CN102485753A - Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity - Google Patents
Human derived heavy chain variable region possessing human vascular endothelial growth factor binding activity Download PDFInfo
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Abstract
The invention provides a human derived heavy chain variable region possessing human vascular endothelial growth factor(VEGF) binding activity. Concretely, the invention provides an antibody single heavy chain variable region which is from fully human derived monoclonal antibody and possesses the human vascular endothelial growth factor binding activity. The single heavy chain variable region can be taken as an antibody fragment, the human VEGF binding activity of complete immunoglobulin can be reserved, the proliferation of human vascular endothelial cells (HUVEC) can be inhibited in dose dependent mode. The invention also provides a method for preparing the antibody fragment and its application.
Description
Technical field
The present invention relates to biological field, relate more specifically to a kind of have VEGF-2 (humanVascular Endothelial Growth Factor, hVEGF) combination active people source variable region of heavy chain and encoding sequence and application.
Background technology
(vascular endothelial growth factor VEGF), claims that also (vascular permeability factor VPF), is by discoveries such as FerraraN in 1989 to the blood vessel permeability factor to VEGF.VEGF is that relative molecular mass is (34～45) * 10
3Homodimer gp, be one of most important vascularization promotor of finding at present, the infiltration and the transfer of tumour had material impact, in the blood vessel generating process of tumour, bring into play important role.
The encoding sox of VEGF is positioned at chromosomal 6q21.3, and total length 14kb contains 8 exons and 7 introns, can form at least 5 kinds of isomer proteins such as VEGF121, VEGF165, VEGF189, VEGF206 and VEGF145.These a few type VEGF biological activitys are close, and VEGF121 and VEGF165 are main isomeric forms in the human cell, and most cell is main with secretion of VEGF 165, is the principal mode that VEGF plays a role.In many isomer, VEGF121, VEGF145 and VEGF165 be ability induction of vascular endothelial cell proliferation and new vessel formation all, but the highest with the activity of VEGF165.
Can effectively suppress tumour and change to the blood vessel phase from the no blood vessel phase through suppressing VEGF and acceptor thereof, thereby suppress its growth, and the probability of reduction cancer metastasis.The listing of the therapeutic antibodies of two kinds of target VEGF of the present approved of FDA, i.e. Avastin and LUCENTIS, the therapeutic antibodies that other has several targets VEGF are just in the clinical experiment of different steps.Avastin has been proved to be for tumours such as the transitivity rectum cancer, nonsmall-cell lung cancer, mammary cancer since listing in 2004 all has significant inhibition effect.
At present nearly all is humanization or chimeric IgG1 antibody by FDA approval listing or the therapeutic antibodies that carrying out clinical experiment, has the shortcoming of big and property two aspects, mouse source of molecular weight.Very difficult penetrate tissue of macromole or combination with hidden are the inner antigenic determinant of antigen.Resist intravital mouse derived components can cause HAMA reaction (Human anti-mouse antibody), when clinical application, cause antibody by degraded and removing rapidly.
Overcome these insufficient main method at present and be make human antibody and antagonist carry out the miniaturized transformation (as, Fab fragment, single-chain antibody, single domain antibody, phage library screen and CDR analog antibody etc.).Wherein, The single domain antibody that is made up of single heavy chain or variable region of heavy chain is proved to be the ability with outstanding entering antigen molecule surface void (Cavity); Have also simultaneously that volume is little, solubility is high, Heat stability is good, advantage such as the refolding ability is strong and the in-vivo tissue penetrance is good, antigen recognition ability and avidity that can higher level reservation complete antibody.
Yet this area still lacks the antibody of the gratifying anti-VEGF of effect up to now, and especially molecular weight is little and do not have the antibody of mouse source property.
Therefore, to press for the new molecular weight of exploitation little and do not have the antibody of the anti-VEGF of mouse source property in this area.
Summary of the invention
It is little and do not have the antibody of the anti-VEGF of mouse source property that one object of the present invention just provides a kind of molecular weight.
Another object of the present invention just provides the method for making and the purposes of said antibody.
In first aspect of the present invention, a kind of monoclonal antibody V is provided
HChain, its complementary determining region CDR have the aminoacid sequence of the CDR of the group of being selected from down:
CDR1 shown in the SEQ ID NO:3,
CDR2 shown in the SEQ ID NO:4 and
CDR3 shown in the SEQ ID NO:5.
In another preference, it has the aminoacid sequence shown in the SEQ ID NO:2.
In another preference, described V
HThe FR district of chain is people's FR district.
In second aspect of the present invention, a kind of recombinant protein that VEGF-2 combines active strand that has is provided, wherein, described recombinant protein has the monoclonal antibody V described in the first aspect present invention
HChain and optional assistance expression and/or the sequence label of purifying.
