CN105820245A - Anti-VEGF antibody - Google Patents

Abstract

The invention discloses an antibody specifically bind to vascular endothelial growth factor (VEGF), especially a heavy-chain antibody, and more especially a single-domain antibody. The invention also discloses a preparation method of the antibody and a treatment purpose thereof.

Description

Anti- VEGF Antibody

Technical field

The present invention relates to antibody and application thereof, specifically, the present invention relates to specific binding VEGF (Vascular Endothelial Growth Factor, VEGF) antibody, particularly heavy chain antibody, more particularly single domain antibody；And the preparation method of described antibody and therapeutic use.

Background technology

Angiogenesis refers to develop from existing blood capillary or blood capillary posterior vein and form new blood vessel, is the complex process of a different kinds of molecules relating to various kinds of cell.Angiogenesis is to promote angiogenesis factor and the complex process of inhibitive factor coordinative role, and the two is in poised state under normal circumstances, and once this balances to break and will activate vascular system, makes angiogenesis excessively or suppression vascular system makes vascular deterioration.

Known numerous disease and angiogenesis associated angiogenesis out of control and undesirable.These diseases include but not limited to the most so-called solid tumor of tumor and liquid (or blood) tumor (such as leukemia and lymphoma), inflammation such as rheumatoid or rheumatic inflammation, especially arthritis (including rheumatoid arthritis), or other chronic inflammatory disease such as chronic asthma, arteriosclerosis or transplanting artery hardening, endometriosis, neovascular diseases of the eye, such as retinopathy (including diabetic retinopathy), age-related macular degeneration, psoriasis, hemangioblastoma, hemangioma, arteriosclerosis.Other is obvious with the disease of angiogenesis associated angiogenesis out of control and undesirable to those skilled in the art.

VEGF, is to have specific heparin binding growth factor to vascular endothelial cell, can in vivo induction of vascular newborn.Including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor.

VEGF-A primarily serves the purpose of promotion vascular endothelial cell proliferation, migration and the formation of tube chamber, also can increase vascular leakage simultaneously, promote that monocyte chemotactic and B cell generate.The biological effect of VEGF-A is dependent on being combined with its specific receptor and mediating, mainly specific receptor Vascular endothelial growth factor receptor-1 in connection And VEGFR-2 (VEGFR-1).Wherein, VEGFR-2 is considered as main VEGFR, and the hypertrophy of vascular endothelial cell is had a major impact by it.VEGFR-2 is by intracellular kinases induction VEGF binding dimer and the receptor needing autophosphorylation, thus strengthens the mitosis (Klettner of cell 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 exons and 7 introns, it is transcribed montage and becomes multiple hypotype, predominantly: VEGF121, VEGF145, VEGF206, VEGF165, VEGF189, these hypotypes have different molecular mass, dissolubility and heparin affinities, wherein VEGF165 is VEGF-A topmost hypotype (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, increase vascular permeability, make Intravascular Thrombus be formed and thrombus occlusion reduces, and suppress neointimal hyperplasia (yellow stars at dawn, Shen Zuguang. the research of VEGF and the application J in tissue repair. China's Reconstructive surgery magazine. 2002 , 160 : 64 – 68)。

Existing VEGF medicine 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 increase the weight of the formation of tissue fibers film.Being clinically used for treating the anti vegf agents of multiple disease (such as age-related macular degeneration) at present, need frequently to carry out ophthalmic injection, cause the potential risk that endophthalmitis occurs, this is the obvious problem that anti-vegf treatment exists.Researcher is had to use huve cell and the fibrocyte of Tenon capsule, observe and cut down monoclonal antibody and the Macugen effect to VEGF different subtype again, result confirms that VEGF-165, VEGF-121 mainly affect angiogenic growth, and VEGF-189 mainly affects fibrosis forming process.Cut down monoclonal antibody again and Lucentis can suppress whole activated VEGF-A hypotype (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 is probably and cuts down monoclonal antibody again and cause Fibrotic reason in some patient's vitreous chamber.

The at present every 4～6 weeks needs repetitive therapy of medicine of anti-vegf, Lucentis is treated the average year injection volume about 6.9 times of the 1st year, is cut down monoclonal antibody about 7.7 (Li X, Hu Y, Sun again X, Zhang J, Zhang M. Bevacizumab for neovascular age-related macular Degeneration in China. Ophthalmology. 2012 Oct., 119 (10): 2087-93), there is the potential risk that endophthalmitis occurs in ophthalmic drug-injection in treatment the most frequently, it is badly in need of exploitation drug effect long, the penetrating absorption of retina more preferable novel antibodies medicine, to extend dosage period, reduce discomfort and risk that drug administration by injection brings to patient.

In addition, current anti-vegf preparation expression and purification complex process, generally there is cost high, the realistic problems such as poor stability, application surface are the widest.

Heavy chain antibody is isolated a kind of antibody from the serum of camellid, only it is made up of heavy chain, its antigen binding domain is only a single domain being connected with Fc district by hinge region, and this antigen binding domain still has a function of conjugated antigen after separating on antibody, therefore referred to as single domain antibody (single-domain Antibody, sdAb) or nano antibody (nanobody).Unlike conventional antibodies, single domain antibody is one and contains about 110 amino acid whose peptide chains, its molecular weight is about the 1/10 of conventional antibodies, this just molecule construction for antibody provide a new method (Muyldermans. Single domain camel antibodies: current status. J Biotechnol 2001, 74:277-302).This kind of single domain antibody has that molecule is little, Heat stability is good, stable under cleaning agent and high concentration uric acid environment, in-vivo tissue good penetrability, solubility are good (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)), easily express, beneficially prokaryotic system is expressed, production cost is low, antigen recognition epi-position unique, and the characteristics such as hiding antigenic site can be identified, 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 area needs the antibody of a kind of drawbacks described above that can overcome existing anti-vegf preparation, such as can specific binding VEGF suppress the single domain antibody of its activity.

Summary of the invention

The invention provides VEGF antibody, its variant or derivant, wherein said antibody comprises variable region of heavy chain, and described variable region of heavy chain comprises: (i) SEQ ID NO:1, SEQ ID NO:2 and CDR1, CDR2 and the CDR3 shown in SEQ ID NO:3 or its functional activity variant；Or (ii) SEQ ID NO:4, SEQ CDR1, CDR2 and CDR3 shown in ID NO:5 and SEQ ID NO:6 or its functional activity variant；Or (iii) SEQ ID NO: 7, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:8 and SEQ ID NO:9 or its functional activity variant；Or (iv) SEQ ID NO:10, SEQ CDR1, CDR2 and CDR3 shown in ID NO:11 and SEQ ID NO:12 or its functional activity variant；Described functional activity variant is and SEQ In ID NO:1-12, the aminoacid sequence of any one has the functional activity variant of at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence iden.

In particular embodiments, the invention provides heavy chain antibody, described antibody is made up of heavy chain, and the variable region of described heavy chain comprises: (i) SEQ ID NO:1, SEQ ID NO:2 and SEQ CDR1, CDR2 and CDR3 shown in ID NO:3；Or (ii) SEQ ID NO: 4, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:5 and SEQ ID NO:6 or its functional activity variant；Or (iii) SEQ ID NO:7, SEQ CDR1, CDR2 and CDR3 shown in ID NO:8 and SEQ ID NO:9 or its functional activity variant；Or (iv) SEQ ID NO: 10, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:11 and SEQ ID NO:12 or its functional activity variant.

On the one hand, the variable region of heavy chain of antibody of the present invention can comprise at least one aminoacid and adds, inserts, lacks and/or replace.On the other hand, the antibody of the present invention can be monoclonal antibody, chimeric antibody or humanized antibody, multi-specificity antibody and/or bi-specific antibody and their fragment.In a specific embodiment, the antibody of the present invention is humanized antibody.

In a specific embodiment, the heavy chain of antibody of the present invention also can contain constant region.In another specific embodiment, the heavy chain of antibody of the present invention is possibly together with Fc fragment.

In some specific embodiments, the antibody of the present invention is heavy chain antibody, i.e. be only made up of heavy chain.In some specific embodiments, the antibody of the present invention is single domain antibody.

It addition, the invention provides and the antibody of reference antibody competition binding VEGF, described reference antibody is any one of above-mentioned antibody.

