CN103204933B - High-stability human anti-VEGF (vascular endothelial growth factor) antibody and application thereof - Google Patents
High-stability human anti-VEGF (vascular endothelial growth factor) antibody and application thereof Download PDFInfo
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Abstract
The invention provides a high-stability human anti-VEGF (vascular endothelial growth factor) antibody and an application thereof. According to computer-aided molecular design and experiment validations, thermal stability transformations are performed in light-and-heavy chain variable regions of one functional anti-VEGF antibody; on the premise of not reducing avidity, a mutant antibody with better thermal stability is obtained; and stacking mutation is performed on each mutant loci, and the thermal stability of an obtained mutant antibody is utmost increased above 7 DEG C as compared with the same of a parent antibody. By the aid of the application of the high-stability human anti-VEGF antibody, high-stability mutant antibody molecules are obtained, storage of medicine of the high-stability human anti-VEGF antibody is benefited, and meanwhile, and experience for reference is provided for stability transformations of other antibodies.
Description
Technical field
The present invention relates to protein engineering field, especially relate to the external thermostability transformation for the treatment of employment source anti-vascular endothelial cell growth factor (VEGF) antibody, particularly, relate to for the anti-VEGF human antibody of a strain, by computer molecular simulateo antagonist, carrying out structure mould builds, and carry out rite-directed mutagenesis to affecting the amino acid of antibody structure stability, and by experiment mutant is carried out the evaluation of avidity and thermostability, screening has obtained the mutant antibody that thermostability obviously improves.
Background technology
Compare with other protein drugs, antibody drug has Stability Analysis of Structures, is easy to expression and purification, is easy to the features such as storage and Half-life in vivo length, and this is also one of its major reason that becomes the leading strength of field of biological pharmacy.Can the structural stability of antibody be to affect one of its important factor that become a good clinical medicine molecule, and good structural stability is the basis that maintains antibody and antigen-specific binding ability.Antibody structure stability study is not only the important component part of protein folding theory in biophysics, is also the problem that must face in therapeutic antibodies performance history.Structural stability not only affects expression, purifying and the storage of antibody, also affects avidity and specificity that it is combined with target antigen in vivo, thereby causes the increase of loss or the toxic side effect of its Biological acdtivity in vivo.
The concern of people's antagonist stability is accompanied by the whole evolution of therapeutic antibodies industry.On affecting the factor of antibody structure stability and causing its structural unstable mechanism and also carried out more deep research.Heat inactivation is the unstable modal form of antibody structure, and evaluating antibody heat resistanceheat resistant deactivation is to evaluate the important indicator of its structural stability.
The thermostability of antibody is from referring in essence, the free energy between the native state of antibody (Native state) and its unfolding state (Unfolded state) poor, i.e. folding free energy.In theory, antibody thermostability is higher, and its heat resistanceheat resistant deactivation is stronger, the concrete important meaning of exploitation of this antagonist.First, the thermostability of antibody is higher, and the probability that its new polypeptide chain produces malfolding (mis-folding) in the in-built timing of cell is lower, thereby solubility expression amount is also higher.Therefore, improve the stability of antibody, can significantly reduce production costs, thereby make medicine be convenient to popularize.Secondly, the research of nearly ten years also shows, the thermostability of antibody is relevant to the tolerance of various proteolytic enzyme to it in vivo.Antibody thermostability is higher, what its structure folded is compacter, and then outside its inner protease cutting site is more not easy to be exposed to, therefore be more not easy by proteasome degradation in vivo, thereby make its in same volume under removing speed remaining effective constituent in vivo more, and this is objectively making in the situation that dosage is identical its Plasma Concentration higher.The more important thing is, the stability of antibody is its guarantee of exercising correct biological function, and antibody stability is higher, and it keeps the bioactive time also longer in vivo.As can be seen here, higher thermostability, is that can a strain therapeutic antibodies finally go on one of clinical and key factor that put on market.In addition, the thermostability of antibody is also vital for character such as its quality guaranteed period and storage conditions.Thermally-stabilised higher, shelf time is under the same conditions also just longer, and also relatively low to preserving the requirement of environment---and this has also reduced storage and the logistics cost of antibody to a certain extent.Therefore, in the situation that character such as guaranteeing affinity of antibody, specificity and expression amount is not affected substantially, improve to the full extent its thermostability, for antibody drug, research and development have important practical significance and using value.
At present, more classical several method for the transformation of antibody stability comprises: the method that CDR transplants, the computer aided molecular design based on crystalline structure, the computer aided molecular design of building based on antibody homology mould, the molecular modification strategy based under protein folding theoretical direction and the molecular modification strategy based on existing structure knowledge etc.In research in the past, for the transformation of antibody stability, successfully report is a lot, and its method adopting is also not quite similar.By the application of one or more above-mentioned methods, all there is the report of successful case.Although these technological methods also do not form ripe technical system, these researchs provide a large amount of materials for more deep understanding antibody structure stability.
Because antibody comprises gently, two chains of heavy chain, so its complicated structure, its integrally-built stability depends on: light chain and heavy chain several aspects such as stability, the interface stability between weight chain and hinge area stability separately, and the stability of weight chain is subdivided into local stability and resistance to overturning.From affecting the analysis of Influential Factors of protein structure stability, affect local stability sexual factor comprise local structural entropy, folding and unfolding free energy, β-bend position and type, special acid as Pro and Gly present position whether rationally, inside and surface amino groups acid environment of living in it is very harmonious and the hydrogen bond of increase key position and other are conducive to constitutionally stable reactive force etc., and the resistance to overturning of molecule is except depending on local stability, also should consider unfolding order and the equimolecular thermokinetics feature of unfolding energy barrier of molecule integral body.These all refer to important thinking and the technological method of the transformation of adpedance body stability.