In another preference, described sequence label comprises the 6His label.
In another preference, described recombinant protein is selected from down group:
(a) has the aminoacid sequence shown in the SEQ ID NO:2;
(b) has the aminoacid sequence shown in the SEQ ID NO:11;
(c) replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID NO:2 or 11 through one or more (like 1-20) amino-acid residue formed, and have VEGF-2 combine active function by (a) or (b) polypeptides derived.
In another preference, described derived sequence is the 4th and is the SEQ ID NO:2 sequence of Ile.
In the third aspect of the invention, a kind of polynucleotide are provided, its coding is selected from down the protein of group:
Monoclonal antibody V described in the first aspect present invention
HChain; Or
Recombinant protein described in the second aspect present invention.
In another preference, described polynucleotide have the dna sequence dna of the group of being selected from down: SEQ ID NO:1 or 10.
In fourth aspect present invention, a kind of carrier is provided, it contains the polynucleotide described in the third aspect present invention.
In fifth aspect present invention, a kind of genetically engineered host cell is provided, it contains the carrier described in the fourth aspect present invention or genome conformity has the polynucleotide described in the second aspect present invention.
Aspect the of the present invention the 6th, a kind of preparation method who prepares recombinant polypeptide is provided, this method comprises:
(a) be fit to cultivate the host cell described in the fifth aspect present invention under the condition of expressing;
(b) from culture, isolate recombinant polypeptide, described recombinant polypeptide is the monoclonal antibody V described in the first aspect present invention
HRecombinant protein described in chain or the second aspect present invention.
Aspect the of the present invention the 7th, a kind of pharmaceutical composition is provided, it contains the monoclonal antibody V described in (i) first aspect present invention
HRecombinant protein described in chain or the second aspect present invention and (ii) pharmaceutically acceptable carrier.
In eight aspect of the present invention, the monoclonal antibody V described in the first aspect present invention is provided
HThe purposes of the recombinant protein described in chain or the second aspect present invention, they are used to prepare compsn, and described compsn is used for (a) and suppresses vascular endothelial cell growth; (b) combine with VEGF-2; Or (c) be used to treat tumour.
In another preference, described compsn is a pharmaceutical composition.
In another preference, described pharmaceutical composition is an injection type.
In another preference; Described pharmaceutical composition is used to prepare the medicine of treating tumour, and described tumour is selected from down group: cancer of the stomach, liver cancer, white blood disease, tumor of kidney, lung cancer, carcinoma of small intestine, osteocarcinoma, prostate cancer, colorectal cancer, mammary cancer, large bowel cancer, prostate cancer, cervical cancer, adrenal tumor or tumor of bladder.
In nineth aspect present invention; A kind of method that suppresses vascular endothelial cell growth or the treatment disease relevant with vascular endothelial cell growth is provided, has comprised step: used the monoclonal antibody V described in the first aspect present invention for the needs object that inhibition maybe need be treated
HPharmaceutical composition described in recombinant protein described in chain or the second aspect present invention or the seventh aspect present invention.
In another preference, the described disease relevant with vascular endothelial cell growth comprises tumour.
In another preference, described to liking Mammals (comprising the people).
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and specifically described each technical characterictic can mutual combination in (like embodiment) hereinafter, thus constitute new or optimized technical scheme.As space is limited, this tired no longer one by one stating.
Description of drawings
Fig. 1 has shown the segmental agarose electrophoresis of VH of being cloned with table 1 primer in instance of the present invention.
(A) obtain the specific PCR amplified band of VH in the position of 350bp.
(B) positive plasmid is identified through Xba I-Xho I and BamH I-Hind III double digestion.1: clone I utilizes Xba I-Xho I enzyme to cut; 2: clone II utilizes Xba I-Xho I enzyme to cut;
(C) 1: clone I utilizes BamH I-Hind III enzyme to cut; 2: clone II utilizes BamH I-Hind III enzyme to cut.
Fig. 2 has shown that antibody VH fragment sequence is analyzed and functional zone are divided.
Fig. 3 has shown rhVVH abduction delivering situation in the BL21 intestinal bacteria in instance of the present invention, wherein utilizes SDS-PAGE to analyze the abduction delivering condition of rhVVH in the BL21 intestinal bacteria.
Each swimming lane is following: 1-2: the cracking supernatant of the BL21 bacterium of plasmid-free and deposition be as negative control, 37 ℃ of inducing culture 3 hours; 3-8: 37 ℃ of abduction deliverings of reorganization bacterium; 9-14: the reorganization bacterium is at 28 ℃ of abduction deliverings; 15-20: the reorganization bacterium is at 32 ℃ of abduction deliverings; 3,4,9,10,15,16 is abduction delivering 1 hour; 5,6,11,12,17,18 is abduction delivering 2 hours; 7,8,13,14,19,20 is abduction delivering 3 hours; 3,5,7,9,11,13,15,17,19 are the cracking supernatant; 4,6,8,10,12,14,16,18,20 are the infusible precipitate after the cracking.M is molecular weight standard (kDa).
