buffers that (lysis buffer is resuspended according to every gram of weight in wet base thalline：With combination Buffer solution dilutes 10X BugBuster Protein Extraction Reagent to 1X, and it is molten to add in 100 μ g/ml of final concentration Bacterium enzyme, 2 μ g/ml nucleases and 1mM PMSF), room temperature middling speed oscillation incubation 1h；

Ii. prepared by protein crude extract：By the sample of cracking in 4 DEG C, 12,000g, 30min is centrifuged, collects supernatant and is used in combination 0.22 μm of membrane filtration.

B) nickel column affinity purification

I. balance columns material：With the ddH of 5 times of column material volumes₂O and equilibration buffer High Affinity Ni-NTA Resin；

Ii. combine：Protein crude extract is mixed with appropriate High Affinity Ni-NTA Resin, concussion is incubated 1h. After incubation, the mixed liquor of crude extract and column material is added in into PD-10 void columns and collects column material, and collected efflux and treat to divide in next step Analysis is used；

Iii. wash miscellaneous：Foreign protein is eluted with the miscellaneous buffer solution of washing of at least 50 column volumes.It (washes miscellaneous process to be contaminated with Bradford Liquid is as indicator：5 μ l wash 200 μ l Bradford dye liquors of miscellaneous liquid addition and see whether to become blue, if becoming blue, continue miscellaneous Albumen wash-out to dye liquor is basically unchanged color, can carry out at this time in next step.)；

Iv. elute：Target protein is eluted with the elution buffer of at least ten column volume.(elution process Bradford Dye liquor judges whether that elution is complete as indicator, method with step iii).

C) desalination/buffer exchange

I. balance：With appropriate flow velocity (0.5ml/min) with 5 times of column volumes in 10 systems of purifier ddH₂The HiTrapTM Desalting columns of O and PBS balances 5ml；

Ii. loading：The protein liquid after appropriate (0.5ml) ni-sepharose purification is added in appropriate flow velocity (0.5ml/min) In HiTrapTM Desalting columns；

Iii. elute：(0.5ml/min) continues to be eluted with the PBS of at least 10 times column volumes under appropriate flow velocity, receives Collect the ultraviolet peak of target where albumen.

6.2.4. endotoxin removal and detection

A) endotoxin removal

I. sample treatment：Ionic strength is adjusted to 0.2 ± 0.5M using 1M sodium chloride before purifying, uses 0.1M hydroxides Sodium or 0.1M salt acid for adjusting pH value are 7.4 ± 0.2；

Ii. activated resin：PD-10Columns is vertically fixed, removes the lid at the top of prepacked column, is added in ToxinEraser TM Endotoxin Removal resins open flow speed controller, protection liquid are made to drain off under the effect of gravity, The regeneration buffer of 5ml is added in, adjusts flow speed controller, keeps flow velocity in 0.25ml/min (about 10 drops/min), it is to be regenerated slow Fliud flushing drains off, and adds 5ml regeneration buffers, repetitive operation is twice, it is ensured that system keeps apyrogeneity (i.e. endotoxin-free) to exist；

Iii. resin is balanced：PD-10Columns activation finishes, and adds in the level pad of 6ml, adjusts flow speed controller, Keeping flow velocity, drain off level pad, then is repeated twice by this operation in 0.5ml/min；

Iv. endotoxin removal：Flow speed controller is closed, is added in sample using no heat source pipette tips, opens controller, control Flow velocity processed is not higher than 0.25ml/min, after effluent volume reaches 1.5ml, no heat source reception pipe is begun to use to receive sample, sample After product drain off, the elution of 1.5ml-3.0ml level pads is added, collects leacheate.Detect sample concentration and level of endotoxin (elution process judges whether to collect complete by the use of Bradford dye liquors as indicator).