The invention still further relates to the nucleotide sequence of encoding such antibodies；Comprise the carrier of these nucleotide sequences；And host cell, the above-mentioned antibody of described host cell expression, and/or comprise these nucleotide sequences or carrier.

Present invention also offers the method preparing antibody, step includes: cultivate above-mentioned host cell under conditions of allowing to express described antibody；With antibody purification from gained cultured products.

The invention still further relates to pharmaceutical composition, comprise the antibody of the present invention and pharmaceutically acceptable excipient.Described pharmaceutical composition also can comprise one or more therapeutical active compound, the such as known anti vegf agents of described therapeutical active compound or antitumor drug.

On the other hand, the invention still further relates to antibody coupling medicine (Antibody-drug conjugate, ADC), it comprises the antibody of the present invention being coupled to other medicament, other medicament described such as chemotherapeutics, growth inhibitor, toxin (the such as enzyme activity toxin of antibacterial, fungus, plant or animal origin, or its fragment) or radiosiotope (i.e. radioconjugates).

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

Moreover, it relates to the method that the antibody of the present invention by giving effective dose regulates VEGF activity.The present invention relates to the method that the antibody of the present invention by giving effective dose to patient in need suppresses angiogenesis.

Present invention also offers a kind for the treatment of and VEGF relevant disease or the method for disease, described method includes the antibody giving at least one present invention of effective dose to patient in need.Described disease or disease include tumor or cancer or ophthalmic diseases.Described tumor or cancer include breast carcinoma, the cerebral tumor, renal carcinoma, ovarian cancer, thyroid carcinoma, pulmonary carcinoma, colorectal cancer, carcinoma of endometrium, angiosarcoma, bladder cancer, embryonal tissue's cancer, tumor colli, glioblastoma, gastric cancer, cancer of pancreas, nasopharyngeal carcinoma etc..Described ophthalmic diseases include macular edema that a variety of causes causes (macular edema that various diseases cause after including diabetic macular edema, postcataract or uveitis etc.), age-related macular degeneration, diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma and other relate to the ophthalmic diseases of new vessels.

It addition, the antibody that the invention still further relates to the present invention is being prepared for regulating the purposes in the active medicine of VEGF；The antibody of the present invention is used for the purposes suppressed in the medicine of angiogenesis in preparation；The antibody of the present invention is used for treatment and the purposes in the medicine of VEGF relevant disease or disease in preparation.

Present invention also offers medicine box, it comprises the antibody of a) present invention, or described pharmaceutical composition；And b) operation instruction.

Accompanying drawing explanation

Fig. 1 is the figure of the SDS-PAGE testing result of the hVEGF165 albumen of purification.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 the 5 non-reduced hVEGF165 of μ g；4th swimming lane is 2 μ g reduction hVEGF165；5th swimming lane is 5 μ g reduction hVEGF165.

Fig. 2 is immunoreation test result, shows that animal creates preferable immunoreation after having injected antigen, and serum titer 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 be the total serum IgE reverse transcription of Fig. 3 is become cDNA after, through the amplification V that PCR obtains_HThe agarose gel electrophoresis purification result of H fragment.

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

Fig. 6 is the figure of phage display library fragment insertion rate detection.By the PCR of 72 Random clones is detected, 69 clones are wherein had to have the insertion of single domain antibody genetic fragment, insertion rate 69/72=95.8%.

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

Fig. 8 is that FASEBA screens special carrier collection of illustrative plates.This carrier is amicillin resistance, containing SASA and 6 × His tag, may be used for the secreting, expressing of antibody.

Fig. 9 is the affinity sequence of antibody after FASEBA screens；9A, 9B, 9C are the affinity ranking results of 3 different batches.Wherein the picture left above: the combination of different clones, the sensing figure dissociated；Top right plot: the combination of different clones, the matrix diagram of dissociation rate；Lower-left figure: different clones are through normalized sensing figure；Bottom-right graph: the sensing figure of the antibody that selected part affinity is higher.

Figure 10 is Receptor Competition the selection result figure, wherein will through expression level screening and affinity sequence and preferred 15 single domain antibodies are used for this screening.Compare with comparison according to this competitiveness result, select wherein 2 prepare, for cell growth inhibition assay for heavy chain antibody.

Figure 11 is the curve chart that HUVEC cell inhibitory effect is tested by heavy chain antibody.Being judged by the degree of variable concentrations antibody on cell Proliferation Ability, 4 heavy chain antibodies are respectively provided with suppression function, and wherein A80887, A80723 and A69458 are the strongest in the suppression function of cellular level.

Figure 12 is the variable region sequences of 4 heavy chain antibodies in embodiment 11.

Figure 13 is blood vessel schematic diagram under Brachydanio rerio intestinal.It is being administered after certain time, is often organizing and take 15 tail Brachydanio rerio at random and take pictures under fluorescence microscope, vascular plexus under intestinal (SIVs) area is carried out quantitative analysis.Comparing employing T-inspection between two groups, multiple-group analysis uses one factor analysis of variance and Dunnett's T-inspection to carry out statistical analysis, and p < 0.05 shows have significant difference, and the drug effect computing formula of anti-angiogenesis is as follows:

Figure 14 is the figure of vessel area under intestinal.It can be seen that as A80887 with Avastin, have significant new vessels inhibitory action.

Figure 15 is the figure of vascular plexus area suppression ratio under intestinal.Avastin is positive control drug, and when equimolar quality (the most about 2.7 pmol), the suppression new vessels formation efficiency of A80887 is 23.2%, hence it is evident that be better than Avastin Both 6.9%(compare, p < 0.001).With the Avastin 1 μ g(6.7 pmol converted by quantity) compare, the suppression ratio of new vessels is had no significant difference.

Detailed description of the invention

The present invention relates to the antibody of specific binding VEGF, its variant or derivant；And the preparation method of described antibody and therapeutic use.Such as, the present invention relates to the heavy chain antibody of specific binding VEGF, be more particularly single domain antibody.Meanwhile, antibody of the present invention exhibits improvements over the excellent effect of prior art Anti-X activity (such as Avastin) in terms of suppression cell proliferation and angiogenesis, as following example further describe.

The single domain antibody of the present invention has the molecular weight less than Fab fragment and total length IgG antibody, general 12-15kD, may be used for building multivalent antibody, and by genetic engineering modified to improve affinity and to extend the half-life, extends the characteristics such as doses at intervals cycle.Compared with common antibody drug, single domain antibody medicine and the binding ability of antigen, more stable under the extreme conditions such as high temperature, gastric acid, protease, and there is the conformational stability of height.Easily inducing complement effector lymphocyte's poison from whole antibody medicine and react different, single domain antibody medicine lacks Fc fragment, will not cause complement effect.Meanwhile, because single domain antibody molecular weight is little, this antibody can have more preferable permeability in ocular tissue and tumor tissues are administered.It is in the stability in protease, extreme temperature and pH environment, high-affinity, makes it oral and other route of administration provides feasibility.

Single domain antibody can carry out scale expression in protokaryon or eukaryotic cell in such as escherichia coli or yeast cells, expression is very big, thus greatly facilitates batch production, is conducive to controlling production cost, is also beneficial to the market prospect of later stage drug development.

Unless defined otherwise herein, should have, to the most relevant scientific and technical terms, the implication that those of ordinary skill in the art are understood.

Term " antibody " was well understood in biology and biomedical sector, it is common that refer to complete antibody and any antibody fragment or its strand.Antibody is by the glycoprotein of the most plasmacytic specialization bone-marrow-derived lymphocyte secretion.It is also referred to as immunoglobulin (Ig), because it contains the apokoinou construction territory being present in numerous protein.Antibody most probable comprises 2 weight (H) chains and 2 light (L) chains or its antigen-binding portion thereof generally connected by disulfide bond.Every heavy chain is by variable region of heavy chain (V_H) and CH composition.Every light chain is equally by variable region (V_L) and constant region composition.Constant region of light chain is made up of a domain C L.V_HAnd V_LDistrict can be further subdivided into hypervariable region, referred to as complementary determining region (CDR), and it is scattered with the more conservative region of referred to as framework region (FR).In some specific embodiments, the antibody of the present invention is only made up of heavy chain.In some specific embodiments, the antibody of the present invention is single domain antibody.