Fig. 4 has shown that rhVVH expresses and the evaluation of purification result.Fig. 4 A and 4B are SDS-PAGE result, and Fig. 4 C is anti-6 * His label Western-blotting detected result.
Each swimming lane is following: (A) 1:rhVVH expresses the cracking supernatant of strain; 2:rhVVH expresses the cracking postprecipitation of strain.(B) sample of sex change purifying collection, 1: stream is worn liquid; 2: washings; The 3:rhVVH elutriant.(C) 1:rhVVH expresses the cracking supernatant of strain; 2:rhVVH expresses the cracking postprecipitation of strain; 3: stream is worn liquid; 4: washings; The 5:rhVVH elutriant.
Fig. 5 has shown that rhVVH combines VEGF activity experiment concentration curve.Fig. 5 A is rhVVH, HVmAb, the ELISA result of VEGF-pAb and negative control.Band 1 to 5 adds according to table 2 concentration.Fig. 5 B is the statistics that ELISA detects OD490.Every hole encapsulates the VEGF of 50ng; The concentration of rhVVH is (serial dilution in 1: 34 times) from 5.926 μ g/mL to 480 μ g/mL; The positive contrast of the anti-people VEGF of rabbit polyclonal antibody, working concentration are that 0.309 μ g/mL is to 25 μ g/mL (serial dilution in 1: 34 times); HVmAb contrasts as another, and working concentration is (serial dilution in 1: 34 times) from 6.173 μ g/mL to 500 μ g/mL; 6 * His label is as negative control, and concentration range is that 6.173 μ g/mL are to 500 μ g/mL (serial dilution in 1: 34 times); The OPD working fluid is a blank.
Fig. 6 has shown that rhVVH is to the HUVEC cell inhibiting activity.Among the figure, (A) the anti-people VEGF of 100ng/mL rabbit polyclonal antibody adds 3ng/mL VEGF.(B) the anti-people VEGF of 100ng/mL rabbit polyclonal antibody.(C) 75ng/mL 6 * His adds 3ng/mL VEGF.(D)75ng/mL?6×His。(E) 0.1% (V/V) PBS adds 3ng/mL VEGF.(F) 0.1% (V/V) PBS; (G) 75ng/mL rhVVH adds 3ng/mL VEGF.(H) 75ng/mL rhVVH; (I) 15ng/mL rhVVH adds 3ng/mLVEGF; (J) 15ng/mL rhVVH; (K) 3ng/mL rhVVH adds 3ng/mL VEGF; (L) 3ng/mL rhVVH.
Fig. 7 has shown that rhVVH suppresses the statistics of HUVEC cell growth.In the HUVEC cell, add hVVH, concentration is from 62.5 to 2000ng/mL, the negative contrast of PBS.
Embodiment
The inventor is through extensive and deep research; Successfully obtained first to the VEGF high specific and molecular weight is very little, the monoclonal antibody fragment in total man source, this monoclonal antibody fragment is the single heavy chain fragment from total man source VEGF monoclonal igm antibody.The people VEGF that this heavy chain of antibody fragment has kept complete Tegeline combines activity, can effectively combine and suppress the growth of vascular endothelial cell with VEGF.Accomplished the present invention on this basis.
Particularly; In order to overcome the big shortcoming of total man VEGF monoclonal igm antibody molecule amount; The inventor has obtained the variable region of heavy chain of this antibody earlier through multiple PCR method, then with this sequence clone to the pET28a expression vector, in intestinal bacteria, carried out abduction delivering.Then, obtained to have 16kDa recombinant antibody fragment--the rhVVH of BA through purifying.
External combination experiment shows that the people VEGF that rhVVH remains with complete Tegeline combines active.Human umbilical vein endothelial cells (HUVEC) proliferation inhibition test shows: the propagation of the inhibition HUVEC that rhVVH can dose-dependently.The above results has disclosed the combined function that this antibody single variable region of heavy chain remains with the hVEGF of complete antibody, in fields such as preventing and/or treating tumour good prospects for application is arranged.
The invention provides a kind of single heavy chain of anti-VEGF monoclonal antibody (or its fragment) of reorganization.
In the present invention; Term " the single heavy chain of anti-VEGF monoclonal antibody ", " albumen of the present invention ", " polypeptide of the present invention ", " heavy chain of antibody of the present invention " or " rhVVH albumen " interchangeable use all refer to have the albumen or the polypeptide of aminoacid sequence (SEQ ID NO:2) of the anti-VEGF monoclonal antibody heavy chain in people source.They can contain or not contain initial methionine.