B) gel method measures endotoxin

I. dilute sample：It needs sample being diluted to suitable concentration according to sensitivity of the limulus reagent (0.25EU/ml) (0.005μg/ml；0.05μg/ml；0.5μg/ml；5μg/ml)；

Ii. endotoxin standard is diluted：Endotoxin standard is got out, redissolves postposition whirlpool with baterial endotoxin test water Oscillator mixing 15min is revolved, then is diluted to debita spissitudo (0.5EU/ml)；

Iii. detect：TAL reagents are taken, it is complete to reagent that 100 μ l baterial endotoxin tests of addition gently shake at least 30s with water Fully dissolved is careful not to cause bubble, is separately added into the 100 following samples of μ l：Positive control (endotoxin standard 0.5EU/ml), Detected sample after negative control (endotoxin-free water), dilution：(1) four concentration samples to be tested in are closed nozzle, are gently shaken It is even, be vertically disposed in 37 DEG C of incubators be incubated 1 it is small when, then take out observation；

Iv. result records：Test tube from constant incubator is gently taken out, is slowly inverted 180 ° of test tube, content is in pipe Real gel, it is indeformable, it is not slid from tube wall as the positive；Other situations are then feminine gender.Positive pipe is the positive, and negative tube is the moon Property, state is identical in same scope, and experiment is effective.

6.2.5. filtration sterilization

0.22 μm of the filter filtered sample of sterile working in Biohazard Safety Equipment, and after appropriate amount of sample is taken to remain Continuous detection.

6.2.6. concentration, purity detecting

A) determination of protein concentration

I. according to the protein amino acid sequence of offer, the absorptivity of the albumen is calculated

Ii. the ultraviolet absorption value (A280) of protein solution is measured

Iii. protein concentration is calculated according to formula：Ultraviolet absorption value (the A of protein concentration=protein solution₂₈₀The suction of)/albumen Backscatter extinction logarithmic ratio

B) purity of protein detects

According to said determination concentration a certain amount of albumen (such as 2 μ g) and the standard protein (such as 2 μ g BSA) of equivalent is taken to go back SDS-PAGE is carried out under former and non reducing conditions, detect purity and confirms concentration.

The HUVEC cell inhibitory effects experiment of 7. single domain antibody of embodiment

7.1. prepared by cell

It will be about 3 × 10⁵HUVEC (ATCC, Cat No.PCS-100-010) cell recovery is inoculated in the culture dish of 10cm And added in fresh culture medium every 3-4 days；When cell fusion degree reaches 85%-95% time-division disk cultures, and add in fresh Culture medium, 7 instead of interior cell be used in Proliferation Ability experiment, be generally fixed to for the 6th generation.

7.2.VEGF the HUVEC Proliferation Abilities experiment induced

M199 buffering liquids (Medium-199 1X Earle's Salts (Invitrogen#11150-059) are used respectively； 10%Fetal Bovine Serum (Gibco, Cat#10100139), heat inactivated；10mM HEPES (Invitrogen,Cat#15630080)；100units/ml Penicillin 100μg/ml Streptomycin (Invitrogen, Cat#10378016)) prepare the antibody samples of 2 × VEGF and various concentration to be measured；By 50 2 × VEGF of μ L It is pre-mixed with the antibody samples of each various concentration to be measured, multiple holes is set on microwell plate, using cell culture medium as the sky of reaction White control, using Avastin as positive control；It will be collected by the cell of trypsase resolution, with M199 buffer solutions clearly simultaneously Cell is resuspended twice, it is 1 × 10 that cell, which is resuspended to cell density,⁵After a cell/ml, each hole 50 μ L of addition of microwell plate are thin Born of the same parents' re-suspension liquid；The microwell plate for adding in cell is placed in culture, 37 DEG C, 5%CO₂Cultivate 96 it is small when；It is used after the completion of cultureLuminescent Cell Viability Assay Kit(Promega,Cat No:G7571) detection is thin Born of the same parents' survival rate, while fluorescence intensity is detected by PHERAStar Plus (BMG Labtech) and records its relative luminous Unit；Production inhibiting rate, growth inhibition ratio %=100* (1-relative light units/largest production are calculated by the following formula Value), wherein associated light unit is the measured value of various kinds sample, and largest production value is relative luminous list when only adding in VEGF Position.Two aspect data of comprehensive maximal percentage inhibition and EC50 choose 4 for building weight from the single domain antibody of 53 prokaryotic expressions Chain antibody and subsequent proliferation inhibition test.

Table 7：The EC50 values of HUVEC cell inhibitory effects experiment are as follows：

The expression of 8. heavy chain antibody of embodiment

By tentatively screening has 4 of certain Proliferation Ability function and 1 screened by Receptor Competition in embodiment 7 Single domain antibody, together with the Fc segments of human IgG1 (SEQ ID NO:20) construct as heavy chain antibody, be cloned into pTT5 carriers Expression and purification and endotoxin removal, detailed process are as follows in HEK293E：

8.1. reagent prepares

With embodiment 6.