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

The present invention includes " variant " of antibody, such as, the variable region of heavy chain of antibody of the present invention can comprise at least one aminoacid and add, inserts, lacks and/or replace, such as 10,20,30,40,50, the most such as 1,2,3,4,5,6,7,8,9,10 aminoacid add, insert, lack and/or replace.

The present invention also includes " derivant " of antibody." derivant " of antibody is the antibody through chemical modification, such as by combining with other chemical part such as Polyethylene Glycol, albumin (such as human serum albumin), phosphorylation and glycosylation.Unless otherwise indicated, otherwise term " antibody " includes its fragment, derivant, variant.

On the one hand, the invention provides VEGF antibody, its variant or derivant, wherein said antibody comprises variable region of heavy chain, and described variable region of heavy chain comprises: (i) SEQ ID NO:1, SEQ ID NO:2 and CDR1, CDR2 and the CDR3 shown in SEQ ID NO:3 or its functional activity variant；Or (ii) SEQ ID NO:4, SEQ CDR1, CDR2 and CDR3 shown in ID NO:5 and SEQ ID NO:6 or its functional activity variant；Or (iii) SEQ ID NO: 7, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:8 and SEQ ID NO:9 or its functional activity variant；Or (iv) SEQ ID NO:10, SEQ CDR1, CDR2 and CDR3 shown in ID NO:11 and SEQ ID NO:12 or its functional activity variant.

Described functional activity variant is to have at least 70% with any one aminoacid sequence in SEQ ID NO:1-12, for example, at least 75%, at least 80%, at least 85%, at least 90%, such as 91%, the functional activity variant of 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence iden.

In particular embodiments, the invention provides heavy chain antibody, described antibody is made up of heavy chain, and the variable region of described heavy chain comprises: (i) SEQ ID NO:1, SEQ ID NO:2 and SEQ CDR1, CDR2 and CDR3 shown in ID NO:3 or its functional activity variant；Or (ii) SEQ ID NO: 4, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:5 and SEQ ID NO:6 or its functional activity variant；Or (iii) SEQ ID NO:7, SEQ CDR1, CDR2 and CDR3 shown in ID NO:8 and SEQ ID NO:9 or its functional activity variant；Or (iv) SEQ ID NO: 10, CDR1, CDR2 and the CDR3 shown in SEQ ID NO:11 and SEQ ID NO:12 or its functional activity variant.

In a specific embodiment, the heavy chain of antibody of the present invention also can contain constant region.In another specific embodiment, the heavy chain of antibody of the present invention is possibly together with Fc fragment.

In some specific embodiments, the antibody of the present invention is heavy chain antibody, i.e. be only made up of heavy chain.In some specific embodiments, the antibody of the present invention is single domain antibody.

In a more particular embodiment, the weight chain variabl area sequence of antibody of the present invention such as SEQ ID NO:34, SEQ ID NO:35, SEQ Shown in ID NO:36, SEQ ID NO:37.

It addition, present invention also offers and reference antibody competition binding VEGF antibody, described reference antibody is any one of above-mentioned antibody.

The invention still further relates to the nucleotide sequence of encoding such antibodies；Comprise the carrier of these nucleotide sequences；And host cell, the above-mentioned antibody of described host cell expression, and/or comprise these nucleotide sequences or carrier." host cell " is the cell for express nucleic acid nucleic acid the most of the present invention.Host cell can be prokaryote, such as escherichia coli, or it can be eukaryote, such as unicellular eukaryote (such as, yeast).

In particular embodiments, described nucleotide sequence such as SEQ ID NO:47, SEQ ID NO:48, SEQ Shown in ID NO:49, SEQ ID NO:50, as following example describe in detail.

Present invention also offers the method preparing antibody, step includes: cultivate above-mentioned host cell under conditions of allowing to express described antibody；With antibody purification from gained cultured products, as following example describe in detail.

The invention still further relates to pharmaceutical composition, its antibody comprising the present invention and pharmaceutically acceptable excipient.Described pharmaceutical composition also can comprise one or more therapeutical active compound, the such as known anti vegf agents of described therapeutical active compound or antitumor drug.

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

Pharmaceutical composition can be prepared according to techniques known in the art.Term " excipient " refers broadly to any component outside one or more active therapeutic ingredient.Excipient can be inert substance, inactive material and/or the material without pharmaceutically active.Excipient can be used as multiple purpose, such as, as carrier, solvent, diluent, tablet auxiliaries, and/or improves administration and/or the absorption of active substance.Being formulated as it is known in the art that of pharmacy activity component and various excipient see for example Remington: The Science and Practice of Pharmacy (such as the 19th edition (1995), and any after version).The limiting examples of excipient is: solvent, diluent, buffer agent, preservative, tonicity contributor, chelating agen and stabilizer.

The antibody of the present invention can give with the form of pharmaceutical composition.It is possible not only to preparation becomes the liquid preparations such as injection, lyophilized formulations, spray, it is also possible to preparation becomes the solid preparations such as capsule.Route of administration can be, such as, intravenous injection, oral or topical administration, such as percutaneous, through conjunctiva, and/or through eyes etc..In particular embodiments, route of administration is oral administration.In another specific embodiment, route of administration is through eyes.

On the other hand, the invention still further relates to antibody coupling medicine, it comprises the antibody of the present invention being coupled to other medicament, other medicament described such as chemotherapeutics, growth inhibitor, toxin (the such as enzyme activity toxin of antibacterial, fungus, plant or animal origin, or its fragment) or radiosiotope (i.e. radioconjugates).

Employing antibody coupling medicine carries out the local delivery of other medicament can be by medicament targeted delivery to tumor, and in tumor intracellular accumulation, and general gives the medicament of these non-couplings and may result in normal cell and need the unacceptable levels of cytotoxicity of tumor cell removed.

Antibody coupling medicine generally comprises the connector unit between drug unit and antibody units.In some embodiments, this joint can cut under the conditions of intracellular, thus the cutting of joint causes drug unit to discharge from antibody in intracellular environment.Joint can be the peptidyl linkers that such as can be cut by intracellular peptidase or protease (including but not limited to: lysosome or Inclusion protease).In some embodiments, the length at least two aminoacid of peptidyl linkers or at least 3 aminoacid.In a specific embodiment, can be Val-Cit joint or Phe-Lys joint by the peptidyl linkers of intracellular protein cleavage.

In other embodiments, connector unit can not cut, and medicine passes through, the degraded of such as antibody and discharge.

It addition, the method that the antibody that the invention still further relates to the present invention by giving effective dose regulates the angiogenesis of (preferably suppression) VEGF activity, suppression mammal.

Moreover, it relates to the method that the antibody of the present invention by giving effective dose regulates VEGF activity.The present invention relates to the method that the antibody of the present invention by giving effective dose to patient in need suppresses angiogenesis.

Present invention also offers a kind for the treatment of and VEGF relevant disease or the method for disease, described method includes the antibody giving at least one present invention of effective dose to patient in need.Described disease or disease include tumor or cancer or ophthalmic diseases.Described tumor or cancer include breast carcinoma, the cerebral tumor, renal carcinoma, ovarian cancer, thyroid carcinoma, pulmonary carcinoma, colorectal cancer, carcinoma of endometrium, angiosarcoma, bladder cancer, embryonal tissue's cancer, tumor colli, glioblastoma, gastric cancer, cancer of pancreas, nasopharyngeal carcinoma etc..Described ophthalmic diseases include macular edema that a variety of causes causes (macular edema that various diseases cause after including diabetic macular edema, postcataract or uveitis etc.), age-related macular degeneration, diabetic retinopathy, central retinal vein occlusion, neovascular glaucoma and other relate to the ophthalmic diseases of new vessels.

Described " patient in need " means any mammal, and described mammal is such as but not limited to people, horse, cattle, cat, mice, rabbit, rat, goat etc..Preferably, described mammal is people.

In some treatment use, the Antibody Combination of the multiple present invention gives.

It addition, the antibody that the invention still further relates to the present invention is being prepared for regulating the purposes in the active medicine of VEGF；The antibody of the present invention is used for the purposes suppressed in the medicine of angiogenesis in preparation；The antibody of the present invention is used for treatment and the purposes in the medicine of VEGF relevant disease or disease in preparation.