The present invention also provides the aminoacid sequence of anti-VEGF monoclonal antibody heavy chain fragment, and has segmental other protein of these heavy chain of antibody or fusion expressed product.Particularly; The present invention includes and have the hypervariable region of containing (complementary determining region; Any protein of heavy chain CDR) or protein conjugate and fusion expressed product (being immune conjugate and fusion expressed product); As long as identical or at least 90% homology of hypervariable region of this hypervariable region and heavy chain of the present invention, preferably at least 95% homology.
Generally; The antigen binding characteristic of antibody can be described by 3 specific zones that are positioned at heavy chain and variable region of light chain, is called hypermutation zone (CDR), should intersegmentally be divided into 4 frame areas (FR); The aminoacid sequence of 4 FR is relatively conservative, does not participate in association reaction directly.These CDR form ring texturees, the βZhe Die that the FR through therebetween forms on space structure each other near, the CDR on CDR on the heavy chain and the corresponding light chain has constituted the antigen binding site of antibody.Can confirm that which amino acid has constituted FR or CDR zone through the aminoacid sequence of antibody more of the same type.
Unexpected is that single heavy chain provided by the present invention (or its fragment) just has the binding ability very strong with VEGF.
The hypervariable region of the V chain of here identifying or complementary determining region (complementarity determiningregion, CDR) interesting especially, because at least partly relate to conjugated antigen in them.Therefore, the present invention includes those the monoclonal antibody light chains and the molecule of weight chain variable chain, as long as its CDR has the homology of (preferably more than 95%, best more than 98%) more than 90% with the CDR that identifies here with band CDR.
The present invention not only comprises complete monoclonal antibody heavy chain, also comprises the fusion rotein that fragment with immunocompetent heavy chain of antibody or heavy chain of antibody and other sequences form.
Therefore, the present invention also comprises fragment, verivate and the analogue of said VEGF heavy chain of antibody.As used herein, term " fragment ", " verivate " are meant biological function or the active polypeptide that keeps said VEGF heavy chain of antibody of the present invention identical basically with " analogue ".Polypeptide fragment of the present invention, verivate or analogue can be that (i) has one or more conservative or substituted polypeptide of non-conservation amino-acid residue (preferred conservative amino acid residue); And so substituted amino-acid residue can be also can not encoded by genetic code; Or (ii) in one or more amino-acid residues, has a polypeptide of substituted radical; Or (iii) mature polypeptide and another compound (such as the compound that prolongs the polypeptide transformation period; Polyoxyethylene glycol for example) merges formed polypeptide; Or (iv) additional aminoacid sequence is fused to this peptide sequence and the polypeptide that forms (like leader sequence or secretion sequence or be used for the sequence or the proteinogen sequence of this polypeptide of purifying, or the fusion rotein that forms with the 6His label).According to the instruction of this paper, these fragments, verivate and analogue belong to the known scope of those skilled in the art.
In the present invention, term " VEGF heavy chain of antibody " refers to have people VEGF and combines active SEQ ID NO:2 polypeptide of sequence.This term also comprises having and variant form VEGF heavy chain of antibody identical function, SEQ ID NO:2 sequence.These variant forms comprise (but being not limited to): one or more (it is individual to be generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of VEGF heavy chain of antibody.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of the coding DNA hybridization of VEGF heavy chain of antibody and the polypeptide or the albumen that utilize the antiserum(antisera) of VEGF antibody heavy chain to obtain.The present invention also provides other polypeptide, as comprises people VEGF heavy chain of antibody or its segmental fusion rotein (fusion rotein shown in SEQID NO:11).Except the polypeptide of total length almost, the present invention has also comprised the fragment of VEGF heavy chain of antibody.Usually, this fragment have the VEGF heavy chain of antibody at least about 50 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
In the present invention; The conservative property of the VEGF heavy chain of antibody " variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID NO:2, has 10 at the most, preferably at the most 8; More preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to Table A and produce.
Table A
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Ash | Lys |
| Ash(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | ?Asn | Asn |
| 6lu(E) | ?Asp | Asp |
| Gly(G) | ?Pro；Ala | Ala |
| His(H) | ?Asn；Gln；Lys；Arg | Arg |
| Ile(I) | ?Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | ?Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | ?Arg；Gln；Asn | Arg |
| Met(M) | ?Leu；Phe；Ile | Leu |
| Phe(F) | ?Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | ?Ala | Ala |
| Ser(S) | ?Thr | Thr |
| Thr(T) | ?Ser | Ser |
| Trp(W) | ?Tyr；Phe | Tyr |
| Tyr(Y) | ?Trp；Phe；Thr；Ser | Phe |
| Val(V) | ?Ile；Leu；Met；Phe；Ala | Leu |
The present invention also provides the polynucleotide molecule of coding said monoclonal antibody heavy chain or its fragment or its fusion rotein.Polynucleotide of the present invention can be dna form or rna form.Dna form comprises the DNA of cDNA, genomic dna or synthetic.DNA can be strand or double-stranded.DNA can be coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can be identical with the coding region sequence shown in the SEQ ID NO:1 or the varient of degeneracy.As used herein, " varient of degeneracy " is meant that in the present invention coding has the protein of SEQ ID NO:2, but with the differentiated nucleotide sequence of coding region sequence shown in the SEQ ID NO:1.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 comprise: the encoding sequence of an encoding mature polypeptide; The encoding sequence of mature polypeptide and various additional code sequence; Encoding sequence of mature polypeptide (with optional additional code sequence) and non-coding sequence.