8.2. method and flow

8.2.1. cell culture

A) after HEK293 suspension cells are taken out from liquid nitrogen or -86 DEG C of refrigerators, it is put into rapidly in 37 DEG C of water-baths, 1 Cell is melted in~2min；

B) cell melted is added in the preheating FreeStyle of 10 times of volumes^TMIn 293Expression Medium, gently Soft reverse mixing, the culture medium that low-speed centrifugal is collected cell and preheated with proper amount of fresh are resuspended；

C) cell of resuspension is transferred in blake bottle and at 37 DEG C, 5%CO₂, cultivate under 110rpm speed conditions.

8.2.2. transfection

A) day before transfection passes on HEK293 suspension cells with proper density, and transfection same day cell density need to reach 1.5~ 2.0×10⁶A cell/ml, cell viability need to be more than 95%；

B) by suitable DNA and PEI with optimal proportion (such as 1：3) in preheating FreeStyle in right amount^TM 293Expression Abundant mixing in Medium stands 10 minutes at room temperature；

C) above-mentioned PEI-DNA mixtures are added in cell culture, softly rotates mixing, continued at 37 DEG C, 5%CO₂, It is cultivated under 110rpm speed conditions；

D) it is interior when 16~24 is small after transfecting, add in the Tryptone N1 of final concentration of 0.5% (w/v)；

E) culture supernatant is collected by centrifugation within the 6th day and with 0.22 μm of membrane filtration after transfecting with to be purified.

8.2.3. protein purification

A) Protein A affinity purifications

I. balance columns material：With the ddH of 5 times of column material volumes₂O and Binding Buffer balance Protein A column material；

Ii. combine：Column material, is loaded on by hatching combination 1h after the expression supernatant after filtering is mixed with column material after incubation In the manual purification columns of PD-10；

Iii. wash miscellaneous：Foreign protein is washed away with the Binding Buffer of at least 30 times column material volumes (to wash miscellaneous process to use Bradford dye liquors are as indicator：5 μ l wash 200 μ l Bradford dye liquors of miscellaneous liquid addition and see whether to become blue, if becoming blue, Continue to wash miscellaneous process to dye liquor and be basically unchanged color, can carry out at this time in next step)；

Iv. elute：It is used in combination with the Elution Buffer elution target proteins of at least 10 times column material volumes Neutralization Buffer adjust pH value, and (elution process is by the use of Bradford dye liquors as indicator, method to 7.0 or so With step iii, judge whether that elution is complete).

B) desalination/buffering fluid exchange：

By albumen HiTrap obtained by affinity purification^TMDesalting columns existIt is replaced in 10 systems of purifier Into PBS buffer solution.Subsequent endotoxin removal, filtration sterilization, concentration, purity testing and etc. with embodiment 6.

8 heavy chain antibody of table, single domain antibody clone correspondence and its variable region amino acid sequence and nucleotide sequence are compiled Number

The HUVEC cell inhibitory effects experiment of 9. heavy chain antibody of embodiment

5 heavy chain antibodies (table 8) are set into 8 gradient dilution concentration altogether, the initial concentration of antibody is 20 μ g/ml, 1:4 ladders Degree dilution, using Avastin (A68467) as control, other experiment conditions are completely the same as embodiment 7.By to various concentration antibody The degree that cell proliferation inhibits judges that 5 heavy chain antibodies have different degrees of inhibition function (Figure 11).

9 heavy chain antibody sample message of table

Although some features of the present invention are illustrated and described herein, those skilled in the art will expect that many is repaiied Change, substitute, change and be equal.It is to be understood, therefore, that the appended claims are intended to fall into true spirit model of the present invention All such modifications and changes within enclosing.