Present invention also offers medicine box, it comprises the antibody of a) present invention, or described pharmaceutical composition；And b) operation instruction.

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

Embodiment 1. Prepared by antigen

The present embodiment for antigen behaviour VEGF165 (Human vascular endothelial Growth 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. 1993 Dec., 4(12):1317-26; 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. 1995 Jun 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. 1996 Mar 29;271 (13): 7788-95), wherein the nucleotide sequence of people VEGF165 antigen as shown in SEQ ID NO:61；The aminoacid sequence of people's VEGF165 antigen is as shown in SEQ ID NO:62.

According to this aminoacid sequence, to its carry out mammal expression codon optimized after, the DNA after being optimized by the way of complete synthesis, after being cloned into carrier for expression of eukaryon pTT5 (being authorized by invention mechanism NRC), be used for preparing transfection level plasmid.Cultivate 7 days after transfection HEK293E cell, be used for Capto post and the HiTrap of hand assembled by centrifugal collecting cell culture supernatant^TMThe two-step solution purification of Q HP, purification also carries out endotoxin process.Protein concentration detection uses UV_280nmLight absorption value detects, and protein endotoxins level uses the detection of LAL method, and the activity of antigen is measured by HUVEC cell proliferation experiment.There are concentration is 1.25 mg/ml, and volume is 22 ml, and total amount is the hVEGF165 albumen of 27.5mg, and level of endotoxin is 0.537 EU/ml (table 1), and SDS-PAGE testing result is shown in Fig. 1, and albumen is in-80 DEG C of preservations.

Table 1 hVEGF165 purifying protein information

Embodiment 2. Animal immune and immunoreaction measurement

1. animal immune

Selection alpaca (Lama pacos) as laboratory animal, 4 different time points, carry out 6 injecting immunes at omoplate and back respectively.PBS is the diluent of antigen, and immunity volume is 1 ml every time, and amount of antigen and adjuvant information are shown in Table 2.The immunoreagent BSA containing final concentration 1 mg/ml, antigen is immune again after mixing with adjuvant Fresh the most before the injection.

Table 2 alpaca immunizing antigen information

Immunologic process design (table 3) different time in four times, respectively at jugular vein blood collection, adds anticoagulant when gathering blood.Take a blood sample 5 ml first, each 15 ml of its excess-three time.After utilizing Ficoll 1.077 reagent (Sangon, Cat. No.:F760014-100) and anticoagulation to carry out gradient centrifugation, separate peripheral blood lymphocyte and carry out the resuspended counting of cell, adding RNAlater (TIANGEN, Cat. No.:DP408-02), preserve in-20 ° of C.The serum that gradient centrifugation obtains also preserves in-20 ° of C.

Table 3 alpaca immunization time calendar

2. immunoreation test

Use elisa (ELISA) respectively to before immunity, third time immunity and the 4th immunity after blood serum sample carry out antigen specific immune reaction test.With NaHCO₃ (pH 9.6) solution dilution immunogen, is coated microwell plate (Corning, Cat.No.:9018), and 4 DEG C overnight.After washing plate four times in washing trigger with PBS-T solution, use 3% BSA confining liquid, close 2 h for 37 DEG C.After PBS-T washes plate tetra-times, the serum of 37 DEG C of night incubation gradient dilutions.After PBS-T washes plate tetra-times, hatch the goat-anti yamma second antibody (Novus of horseradish peroxidase-labeled Biologicals, Cat.No.:NB7242).Use TMB to develop the color 10 min, add 1M HCl color development stopping.System after reaction terminating uses MK3 (Thermo) microplate reader to detect the light absorption value at 450 nm.Be may determine that by the reaction result of ELISA, animal creates preferable immunoreation after having injected proteantigen, and serum titer is about 1:100k (Fig. 2).

Embodiment 3. Antibody phage libraries builds

3.1. RNA extracts

According to cell number, the TRIzol reagent of corresponding volume is added in the peripheral blood lymphocyte separated, after cell has cracked, according to operating instruction (Invitrogen, the Cat. of TRIzol Plus RNA purification system No.:12183-555) extraction completing total serum IgE separates.By the quality of agarose gel electrophoresis detection total serum IgE, and light absorption method is utilized to measure the concentration of RNA.According to measurement result, obtain 105.6 μ g total serum IgE altogether.RNA form on agarose gel electrophoresis figure is complete (Fig. 3), and quality meets the demand of library construction.

3.2. reverse transcriptional PCR

According to SuperScriptTM III First-Strand Synthesis System uses technical instruction (Invitrogen, Cat. No.:18080-051), uses Oligo (dT) total serum IgE reverse transcription is become cDNA by 20 primers.According to the sequence signature of camel antibodies, select specific forward primer and reverse primer for the amplification (A. of VHH 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 particular sequence of primer is shown in Table 4.By the first round PCR to cDNA, the isolated and purified fragment containing VHH being about 600bp of molecular weight according to PCR primer, take turns PCR amplification by second the most again obtain the fragment of VHH and introduce the Sfi I restriction enzyme site that two recognition sequences are different simultaneously at DNA fragmentation two ends, obtain the VHH fragment (Fig. 4) of 101 μ g gel-purified altogether.

Table 4. primer sequence information and PCR amplification effect

3.3 library construction

The V obtained will be expanded with different batches cell and different primers_HH fragment mixes, and then utilizes restricted enzyme Sfi I to carry out enzyme action.Carry out separating by 2% agarose gel electrophoresis, purification the V after obtaining enzyme action_HH；Meanwhile, utilize restricted enzyme Sfi I that phagemid vector (Fig. 5) is carried out enzyme action, carry out separating by 1.5% agarose gel electrophoresis, purification the carrier after obtaining enzyme action.The V after enzyme action is measured by light absorption method_HAfter the concentration of H and phagemid vector, being respectively 1:3 according to carrier/fragment mol ratio, 1:5,1:10 mix, and carry out the preparation of same volume coupled reaction system, be overnight attached in 16 DEG C after adding T4 ligase (NEB, Cat. No.:M0202L).Linked system carries out phenol/chloroform, chloroform, ethanol precipitation in order, and the product that connects obtaining purification carries out the mensuration of concentration by light absorption method.Product for 3 different linked systems acquisitions all carries out the electricity conversion of equivalent DNA and TG1 electricity transformed competence colibacillus, calculated the library i.e. size of transformation efficiency of 3 linked systems by spread plate and gradient dilution method, random picking positive colony send survey detection library diversity.The system selecting transformation efficiency the highest connects in a large number and converts, and adds up storage capacity.Showing according to plate count result, library storage capacity is about 1.8 × 10⁸(table 5).

Table 5 single domain antibody phage display library storage capacity calculates

Library bacterium colony is carried out Random clones PCR, it is seen that the fragment insertion rate in library is 95.8% (Fig. 6), the positive colony wherein having Insert Fragment is checked order, by the aminoacid sequence in single domain antibody CDR region territory is compared, it is seen that library diversity is good (Fig. 7).Use the 2YT culture medium containing 100 μ g/ml ampicillin and 2% glucose to carry out microorganism collection, 5,000g centrifugal segregation products of cellular metabolism the overnight flat board of coating, and use same medium re-suspended cell, be divided into two parts to carry out library storage.

Embodiment 4. Phage display and screening

4.1. prepared by antigen biotinylation complex

Operating process explanation according to EZ-Link Sulfo-NHS-LC-Biotinylation test kit (Pierce, Cat. No.:21335), carries out biotinylation labelling by hVEGF165 albumen.Degree by HABA experiment detection protein biotinylation labelling.The hVEGF165 of biotinylation labelling and the magnetic bead of 0.5 ml M-280 Streptavidin are mixed (Invitrogen, Cat. No.:112.06D), 4 DEG C of night incubation, then by magnetic frame separation magnetic bead, and fail with PBS solution eluting and the biotinylated protein of magnetic bead combination, be used for preparing antigen magnetic bead coupled complex.Biotinylation reacts and obtains 0.52 after purification Mg hVEGF165 albumen, HABA experiment display biotin coupling level is 6 moles of biotin molecules of every mole of albumen coupling.