Term " polynucleotide of coded polypeptide " can be the polynucleotide that comprise this polypeptide of encoding, and also can be the polynucleotide that also comprise additional code and/or non-coding sequence.
The invention still further relates to and above-mentioned sequence hybridization and two sequences between have at least 50%, preferably at least 70%, the polynucleotide of at least 80% homogeny more preferably.The present invention be more particularly directed under stringent condition and the interfertile polynucleotide of polynucleotide according to the invention.In the present invention, " stringent condition " is meant: (1) than hybridization under LIS and the comparatively high temps and wash-out, like 0.2 * SSC, and 0.1%SDS, 60 ℃; Or (2) hybridization the time is added with denaturing agent, like 50% (v/v) methane amide, 0.1% calf serum/0.1% Ficoll, 42 ℃ etc.; Or (3) only at the homogeny between the two sequences at least more than 90%, be more preferably 95% and just hybridize when above.And the polypeptide of interfertile polynucleotide encoding has identical biological function and activity with the mature polypeptide shown in the SEQ ID NO:2.
The Nucleotide full length sequence of monoclonal antibody heavy chain of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.A kind of feasible method is to synthesize relevant sequence, especially fragment length more in short-term with artificial synthetic method.Usually, through first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.In addition, also can the encoding sequence of heavy chain be merged with expression label (like 6His), form fusion rotein.
In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
At present, can be fully obtain the dna sequence dna of code book invention albumen (or its fragment, or derivatives thereof) through chemosynthesis.Can this dna sequence dna be introduced in various existing dna moleculars as known in the art (or like carrier) and the cell then.In addition, also can will suddenly change and introduce in the protein sequence of the present invention through chemosynthesis.
The invention still further relates to the carrier that comprises above-mentioned suitable dna sequence dna and suitable promotor or control sequence.These carriers can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS7,293 cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
The present invention also provides a kind of compsn.In preference, described compsn is a pharmaceutical composition, and it contains above-mentioned monoclonal antibody heavy chain or its active fragments or its fusion rotein, and pharmaceutically acceptable carrier.Usually, can these materials are formulated in nontoxic, inert and the pharmaceutically acceptable aqueous carrier medium, wherein pH is about 5-8 usually, and preferably pH is about 6-8, although the pH value can be with being changed to some extent by preparation Substance Properties and illness to be treated.The pharmaceutical composition for preparing can carry out administration through conventional route, comprising (but being not limited to): in the knurl, intraperitoneal, intravenously or topical.
Pharmaceutical composition of the present invention can directly be used to suppress the growth of vascular endothelial cell, thereby can be used for prevention and treatment tumour.In addition, also can use the other treatment agent simultaneously.
Pharmaceutical composition of the present invention contains above-mentioned monoclonal antibody (or its conjugate) and the pharmaceutically acceptable carrier or the vehicle of the present invention of safe and effective amount (like 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%).This type carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as injection, solution should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, polypeptide of the present invention also can use with the other treatment agent.
When making pharmaceutical composition; Be that immune conjugate with safe and effective amount is applied to Mammals; Wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight; And in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
Major advantage of the present invention is:
Adopt the rhVVH that the present invention obtained; The combination that not only has very high hVEGF is active; And because its molecular weight has only 16kD; Can avoid the big defective of complete antibody molecular weight, penetrate tissue gets into antigen molecule surface void (Cavity) more easily, has also simultaneously that volume is little, solubility is high, Heat stability is good, the refolding ability is strong and be easy to advantage such as a large amount of acquisitions.
Below in conjunction with specific embodiment, further set forth the present invention.Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989), or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber be weight percentage and parts by weight.
Embodiment
Material
The pMD19-T carrier is available from TaKaRa company; The pET28a expression vector is available from Novagen company; DH5 α intestinal bacteria and BL21 (DE3) intestinal bacteria are conventional commercially available bacterial classification; (Humanumbilical vein endothelial cells, HUVEC) the CRL-1730 cell strain is available from ATCC for Human umbilical vein endothelial cells.