Sequence table

<110>Yisheng Biological Pharmaceutical Co., Ltd., Shuhai

<120>Anti-VEGF antibody

<160> 31

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213>Alpaca (Lama pacos)

<400> 1

Val Pro Asp Met His

1 5

<210> 2

<211> 16

<212> PRT

<213>Alpaca (Lama pacos)

<400> 2

Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 3

<211> 13

<212> PRT

<213>Alpaca (Lama pacos)

<400> 3

Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr

1 5 10

<210> 4

<211> 5

<212> PRT

<213>Alpaca (Lama pacos)

<400> 4

Ala Tyr Asn Met Gly

1 5

<210> 5

<211> 17

<212> PRT

<213>Alpaca (Lama pacos)

<400> 5

Ala Ile Asn Trp Ser Gly Ile Ser Thr Tyr Tyr Thr Asp Ser Val Lys

1 5 10 15

Gly

<210> 6

<211> 14

<212> PRT

<213>Alpaca (Lama pacos)

<400> 6

Asn Arg Gly Gly Asn Tyr Glu Lys Val Tyr Leu Tyr Asn Asn

1 5 10

<210> 7

<211> 5

<212> PRT

<213>Alpaca (Lama pacos)

<400> 7

Thr Tyr Thr Ile Gly

1 5

<210> 8

<211> 17

<212> PRT

<213>Alpaca (Lama pacos)

<400> 8

Gly Ile Thr Trp Gly Gly Gly Ile Ile Asp Ser Ile Asp Ser Met Lys

1 5 10 15

Gly

<210> 9

<211> 16

<212> PRT

<213>Alpaca (Lama pacos)

<400> 9

Gly Arg Asn Thr Gly Gly Tyr Thr Arg Leu Trp Arg Ser Tyr Asp Tyr

1 5 10 15

<210> 10

<211> 121

<212> PRT

<213>Artificial sequence (RenGongXuLie)

<400> 10

Gly Val Gln Leu Val Glu Ser Gly Gly Gly Trp Val Gln Ala Gly Asp

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Ile Gln Val Thr Val Ser Ser

115 120

<210> 11

<211> 121

<212> PRT

<213>Artificial sequence (RenGongXuLie)

<400> 11

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Pro Tyr Val Pro

20 25 30

Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val

35 40 45

Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly

100 105 110

Gln Gly Ile Gln Val Thr Val Ser Ser

115 120

<210> 12

<211> 123

<212> PRT

<213>Artificial sequence (RenGongXuLie)

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Asp Ser Gly Arg Thr Phe Gly Ala Tyr

20 25 30

Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Asn Trp Ser Gly Ile Ser Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Cys

65 70 75 80

Leu Gln Met Asn Asn Leu Ser Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asn Arg Gly Gly Asn Tyr Glu Lys Val Tyr Leu Tyr Asn Asn

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 13

<211> 123

<212> PRT

<213>Artificial sequence (RenGongXuLie)

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Gly Ala Tyr

20 25 30

Asn Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val

35 40 45

Ala Ala Ile Asn Trp Ser Gly Ile Ser Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Asn Leu Ser Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asn Arg Gly Gly Asn Tyr Glu Lys Val Tyr Leu Tyr Asn Asn

100 105 110

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120

<210> 14

<211> 125

<212> PRT

<213>Artificial sequence (RenGongXuLie)

<400> 14

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Asn Phe Arg Thr Tyr

20 25 30

Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ile

35 40 45

Val Gly Ile Thr Trp Gly Gly Gly Ile Ile Asp Ser Ile Asp Ser Met

50 55 60

Lys Gly Arg Ala Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys

85 90 95

Ala Ala Gly Arg Asn Thr Gly Gly Tyr Thr Arg Leu Trp Arg Ser Tyr

100 105 110

Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 15

<211> 363

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 15

ggggtgcagc tggtggagtc tgggggaggt tgggtgcagg ctggggactc tctgagactc 60

tcctgtacag cctctggaag cataccatat gtccctgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgct 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatcta 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttg cattgaagct tgtggaatac tggggccagg ggatccaggt caccgtctcc 360

tca 363

<210> 16

<211> 363

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 16

caggtaaagc tggaggagtc tgggggaggg ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag cataccatat gtccctgaca tgcactggta ccgccaggct 120

ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgct 180

gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatcta 240

caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300

tcgagtgttg cattgaagct tgtggaatac tggggccagg ggatccaggt caccgtctcc 360

tca 363

<210> 17

<211> 369

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 17

caggtacagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtacag actctgggcg caccttcggt gcttataaca tgggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tgtagcagct attaactgga gtggtattag tacatactat 180

acagactccg tgaagggccg attcactatc tccagagaca acgccaagaa cacggtgtgt 240

ctgcaaatga acaacctgag ccctgaggac acggccgttt attactgtgc agcaaatcgg 300

ggtggtaatt acgaaaaggt ctatctctac aacaactggg gccaggggac ccaggtcacc 360

gtctcctca 369

<210> 18

<211> 369

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 18

caggtacagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtacag cctctgggcg caccttcggt gcttataaca tgggctggtt ccgccagact 120

ccagggaagg agcgtgagtt tgtagcagct attaactgga gtggtattag tacatactat 180

acagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240

ctgcaaatga acaacctgag ccctgaggac acggccgttt attactgtgc agcaaatcgg 300

ggtggtaatt acgaaaaggt ctatctctac aacaactggg gccaggggac ccaggtcacc 360

gtctcctca 369

<210> 19

<211> 375

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 19

gctgtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60

tcctgtgcag cctctggact caacttcagg acctatacca taggctggtt ccgccaggct 120

ccagggaagg agcgtgagtt tatagttggt attacttggg gtggtggtat catagactcc 180

atagactcca tgaagggccg cgccaccatc tccagagaca acgccgagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attcttgtgc agcaggcagg 300

aacacaggag gctacacacg actgtggcga agctatgact actggggcca ggggacccag 360

gtcaccgtct cctca 375

<210> 20

<211> 232

<212> PRT

<213>People (Homo sapiens)

<400> 20

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 21

<211> 498

<212> DNA

<213>People (Homo sapiens)

<400> 21

gcacccatgg cagaaggagg agggcagaat catcacgaag tggtgaagtt catggatgtc 60

tatcagcgca gctactgcca tccaatcgag accctggtgg acatcttcca ggagtaccct 120

gatgagatcg agtacatctt caagccatcc tgtgtgcccc tgatgcgatg cgggggctgc 180

tgcaatgacg agggcctgga gtgtgtgccc actgaggagt ccaacatcac catgcagatt 240

atgcggatca aacctcacca aggccagcac ataggagaga tgagcttcct acagcacaac 300

aaatgtgaat gcagaccaaa gaaagataga gcaagacaag aaaatccctg tgggccttgc 360

tcagagcgga gaaagcattt gtttgtacaa gatccgcaga cgtgtaaatg ttcctgcaaa 420

aacacagact cgcgttgcaa ggcgaggcag cttgagttaa acgaacgtac ttgcagatgt 480

gacaagccga ggcggtga 498

<210> 22

<211> 165

<212> PRT

<213>People (Homo sapiens)

<400> 22

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

115 120 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

130 135 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys

145 150 155 160

Asp Lys Pro Arg Arg

165

<210> 23

<211> 45

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 23

gcccagccgg ccatggccsm bgtrcagctg gtggaktctg gggga 45

<210> 24

<211> 45

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 24

gcccagccgg ccatggccca ggtaaagctg gaggagtctg gggga 45

<210> 25

<211> 45

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 25

gcccagccgg ccatggccca ggctcaggta cagctggtgg agtct 45

<210> 26

<211> 41

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 26

gcccagccgg ccatggccga ggtgcagctg gtggagtgtg g 41

<210> 27

<211> 21

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 27

cgccatcaag gtaccagttg a 21

<210> 28

<211> 29

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 28

ggggtacctg tcatccacgg accagctga 29

<210> 29

<211> 34

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 29

catgtgtaga ctcgcggccc agccggccat ggcc 34

<210> 30

<211> 47

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 30

catgtgtaga ttcctggccg gcctggcctg aggagacggt gacctgg 47

<210> 31

<211> 42

<212> DNA

<213>Artificial sequence (RenGongXuLie)

<400> 31

catgtgtaga ttcctgcggc cgctgaggag acggtgacct gg 42

Claims

1. a kind of anti-VEGF antibody, it is characterised in that：It is made of heavy chain variable region and Fc segments, the amino acid sequence of Fc segments Such as SEQ ID NO：Shown in 20, the amino acid sequence such as SEQ ID NO of heavy chain variable region：Shown in 14.

2. a kind of nucleic acid encodes the heavy chain variable region in claim 1, sequence such as SEQ ID NO：Shown in 19.

3. a kind of carrier, it is characterised in that contain the nucleic acid described in claim 2.

4. a kind of host cell, express the anti-VEGF antibody of claim 1 or it includes the nucleic acid described in claim 2 or Carrier described in claim 3.

5. a kind of pharmaceutical composition, the composition includes the anti-VEGF antibody of claim 1 and pharmaceutically acceptable figuration Agent.