4.2. phage library recovery

Take about 100 μ l The phage library liquid storage of (MOI is about 20) is inoculated in 2YT culture medium, 225 rpm, cultivates for 30 DEG C, is cultivating to exponential phase (OD₆₀₀=0.5) addition M13KO7 helper phage (NEB, Cat. No.:N0315S) time, 225 Rpm, 30 DEG C of incubated overnight.Centrifugal collection phage, mixes culture supernatant and PEG/NaCl solution and centrifugation phage, by repeatedly centrifugal and resuspended, is finally suspended in the PBS of 1-2ml by the phage particle that recovery obtains.The titer of the phage library that recovery obtains is calculated, it is thus achieved that 3.15 × 10 by finite gradient dilution method¹³The library of pfu/ml.

4.3. for target protein phage selection

Take ~ 2 × 10¹¹The phage of pfu carries out room temperature hatch as the input of the first round and the antigen magnetic bead coupled complex of 10ul, and gentle mixing 2 hours on gyroscope；By magnetic frame by Beads enrichment, the phage not being combined with magnetic bead is washed away, needs to carry out 7 times to the eluting of the resuspended of magnetic bead and non-specific binding；The most resuspended magnetic bead is added TEA solution, by the bacteriophage elution combined with antigen magnetic bead coupled complex and separate, is rapidly added Tris-HCl buffer and is neutralized；The output of phage after being screened by the finite gradient dilution method calculating first round, the phage simultaneously afforded the first round carries out incubated overnight and amplification, design parameter and process and describes identical with the recovery of library before this.Second takes turns screening will be with ~ 10¹¹Library after the first round output Phage amplification of pfu is as input, and the antigen magnetic bead coupled complex of 1ul is hatched and screened, and specific operation process is identical with first round screening with parameter.

4.4. Phage-ELISA is identified

Picking takes turns the monoclonal plaque on the overnight flat board that screening phage output calculates for second, puts into every hole and contains 96 hole depth orifice plates of 500 μ l 2YT culture medium, 225 rpm, cultivates for 30 DEG C, is cultivating to exponential phase (OD₆₀₀=0.5) addition M13KO7 helper phage (NEB, Cat. No.:N0315S) time, 225 Rpm, 30 DEG C of incubated overnight.Centrifugal collection thalline, take supernatant, add and be coated in advance in hVEGF165 the microwell plate closed, with HRP/ anti-M13 monoclonal antibody (GE Healthcare, Cat. No.:27-9421-01) detect as second antibody, other ELISA operating parameter is identical with immunoreation detection, according to the positive rate of light absorption value assessment output.The positive phage clones of the part of random picking with antigen recognition is carried out V_HThe order-checking of H fragment, by sequence alignment and analysis, infers the clonal diversity by phage display output.The phage display determining the need for carrying out more round by the multiformity of positive rate and sequence screens.The phage clone positive is more than 50%, and meets diversity requirement.Therefore second is selected to take turns output phage clone sdAb gene constructed FASEBA library, for further colony screening.

Table 6 phage washes in a pan sieve and ELISA detection

Embodiment 5. FASEBA Screening

5.1. FASEBA library construction

Extract last phage DNA taking turns phage display output, by PCR amplification coding V_HThe fragment of H is cloned in the FASEBA carrier of patent by connection, and all structure clonal structures are V_HH linker SASA 6 × His (Fig. 8).Connect product and will be transformed into TG1 thalline.

5.2. FASEBA screens

5.2.1. sample preparation and expression assessment

From the FASEBA library built, random picking monoclonal, puts into every hole and contains 96 hole depth orifice plates of 500 μ l 2YT culture medium, is cultivating to OD₆₀₀During=0.6-0.8, add IPTG overnight abduction delivering.Centrifugal collect thalline after remove 100 μ l culture supernatant of clarification, join and be coated in advance in BSA the microwell plate closed, use the mouse-anti His monoclonal antibody of HPR labelling to detect (GenScript, Cat. as second antibody No.: A00186)；Meanwhile, take aliquot of culture supernatant and join and be coated in advance in hVEGF165 albumen the microwell plate closed, use the mouse-anti His monoclonal antibody of HPR labelling to detect (GenScript, Cat. No.:A00186) as second antibody, with OD₄₅₀Light absorption value assesses the expression of different clone.Different batches totals over 5000 monoclonals and is used for expression and the screening of antigen adhesion, by higher for expression and with antigen have 138 positive colonies of high-bond be used for affinity sequence and follow-up screening.

5.2.2. Chip prepares

According to BIAcore T200 equipment operating manual, BSA albumen is fixed on CM5 (GE by standard coupling procedure flow process 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 (0.01 M HEPES [pH 7.4], 0.15 M NaCl, 3 mM EDTA and 0.005% [v/v] surfactant P20) it is equipment running buffer, flow velocity is 10 ml/min.First the injection 0.4 M more than 7 minutes 1-ethyl-3-(dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1M N-hydroxy-succinamide (NHS) (1:1), for activating Sensor Chip CM 5 surface, then within 7 minutes, be diluted in 20 μ g/ml BSA protein solutions of 10 mM sodium acetates (pH4.5) by injection, unconjugated activated sites point is closed by the 1M ethylaminoethanol (pH8.5) injected the most again 7 minutes.Aforesaid operations obtains BSA coupling reaction level at 327 reacton (resonance units, RU)。

5.2.3. The affinity sequence of anti-hVEGF165 single domain antibody

By sdAb-SASA clonal expression supernatant mentioned above, through 96 hole screen plate (Pall, Cat. No.:PN5045) at 4 DEG C, with 4000g centrifugal filtration 5 minutes to remove antibacterial and other granule, with HBS-EP solution dilution detection sdAb antibody and sequentially pass through BSA coupling chip surface.Ordination process includes that following four step: a. uses the single domain antibody of the chip capture SASA coupling of fixing BSA；B. hVEGF165 is injected so that it is have the chip surface of single domain antibody to be combined with capture；C. inject running buffer, and monitor stage 300 s that dissociates；D. the chip surface to coupling BSA injects 10 mM glycine/HCl (pH 2.0), and 30 μ l/min, 30 s live again.Each chip taken turns captures antibody, antigen combines, antigen dissociates, BSA chip surface is required to regeneration.With concentration 200 The purification SASA albumen of nM flows through BSA chip surface lives again as control test chip the inspection of effect.138 clone point 3 batches are ranked up, with clone A10981 as reference, 6 A10981 repeated clonings concordance between different batches is fine, 400s dissociation degree about ~ 20%, wherein 93 clone's dissociation rates slower than A10981 (Fig. 9), picking checks order.Choosing wherein 53 single domain antibodies to test for prokaryotic expression and with cell growth inhibition assay, wherein 15 single domain antibodies are for the most competitive screening.

5.2.4. HVEGFR2 competitiveness is screened

Receptor Competition screening will be used for through the screening of expression level and affinity preferred 15 single domain antibodies of sequence, can blocking antigen hVEGF165 and its receptor hVEGFR binding antibody to obtaining.Detailed process is as follows:

A. the fixing method (same to 5.2.2) of amino coupled is passed through by hVEGFR2 proteopexy at CM5 chip surface；B. injection hVEGF165 albumen, observes and combines feature, at binding curve close to stopping injection time saturated；C. on the chip surface that hVEGF165 combines, inject different single domain antibodies and observation combines feature, inject the medicine Avastin of the anti-vegf listed as comparison simultaneously；If d. the epi-position of antibodies VEGF165 is just VEGF and VEGFR2 combines epi-position, antibody can not be combined with VEGF again, or is competed by the VEGF combining VEGFR2, and binding signal will be significantly less than VEGF itself；If it is different or unrelated that the epi-position of antibodies VEGF165 combines epi-position with VEGF&VEGFR2, the VEGF165 that combination has been combined by antibody with receptor, the binding signal of generation will be apparently higher than VEGF itself.Compare (Figure 10) with comparison according to competitive result, select 2 preferable clones therein to prepare for cell growth inhibition assay for heavy chain antibody.