Restriction enzyme, LA Taq polysaccharase and RNA LA PCR
TMKit (AMV) Ver.1.1 reverse transcription test kit is available from Takara company; DMEM (High glucose) substratum, 0.25% trypsinase are available from Invitrogen company; RPMI 1640 substratum, foetal calf serum are available from PAA company; Gel reclaims test kit, the big extraction reagent kit of plasmid available from Qiagen company; The T4DNA ligase enzyme is available from NEB company; His affinitive layer purification test kit is available from Novagen company; The anti-mouse SA of mouse-anti His antibody and HRP mark is available from Shanghai Xinbainuo Biology Science Co., Ltd; People VEGF standard substance are available from Sigma company; The how anti-rabbit anteserum of VEGF (NB100-698) is available from Novus company; Mouse-anti people λ chain antibody is available from Santa Cruz company; Costar 96 hole enzyme plates are available from Corning company; (6 * His) (BS-0287P) are available from Beijing Bo Aosen Bioisystech Co., Ltd for the polyhistidyl label protein; CCK-8 viable count test kit is available from Japanese colleague's chemistry institute; The PCR primer is synthetic to be accomplished by Shanghai Invitrogen company with dna sequencing.
The clone of embodiment 1 variable region of heavy chain
The total RNA of hybridoma V75 (CCTCC NO:C200623) extracts and reverse transcription is carried out according to the test kit specification sheets.(Heavy chain variable region, VH) the used PCR primer of amplification is seen table 1 to carry out the human immunoglobulin heavy chain variable region.The PCR reaction conditions is: 94 ℃ of preparatory sex change 3min; 94 ℃ of sex change 30s, 69 ℃ of annealing 30s, 72 ℃ are extended 30s, circulate 30 times; Last 72 ℃ are extended 7min.The PCR product of expection size through 1.5% agarose gel electrophoresis after, recovery, be cloned in the pMD19-T carrier.
The employed universal primer of table 1 amplification VH
Attempt confirming that primer VH5-1 and primer VH3-3 pairing can efficiently amplify the VH fragment (Figure 1A) of about 350bp through the primer pairing.This fragment is inserted into the pMD19-T carrier, obtains among the carrier pMD19-T/VH, enzyme is cut and is identified correct (Figure 1B).Show that through order-checking the fragment of being cloned belongs to human antibody heavy chain's gene family.
Further the VH sequence that obtains is carried out the function fragment analysis through common tools such as NCBI IgBLAST, the functional area that has obtained in the VH district is divided (see figure 2).This fragment belongs to people VH1 family.Structure to FR1, FR2 and FR3 is analyzed, and the result shows that these FR districts are consistent with human antibody heavy chain's FR district, and this shows that this antibody belongs to people (source) antibody.
The segmental nucleotide sequence of VH is shown in SEQ ID NO:1, and its aminoacid sequence is shown in SEQ ID NO:2, and wherein each FR and CDR are following:
| Position among the SEQ ID NO:2 | The sequence of CDR | |
| FR1 | 1-30 | See Fig. 2 |
| CDR1 | 31-35 | GYYMH(SEQ?ID?NO：3) |
| FR2 | 36-49 | See Fig. 2 |
| CDR2 | 50-66 | WINPNSGGTNYAQKFQG(SEQ?ID?NO：4) |
| FR3 | 67-98 | See Fig. 2 |
| CDR3 | 99-105 | DHGPFDY(SEQ?ID?NO：5) |
| FR4 | 106-116 | See Fig. 1 |
The abduction delivering and the purifying of embodiment 2 recombinant proteins
With amplified production among the embodiment 1 or carrier pMD19-T/VH is template, carries out pcr amplification with VH-forward primer shown in the table 1 and VH-reverse primer (having Nde I and Hind III restriction enzyme site respectively).With amplified production (VH fragment) with Hind III and Nde I double digestion; Be connected with the pET28a carrier of Nde I double digestion with Hind III; Thereby utilize Hind III and Nde I restriction enzyme site with in the directed pET28a of insertion of the VH fragment expression vector, form recombinant plasmid pET-VH.
Change pET-VH over to conventional e. coli bl21 recipient bacterium, grope best abduction delivering temperature (28 ℃, 32 ℃ and 37 ℃) and abduction delivering time (1h, 2h and 3h), express having obtained the rhVVH recombinant protein.
The aminoacid sequence of rhVVH recombinant protein is shown in SEQ ID NO:11; Wherein, The 22-137 position is the aminoacid sequence of the variable region of heavy chain of VEGF humanized antibody, and the 1-21 position is the restriction enzyme site of 6His label and blood coagulating protein enzyme, the sequences such as 6His label of 138-150 position for adding.
The dna encoding sequence of rhVVH recombinant protein is shown in SEQ ID NO:10, and wherein the 64-411 position is the ORF district of the variable region of heavy chain of VEGF humanized antibody.