Embodiment 6 : the preparation of single domain antibody

The prokaryotic expression of single domain antibody, purification, 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

Glycerol, Sigma G5516-500ML

IPTG, Amresco 0487-100G

VB1, Aladdin AR 1099302

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

IPTG mother solution: 1 M, 0.22 μm filter filtration treatment, 1-2 ml subpackage, 20 DEG C frozen (3 months effect duration)；

MgCl₂Mother solution: 1 M, 121 DEG C of moist heat sterilization 30 min, 1-2 ml subpackages, deposit (6 months effect duration) for 4 DEG C；

CaCl₂Mother solution: 1 M, 0.22 μm filter filtration treatment, 1-2 ml subpackage, deposit (6 months effect duration) for 4 DEG C；

VB1 mother solution: 50 mg/ml, 0.22 μm filter filtration treatment, 1-2 ml subpackage, deposit (6 months effect duration) for 4 DEG C；

Glucose mother liquid: 20% (W/V), 0.22 μm filter filtration treatment, deposit (3 months effect duration) for 4 DEG C；

Glycerol mother solution: 50% (V/V), 121 DEG C of moist heat sterilization 30 min, deposit (6 months effect duration) for 4 DEG C；

Casein acid hydrolysate mother solution: 4%, 121 DEG C of moist heat sterilization 30 min, room temperature deposits (3 months effect duration)；

10 Χ M9 saline solution: 6% Na₂HPO₄ (W/V) 、3% KH₂PO₄ (W/V) 、1% NH₄Cl (W/V) 、0.5%；

NaCl (W/V), 121 DEG C of moist heat sterilization 30 min, room temperature deposits (3 months effect duration)；

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

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

6.1.2. Protein purification reagent and equipment

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

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

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

5 mg/ml lysozyme (Lysozyme): generally every gram weight in wet base thalline is with 100 μ l (raw emerging biology, L0005-10)；

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

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

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

ÄKTA purifier 10 (GE Healthcare)；

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

In conjunction with buffer: 20 mM Na₂HPO₄, 0.5 M NaCl, 20 mM imidazoles, pH 7.4；

Wash miscellaneous buffer: 20 mM Na₂HPO₄, 0. 5 M NaCl, 40 mM imidazoles, pH 7.4；

Elution buffer: 20 mM Na₂HPO₄, 0. 5 M NaCl, 300 MM imidazoles, pH 7.4；

1 Χ PBS:137 MM NaCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4.

6.1.3. Endotoxin removal reagent and equipment

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

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

ToxinEraser TM Equilibration Buffer(GenScript M01054)；

Gel method endotoxin detection kit (GenScript, L00451)；

Tachypleus amebocyte lysate (Tachypleus Amebocyte Lysate is called for short TAL sensitivity 0.25 EU/ml, company limited of tachypleus amebocyte lysate trial (demonstration) plant of Xiamen City)；

0.1 M NaOH (prepares without heat source water)；

0.1 M hydrochloric acid (is prepared without heat source water)；

37 DEG C of constant incubators.

6.1.4. Filtration sterilization reagent and equipment

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

6.1.5. Determination of protein concentration and detectable and equipment

Nanodrop 2000 spectrophotometer (Thermo)；

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

MOPS Running Buffer Powder (GenScript, M00138)；

Loading Buffer (5 Χ, non-reduced): 0.25 M Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, 7.8%DTT；

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

6.2. Method and flow process

6.2.1. prepared by bacterial strain

A) convert: the prokaryotic expression plasmid being built into single domain antibody gene is proceeded to bacterial strain TG1 by chemical conversion or electricity, is coated on the flat board of 2YT amicillin resistance, 37 DEG C of constant temperature incubated overnight；

B) picking monoclonal: add final concentration 200 μ g/ml ampicillin and 2% (W/V) glucose in the 2YT culture medium of 10 ml；Fully being burnt on alcohol burner by tweezers, grip 10 μ l sterilizing rifle heads after cooling, from converting 1 monoclonal of picking flat board, put into culture medium, 225 rpm, 37 DEG C, constant temperature culture is overnight.

6.2.2. Switching induction

A) preparation M9 culture medium: to 1X M9 Saline solution adds final concentration 0.2% (W/V) glucose, 1 mM MgCl₂、0.1 mM CaCl₂, 0.4% (W/V) Casein acid hydrolysate, 5 mg/L VB1,200 μ g/ml ampicillin, put the preheating of 37 DEG C of shaking tables；

B) switching: incubated overnight bacterium solution taken out from shaking table, by 1:100 switching system, takes incubated overnight product 4000 rpm and is centrifuged 10 Min, resuspended with fresh 1X M9 saline solution, 4000 Rpm is centrifuged 10 min, the most resuspended after be forwarded in the culture medium of preheating, rearmounted 37 DEG C of shaking tables, 225 rpm cultivate 24 h；

C) induction: add final concentration 1X TB culture medium and the IPTG of final concentration of 1 mM and the ampicillin of 200 μ g/ml in M9 culture medium, 225 rpm, cultivate 48 h (24 h add the most final concentration of 200 μ g/ml ampicillin) for 25 DEG C；

D) sample is received: after induction terminates, by overnight culture subpackage to Centrifuge Cup, 4 DEG C, 11,000 rpm, centrifugal 15 Min, collects thalline.

6.2.3. Protein purification

A) prepared by BugBuster cracking process sample

I. cracking: express precipitation and add the resuspended (lysis buffer: with combining buffer dilution 10X BugBuster of 5 ml lysis buffers according to every gram of weight in wet base thalline Protein Extraction Reagent to 1X, and add final concentration 100 μ g/ml lysozyme, 2 μ g/ml nucleases and 1 mM PMSF), room temperature middling speed oscillation incubation 1 h；

Ii. prepared by protein crude extract: by the sample of cracking in 4 DEG C, and 12, 000 g, centrifugal 30 min, collect supernatant and with 0.22 μm membrane filtration.

B) nickel post affinity purification

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

Ii. combining: mixed with appropriate High Affinity Ni-NTA Resin by protein crude extract, 1 h is hatched in concussion.After hatching end, the mixed liquor of crude extract and post material is added PD-10 void column and collects post material, and collect effluent and treat that next step is analyzed and use；

Iii. wash miscellaneous: that uses at least 50 column volumes washes miscellaneous buffer solution elution foreign protein.(wash miscellaneous process Bradford dye liquor as indicator: 5 μ l wash miscellaneous liquid and add 200 μ l Bradford dye liquors and see whether to become indigo plant, if becoming indigo plant, then proceed foreign protein and are eluted to dye liquor and are basically unchanged color, now can carry out next step.)；

Iv. eluting: with the elution buffer eluting target protein of at least 10 column volumes.(, with Bradford dye liquor as indicator, method is with step iii, it may be judged whether eluting is complete for elution process).

C) desalination/buffer exchange

I. balance: with suitable flow velocity (0.5 ml/min) ddH of 5 times of column volumes in KTA purifier 10 system₂O and PBS balances the HiTrapTM of 5 ml Desalting post；

Ii. loading: the protein liquid after appropriate (0.5 ml) ni-sepharose purification is added HiTrapTM with suitable flow velocity (0.5 ml/min) In Desalting post；

Iii. eluting: (0.5 ml/min) continues to carry out eluting with the PBS of at least 10 times of column volumes under suitable flow velocity, collects the target ultraviolet peak at albumen place.

6.2.4. Endotoxin removal and detection

A) endotoxin removal

I. sample treatment: use 1 M sodium chloride regulation ionic strength to 0.2 ± 0.5 M before purification, use 0.1 M sodium hydroxide or 0.1 M salt acid for adjusting pH value 7.4 ± 0.2；

Ii. activated resin: PD-10 Columns is vertically fixed; remove the lid at prepacked column top; add ToxinEraser TM Endotoxin Removal resin; open flow speed controller; protection liquid is made to drain off under gravity; add the regeneration buffer of 5 ml, regulate flow speed controller, keep flow velocity 0.25 Ml/min (about 10/min) , buffer to be regenerated drains off, and adds 5 ml regeneration buffer, repetitive operation twice, it is ensured that system keeps apyrogeneity (i.e. without endotoxin) to exist；

Iii. balance resin: PD-10 Columns activation is complete, add the level pad of 6 ml, regulate flow speed controller, keep flow velocity 0.5 Ml/min, drain off level pad, then is repeated twice by this operation；

Iv. endotoxin removal: closed by flow speed controller, uses and is added by sample without thermal source rifle head, open controller, and coutroi velocity is not higher than 0.25 Ml/min, after effluent volume reaches 1.5 ml, begins to use and accepts sample without thermal source reception pipe, after sample drains off, add 1.5 Ml-3.0 ml level pad drip washing, collects leacheate.Detection sample concentration and level of endotoxin (elution process with Bradford dye liquor as indicator, it may be judged whether collect completely).