Utilize the expression and the purification result of SDS/PAGE and Western-blot analysis purposes albumen (rhVVH recombinant protein).Western blotting one is anti-to be mouse-anti 6 * His monoclonal antibody, and two anti-ly are the anti-mouse IgG antibody of HRP mark.
Carry out the purifying of rhVVH recombinant protein with reference to the His Bind purification kit explanation of Novagen company.The main agents prescription is: binding buffer liquid is the 5mmol/L imidazoles, 0.5mol/L NaCl, and 20mmol/LTris-HCl, pH 7.9; Lavation buffer solution is the 60mmol/L imidazoles, 0.5mol/L NaCl, and 20mmol/LTris-HCl, pH 7.9; Elutriant is the 1mol/L imidazoles, 0.5mol/L NaCl, and 20mmol/L Tris-HCl, pH 7.9).
The sex change purification process of rhVVH is: thalline is through ultrasonic degradation (interval 30s repeats 5 times behind the ultrasonic 30s for intensity 80, interval 2s), the centrifugal collection inclusion body deposition of 5000g.Binding buffer liquid dissolving inclusion body with containing 6mol/L urea behind the 0.45 μ m membrane filtration, carries out Ni-NTA chromatographic column purifying.The main agents prescription is: binding buffer liquid is the 5mmol/L imidazoles, 0.5mol/L NaCl, and 20mmol/LTris-HCl, 6mol/L urea, pH 7.9; Lavation buffer solution is the 60mmol/L imidazoles, 0.5mol/LNaCl, and 20mmol/L Tris-HCl, 6mol/L urea, pH 7.9; Elutriant is the 1mol/L imidazoles, 0.5mol/L NaCl, and 20mmol/L Tris-HCl, 6mol/L urea, pH 7.9.
Under 4 ℃ of conditions, the rhVVH that the sex change purifying is obtained is successively through containing 4,2,1,50 times of volume PBS dialysis renaturation of 0mol/L urea.At last that renaturation is good rhVVH is through 0.22 μ m membrane filtration degerming, 4 ℃ of preservations.
The result shows, changes recombinant plasmid pET-VH over to the BL21 intestinal bacteria, and the best inductive condition that non-sex change is expressed is: during OD600 ≈ 0.6, add 1mmol/L IPTG, induce 3h (Fig. 3 sample 19) for 32 ℃.
The reorganization rhVVH albumen that the specific band of about 16kDa size really reaches for institute's strap segment table among the pET-28a has been verified in Western blotting experiment (Fig. 4 C sample 1,2), and size is accord with expectation also.
The binding ability of embodiment 3 antibody and VEGF
The proteic VEGF binding ability of reorganization rhVVH is undertaken by following method: with the people VEGF standard substance coated elisa plate of 0.5 μ g/mL, and every hole 100 μ L.RhVVH is done doubling dilution; With the VEGF monoclonal antibody (monoclonal antibody that V75 hybridoma cell line CCTCC NO:C200623 is produced; Abbreviate HVmAb as; Can be referring to one Chinese patent application 200610116318.1)) and VEGF how anti-as positive controls, pET28a empty carrier extract is as negative control group (NC).Each organizes weaker concn, used antibody situation such as table 2, every group of 3 repetitions.With the OPD colour developing, measure the absorbance value of each sample, calculating mean value and variance at last at the 490nm place.
Table 2 diluted sample and antibody service condition
The result shows, used one anti-inconsistent with enzyme labelled antibody although experiment is respectively organized, the antigen antibody reaction effect is not quite similar, and visible from Fig. 5: compare with the complete IgM monoclonal antibody that derives from the V75 hybridoma (HVmAb group), rhVVH still has good VEGF binding ability, 0D
490Have concentration gradient preferably.
(Human Vascular Endothelial cell HUVEC) to the 100mm petridish, to contain high sugared DMEM culture medium culturing to 90% density of 10%FBS, counts after 0.25% trysinization, according to every hole 6 * 10 the recovery human vascular endothelial
3Individual cell shop is to 24 orifice plates.After treating the 8h cell attachment, add different concns recombinant antibodies rhVVH respectively.Positive control is that the anti-people VEGF of rabbit is how anti-, and 6 * His polyhistidyl label protein and PBS be as negative control, every group of 3 repetitions.Use CCK-8 kit measurement viable cell quantity after 2～4 days, measure 450nm place absorbancy.
The result shows, adds 48h observation of cell behind the rhVVH, the visible notable difference (Fig. 6) of cell quantity, and 75ng/mL rhVVH experimental group (Fig. 6 H) has shown the effect with the approaching inhibition HUVEC cell proliferation of positive control (Fig. 6 B).Compare with adding VEGF group (Fig. 6 G), add recombinant antibodies rhVVH and demonstrate antagonistic action VEGF.