B) gel method measures endotoxin

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

Ii. endotoxin standard is diluted: get out endotoxin standard, with baterial endotoxin test water redissolution rearmounted turbula shaker mixing 15 Min, then it is diluted to debita spissitudo (0.5 EU/ml)；

Iii. detection: take TAL reagent, add 100 μ l baterial endotoxin test water to shake at least 30 s gently and be completely dissolved to reagent, it is careful not to cause bubble, it is separately added into the 100 following samples of μ l: detected sample after positive control (endotoxin standard 0.5 EU/ml), negative control (without endotoxin water), dilution: four concentration testing samples in (1), close the mouth of pipe, shake up gently, it is vertically disposed in 37 DEG C of incubators to hatch 1 hour, then takes out observation；

Iv. result record: taken out gently from constant incubator by test tube, is slowly inverted 180 ° of test tube, and in pipe, content is in reality gel, indeformable, not from tube wall landing be the positive；Other situations are then negative.Positive pipe is the positive, and negative tube is negative, and in same scope, state is identical, and experiment is effectively.

6.2.5. Filtration sterilization

By the filter filtered sample of 0.22 m in the case of sterile working in Biohazard Safety Equipment, and take appropriate amount of sample and remain subsequent detection.

6.2.6. Concentration, purity detecting

A) determination of protein concentration

I. according to the protein amino acid sequence provided, the specific absorbance of this albumen is calculated

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

Iii. protein concentration is calculated according to formula: the ultraviolet absorption value (A of protein concentration=protein solution₂₈₀)/ The specific absorbance of albumen

B) purity of protein detection

Take the standard protein (such as 2 g BSA) of a certain amount of albumen (such as 2 g) and equivalent according to said determination concentration under reduction and non reducing conditions, carry out SDS-PAGE, detection purity and confirmation concentration.

Embodiment 7. Single domain antibody HUVEC Cell inhibitory effect is tested

7.1. prepared by cell

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

7.2. The HUVEC Proliferation Ability test of VEGF induction

Use M199 buffering liquid respectively (Medium-199 1X Earle's Salts (Invitrogen # 11150-059); 10% Fetal Bovine Serum (Gibco, Cat# 10100139), heat inactivated; 10 mM HEPES (Invitrogen, Cat# 15630080);100 units/ml Penicillin 100 µg/ml Streptomycin (Invitrogen, Cat# 10378016) 2 × VEGF and the antibody samples of variable concentrations to be measured) are prepared；By 50 L 2 × VEGF and the antibody samples premixing of each variable concentrations to be measured, microwell plate arranges multiple hole, using cell culture medium as the blank of reaction, using Avastin as positive control；The cell that clear up through trypsin is collected, and re-suspended cell clear with M199 buffer twice, by resuspended for cell to cell density be 1 × 10⁵After individual cell/ml, each hole of microwell plate adds 50 L cell re-suspension liquid；The microwell plate adding cell is placed in cultivation, 37 ° of C, 5% CO₂Cultivate 96 hours；Cultivation uses CellTiter-Glo after completing Luminescent Cell Viability Assay Kit (Promega, Cat No:G7571) detection cell survival rate, detects fluorescence intensity by PHERAStar Plus (BMG Labtech) simultaneously and records its relative light units；Being calculated by below equation and produce suppression ratio, growth inhibition ratio %=100* (1 relative light units/largest production value), wherein associated light unit is the measured value of various kinds sample, relative light units when largest production value is only to add VEGF.Comprehensive maximal percentage inhibition and EC50 two aspect data, choose 2 for building heavy chain antibody and follow-up proliferation inhibition test from the single domain antibody of 53 prokaryotic expressions.

The EC50 value of table 7:HUVEC cell inhibitory effect test is as follows:

Embodiment 8. The expression of heavy chain antibody

2 that Preliminary screening in embodiment 7 is had certain Proliferation Ability function and 2 single domain antibodies screened through Receptor Competition, Fc fragment (SEQ ID NO:60) with human IgG1 constructs as heavy chain antibody together, being cloned into pTT5 carrier expression and purification and endotoxin removal in HEK293E, detailed process is as follows:

8.1. reagent prepares

With embodiment 6.

8.2. method and flow process

8.2.1. cell is cultivated

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

B) cell melted is added the preheating FreeStyle of 10 times of volumes^TM In 293 Expression Medium, gentle inversion mixes, and low-speed centrifugal collects cell and the culture medium with proper amount of fresh preheating is resuspended；

C) resuspended cell is proceeded in culture bottle and at 37 DEG C, 5%CO₂, cultivate under 110 rpm speed conditions.

8.2.2. Transfection

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

B) by appropriate DNA Yu PEI with optimal proportion (such as 1:3) in preheating FreeStyle in right amount^TM 293 Expression Medium fully mix, left at room temperature 10 minutes；

C) above-mentioned PEI-DNA mixture is added in cell culture, softly rotate mixing, continue at 37 DEG C, 5%CO₂, 110 Cultivate under rpm speed conditions；

D), after transfection in 16 ~ 24 hours, final concentration of 0.5% is added (w/v) Tryptone N1；

E) within after transfection the 6th day, it is centrifuged collection culture supernatant and with 0.22 μm membrane filtration with to be purified.

8.2.3. Protein purification

A) Protein A affinity purification

I. balance columns material: with the ddH of 5 times of post material volumes₂O and Binding Buffer balances Protein A post material；

Ii. combine: the expression supernatant after filtration is hatched after mixing with post material and combined 1 h, is loaded in the manual purification column of PD-10 by post material after hatching end；

Iii. wash miscellaneous: with the Binding of at least 30 times of post material volumes Buffer wash away foreign protein (wash miscellaneous process Bradford dye liquor as indicator: 5 μ l wash miscellaneous liquid add 200 μ l Bradford dye liquors see whether become indigo plant, if becoming indigo plant, then proceed to wash miscellaneous process and be basically unchanged color to dye liquor, now can carry out next step)；

Iv. eluting: with the Elution of at least 10 times of post material volumes Buffer eluting target protein also regulates pH value to about 7.0 (, as indicator, method is with steps iii, it may be judged whether eluting is complete for elution process Bradford dye liquor) with Neutralization Buffer.

B) desalination/buffer exchange:

By affinity purification gained albumen HiTrap^TMDesalting post is replaced in PBS in KTA purifier 10 system.The steps such as follow-up endotoxin removal, filtration sterilization, concentration, purity testing are with embodiment 6.

Table 8 heavy chain antibody, single domain antibody clone's corresponding relation and variable region amino acid sequence thereof and nucleotides sequence column number

Embodiment 9. Heavy chain antibody HUVEC Cell inhibitory effect is tested

4 heavy chain antibodies (table 8) are arranged 8 gradient dilution concentration altogether, and the initial concentration of antibody is 20 μ g/ml, 1:4 gradient dilution, and using Avastin (A68467) as comparison, other experiment condition is completely with embodiment 7.By judging the degree of variable concentrations antibody on cell Proliferation Ability, 13 heavy chain antibodies all have suppression function (Figure 11) in various degree, and wherein A80887, A80723 and A69458 are the strongest in the suppression function of cellular level.Specifically, for A69458, it is 10 in antibody concentration^-3During ~ 1 μ g/ml, its inhibition is substantially better than comparison；For A80723, it is 10 in antibody concentration^-3~10^-1During μ g/ml, its inhibition is substantially better than comparison；For A80887, it is 10 in antibody concentration^-2During ~ 1 μ g/ml, its inhibition is substantially better than comparison.

Table 9 heavy chain antibody sample message

Embodiment 10. Zebra fish model evaluates the anti-angiogenesis effect of antibody of the present invention

The molecular weight of testing sample (A80887) is about 75 KDa, concentration is 5.1 mg/ml, and-80 DEG C of subpackages preserve, and 1 × PBS (pH7.4) dilution, is placed in sample during injection and preserves on ice before use.Experimental apparatus used includes with reagent: microinjection instrument (IM300, Narishige)；Anatomic microscope (SMZ645, Nikon company)；Power focus continuous zoom fluorescence microscope (AZ100, Nikon company)；6 orifice plates (Nest Biotech)；MESAB(Sigma)；Bevacizumab (trade name Avastin, Roche).

The breeding of transgenic blood vessel fluorescence zebrafish embryo is carried out in the way of natural paired cross.Copulation prepares 4 ~ 5 pairs of Adult Zebrafishs every time, and averagely every pair can be produced 200 ~ 300 embryos.Embryo is cleared up (removing dead embryo) at after fertilization 6 hours (i.e. 6 hpf) and 24 hpf, and select suitable embryo (Kimmel, 1995) according to the stage of development of embryo.Embryo's (fish culture water water quality: adding 200 mg Instant Ocean in every 1L reverse osmosis water, electrical conductivity is 480 ~ 510 S/cm is hatched with fish culture water under the conditions of 28 DEG C；PH is 6.9 ~ 7.2；Hardness is 53.7 ~ 71.6 mg/L CaCO₃).Because embryo can obtain nutrient substance from the yolk sac of self, so (9dpf) need not feeding in after fertilization 9 days.After having tested, with tricaine methanesulfonic acid, the Brachydanio rerio of each stage of development carried out over-exposure process, thus Brachydanio rerio anesthesia is put to death.All experimental implementation all perform in strict accordance with International Laboratory Animal assessment and accreditation committee (AAALAC) standard.

Using microinjection instrument, (to be equivalent to human vein be administered) in maximum concentration, maximum injection volume injection to transgenic blood vessel fluorescence Brachydanio rerio blood circulation, there is not dead and obvious abnormal phenotype in sample.According to above-mentioned experimental result, 1/10 maximum injection dosage (maximum concentration × maximum injection volume), 1/3 maximum injection dosage and 3 dosage of maximum injection dosage are selected to detect, positive controls (Avastin), solvent control group (PBS) and blank group are set simultaneously, often organize and all process 30 tail Brachydanio rerio.It is being administered after certain time, is often organizing and take 15 tail Brachydanio rerio at random and take pictures under fluorescence microscope, vascular plexus under intestinal (SIVs) area is carried out quantitative analysis.Comparing employing T-inspection between two groups, multiple-group analysis uses one factor analysis of variance and Dunnett's T-inspection to carry out statistical analysis, and p < 0.05 shows have significant difference, and the drug effect computing formula of anti-angiogenesis is as follows:

In whole experimentation, all there is not Brachydanio rerio death or abnormal phenotype in all experimental grouies；Blank group and solvent control group no difference of science of statistics (p > 0.05)；Positive controls (Avastin) compares with solvent control group and has significant difference (p < 0.05)。

Groping experiment by dosage, A80887 injection maximum concentration is 5.1 mg/ml, has no that dead and obvious abnormal phenotype occurs in Brachydanio rerio.Therefore, select 40.8 ng (0.544 pmol), 136 ng (1.81 pmol), 408 Ng (5.44 pmol) is for being administered accumulated dose；.

Test result indicate that, A80887 dosage is 20.4 ng (0.272 Pmol), 68 ng (0.907pmol) and 204 ng (2.72 pmol) time, new vessels formed suppression ratio be followed successively by 10.9% (p>0.05), 18.5% (p<0.05) and 23.2% (p<0.001).Avastin dosage is that the vascularization suppression ratio of 400 ng (2.68 pmol) and 1 μ g (6.7 pmol) is respectively 6.9% (p>0.05) and 19.5% (p<0.01), and both compare, p<0.05.When equimolar quality (the most about 2.7 pmol), the suppression new vessels formation efficiency of A80887 is 23.2%, hence it is evident that be better than Avastin 6.9% (both compare, p < 0.001).And Avastin 1 μ g (6.7 pmol) and A80887 20.4 ng (0.272 pmol), 68 ng (0.907pmol) and 204 Ng (2.72 pmol) compares, and the suppression ratio of new vessels is had no significant difference.Initial data see table.

Table 10. A80887 inhibitory action to blood vessel

Brachydanio rerio vascularization model carries out pharmacodynamic evaluation and the checking acquisition of medicine novel targets is widely recognized as.At present, branched entrance preclinical laboratory (Pre-clinical Trial) or the cancer therapy drug (including obtaining the medicine of FDA approval listing) in clinical trial (Clinical Trial) stage, such as Vatalanib (Novartis) (Chan, 2002), Thalidomide (Celgene) (Yabu, 2005), Compound 6 (TargeGen) (Murphy, 2010), Rosuvastatin (Wang 2010), Solenopsin (Eli Lilly) (Arbiser, 2007) etc., the most successfully utilize Brachydanio rerio vascularization inhibition to carry out effective checking.Being generally selected vascular plexus or body intersegmental blood vessel under transgenic blood vessel fluorescence Brachydanio rerio intestinal and evaluate the impact that new vessels is formed by compound, under the quantitative intestinal of this project alternatives, the method for vascular plexus area evaluates the impact that new vessels is formed by sample A80887.

Avastin is the human monoclonal IgG1 antibody of a kind of restructuring, by combining VEGF (VEGF) and stoping it to combine with the receptor (Flt-1 and KDR) of endothelial cell surface.This research selects Avastin (molecular weight 149 kDa) as the positive control drug of macromole class anti-angiogenesis.Experimental result display Avastin has preferable new vessels inhibition, and therefore, it is reliable for evaluating this model with Avastin as positive control drug.

In sum, 1. A80887 Yu Avastin all has obvious inhibitory action to the formation of Brachydanio rerio new vessels.2. in equimolar quality (about 2.7 Pmol), time, the effect that A80887 suppression new vessels is formed is substantially better than Avastin.3., when the dosage of A80887 is respectively 20.4 ng, 40.8ng, 68 ng, 136 ng, 204 ng and 408 ng, respectively may be about 81.6,163.2,272,544,816 and 1632 g/kg body weight relative to human administration's reference dose.

Although some feature of the present invention is explained the most in this article and describes, but those skilled in the art are it is appreciated that many is revised, substitutes, changes and is equal to.It is to be understood, therefore, that appended claims is intended to fall into these type of modifications and changes all within true spirit of the present invention.

Claims (10)

1. a VEGF antibody, wherein said antibody comprises variable region of heavy chain, and described variable region of heavy chain comprises:

(i) SEQ ID NO:1, SEQ ID NO:2 and CDR1, CDR2 and the CDR3 shown in SEQ ID NO:3 or its functional activity variant；Or

(ii) SEQ ID NO: 4、SEQ ID CDR1, CDR2 and CDR3 shown in NO:5 and SEQ ID NO:6 or its functional activity variant；Or

(iii) SEQ ID NO: 7、SEQ ID CDR1, CDR2 and CDR3 shown in NO:8 and SEQ ID NO:9 or its functional activity variant；Or

(iv) SEQ ID NO: 10、SEQ ID CDR1, CDR2 and CDR3 shown in NO:11 and SEQ ID NO:12 or its functional activity variant.

2. the VEGF antibody of claim 1, wherein said heavy chain is possibly together with Fc fragment.

3. the VEGF antibody any one of claim 1-2, wherein said antibody is made up of heavy chain.

4. the VEGF antibody of claim 1, wherein said antibody is single domain antibody.

5. the VEGF antibody any one of claim 1-2, the weight chain variabl area sequence of wherein said antibody is as shown in SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37.

6. the nucleotide sequence separated, its coding VEGF antibody any one of claim 1-5.

7. the nucleotide sequence of claim 6, it is such as SEQ ID NO: 47、SEQ ID NO: 48、SEQ ID NO: 49, shown in SEQ ID NO:50.

8. a carrier, it comprises the nucleotide sequence of claim 6 or 7.

9. a host cell, it is expressed the VEGF antibody any one of claim 1-5, and/or comprises nucleotide sequence or the carrier of claim 8 of claim 6 or 7.

10. a pharmaceutical composition, described compositions comprises the VEGF antibody any one of claim 1-5 and pharmaceutically acceptable excipient.