Amplify the administration concentration repeated experiments, after cultivating 120h, use the CCK-8 test kit that viable count is measured (Fig. 7), the result shows that recombinant antibodies rhVVH has restraining effect to the HUVEC cell, and restraining effect and concentration positive correlation.
The preparation and the activity of embodiment 5 antibody variation bodies
Press the method described in the embodiment 4,, add to by 15ng/mL or 75ng/mL consumption (interpolation simultaneously or do not add 3ng/mL VEGF) and to contain 6 * 10 the rhVVH antibody variation body of preparation
3In 24 orifice plates of individual HUVEC cells/well, observe its effect to the HUVEC cell.
The result shows that growth has restraining effect to this rhVVH antibody variation body to the HUVEC cell, and restraining effect and concentration positive correlation.In addition, this rhVVH antibody variation body has antagonistic action to VEGF.
Embodiment 6 pharmaceutical compositions
Be prepared as follows the pharmaceutical composition that contains recombinant antibodies rhVVH, its formulation is an injection, can be used for intravenous administration.
Get the recombinant expressed antibody rhVVH of embodiment 2 preparations; Add injection saline water, with the sterilizing filter degerming of 0.22uM, injection liquid is divided in (50ml/ bottle) in the bottle behind the mixing; Packing is good subsequent use, and wherein every 50ml injection solution contains recombinant monoclonal antibody 50mg.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. monoclonal antibody V
HChain is characterized in that, its complementary determining region CDR has the aminoacid sequence of the CDR of the group of being selected from down:
CDR1 shown in the SEQ ID NO:3,
CDR2 shown in the SEQ ID NO:4 and
CDR3 shown in the SEQ ID NO:5.
2. monoclonal antibody V as claimed in claim 1
HChain is characterized in that, it has the aminoacid sequence shown in the SEQ ID NO:2.
3. one kind has the recombinant protein that VEGF-2 combines active strand, it is characterized in that, described recombinant protein has monoclonal antibody V as claimed in claim 1
HChain and optional assistance expression and/or the sequence label of purifying.
4. recombinant protein as claimed in claim 3 is characterized in that, described recombinant protein is selected from down group:
(a) has the aminoacid sequence shown in the SEQ ID NO:2;
(b) has the aminoacid sequence shown in the SEQ ID NO:11;
(c) replacement, disappearance or the interpolation of the aminoacid sequence shown in SEQ ID NO:2 or 11 through one or more (like 1-20) amino-acid residue formed, and have VEGF-2 combine active function by (a) or (b) polypeptides derived.
5. polynucleotide is characterized in that, its coding is selected from down the protein of group:
The described monoclonal antibody V of claim 1
HChain; Or
The described recombinant protein of claim 3.
6. a carrier is characterized in that, it contains the described polynucleotide of claim 5.
7. a genetically engineered host cell is characterized in that, it contains and is integrated with the described polynucleotide of claim 5 in described carrier of claim 6 or the genome.
8. a preparation method who prepares recombinant polypeptide is characterized in that, this method comprises:
(a) be fit to cultivate the described host cell of claim 7 under the condition of expressing;
(b) from culture, isolate recombinant polypeptide, described recombinant polypeptide is the described monoclonal antibody V of claim 1
HThe described recombinant protein of chain or claim 3.
9. a pharmaceutical composition is characterized in that, it contains the described monoclonal antibody V of (i) claim 1
HDescribed recombinant protein of chain or claim 3 and (ii) pharmaceutically acceptable carrier.
10. described monoclonal antibody V of claim 1
HThe purposes of the described recombinant protein of chain or claim 3 is characterized in that, is used to prepare compsn, and described compsn is used for (a) and suppresses vascular endothelial cell growth; (b) combine with VEGF-2; Or (c) be used to treat tumour.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109021103A (en) * | 2017-06-12 | 2018-12-18 | 上海睿智化学研究有限公司 | Antibody of human vessel endothelium growth factor resisting and its preparation method and application |
| CN110305213A (en) * | 2018-11-09 | 2019-10-08 | 上海复旦张江生物医药股份有限公司 | A kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109021103A (en) * | 2017-06-12 | 2018-12-18 | 上海睿智化学研究有限公司 | Antibody of human vessel endothelium growth factor resisting and its preparation method and application |
| CN110305213A (en) * | 2018-11-09 | 2019-10-08 | 上海复旦张江生物医药股份有限公司 | A kind of anti-B7-H3 antibody and preparation method thereof, its conjugate and application |
| CN110305213B (en) * | 2018-11-09 | 2023-03-10 | 泰州复旦张江药业有限公司 | anti-B7-H3 antibody, preparation method thereof, conjugate thereof and application thereof |
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