Antibody molecule is the more special protein molecular of a class, and its structure is except heavy chain CDR3 variation is huge, and the structural changes of other 5 GeCDR districts and framework region is less, and most of antibody has more similar standardized structural feature in the part except HCDR3.This constitutional features of antibody, may for Computer-Aided Drug Design (Computer Aided Drug Design, CADD) is provided for the molecular modification of antibody.At present, to improve the successful case of antibody structure stability a lot of for the method by computer aided molecular design.From essence, CADD is that molecular simulation (molecular modeling) method is in the integrated application of pharmacy field.Along with improving and the progress of technology of molecular simulation theory, molecule simulation method is used in the middle of the mutual identification of protein structure-functional relationship, protein and part and the research work of medicinal design just more and more.Now, molecule simulation method has become in some aspects experimental study and has been difficult to alternative means.According to the theoretical basis of molecule simulation method, it roughly can be divided three classes, based on Newtonian mechanics, based on quantum mechanics and the molecule simulation method based on knowledge.Wherein the molecule simulation method based on Newtonian mechanics comprises that molecular dynamics simulation (molecular dynamics simulation), molecular docking (molecular docking) and homology mould build various classical ways such as (homology modeling).Its advantage is that theoretical basis is sturdy, and computing velocity is very fast, and calculation result is reliable in most cases, therefore becomes the main stream approach in current molecular simulation field.
The structure that Computer-aided Molecular is modeled as understanding antibody and mixture provides good platform support, in recent years, by Computer-aided Molecular countermeasures simulation body molecule, design or transform, especially it is more and more that antagonist carries out the successful report of external affinity maturation, prove that this technology has important effect in antibody development field, the method that even starts in the world to attempt screening by computer simulation obtains needed target antibody.Yet, because the antibody drug exploitation of China is started late, relevant technological method system is also in the initial stage of setting up, and the rare report that utilizes computer molecule molecular designing to carry out antibody molecule optimization, especially not yet finds to use it for the report that antibody stability is transformed.
Applicant is engaged in the R&D work of original therapeutic antibodies always, by the large capacity people antibody resources bank technology certainly building, has obtained some original candidate's antibody kinds.In the research of VEGF target antigen therapeutic antibodies, applicant obtained a strain have in vitro with rhuMAb-VEGF in and active suitable antibody molecule, be expected to become brand-new anti-VEGF drug candidate.But its structural stability is poor.Therefore need to obtain the mutant antibody molecule that stability is obviously improved, for it, further developing anti-VEGF medicine lays the foundation, also wish to improve the theoretic knowledge of antagonist molecular physics structural stability and area of computer aided SARS drug design in deficiency and the defect in this field, for the transformation of other antibody drugs provides technology and theoretical support simultaneously.
Summary of the invention
The VEGF antibody and the active fragments thereof that the object of the present invention is to provide thermostability obviously to improve.
Second object of the present invention is to provide the application of VEGF antibody.
The present invention uses computer molecular simulateo to carry out stability transformation in all directions to the variable region of a strain thermostability and the very poor anti-VEGF function antibody of external shelf-stability, by avidity and stability, screen, finally obtained a plurality of avidity substantially constant, but light, the heavy chain mutational site this antibody stability to positive acting, and further confirm that these mutational sites can further suddenly change by stack, antibody thermostability is further improved.Final its thermostability improves more than 7 ℃ compared with parental antibody.
Antibody provided by the invention, various antibody formations are all included.As, VEGF antibody can be whole antibody or antibody fragment.And then antibody can be labeled detection label, be fixed on solid phase or crosslinked allos mixture (as cytotoxic substance).Use can be diagnosed or treat to antibody.When diagnostic use, the invention provides a kind of method that vegf protein exists that detects.For this purposes, the invention provides a test kit, comprise antibody and for detection of technical specification.
People provided by the invention source VEGF antibody, its variable region of light chain containing just like the aminoacid sequence shown in SEQ ID No.1, its variable region of heavy chain is containing just like the aminoacid sequence shown in SEQ ID No.2; Or
Its variable region of light chain contains just like the aminoacid sequence shown in SEQ ID No.7, and its variable region of heavy chain contains just like the aminoacid sequence shown in SEQ ID No.2.
Wherein, aminoacid sequence shown in SEQ ID No.1 be the people source VEGF antibody Amv6(that researches and develops in advance of contriver it is light, the aminoacid sequence of variable region of heavy chain and encoding gene are shown in respectively SEQ ID No.3,4 and SEQ ID No.9,11, the correlated series of Amv6 antibody also can be referring to Chinese patent ZL201210533178.3) the amino acid mutation sequence of variable region of light chain; Aminoacid sequence shown in SEQ ID No.2 is the amino acid mutation sequence of the variable region of heavy chain of Amv6 antibody; Aminoacid sequence shown in SEQ ID No.7 is that its variable region of light chain encoding gene of people source VEGF antibody Amv4(that contriver researches and develops is in advance shown in SEQ ID No.10) the amino acid mutation sequence of variable region of light chain.
Further, the VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.5, and the amino acid of its variable region of heavy chain is as shown in SEQ ID No.4.Wherein the aminoacid sequence shown in SEQ ID No.5 is the variable region of light chain after the mutational site stack found of the present invention, these mutational sites comprise: these mutational sites comprise: L21 sports I, V29 sports I, G52 sports A, S54 sports N, T57 sports P, and A61 sports D, and G97 sports P; Aminoacid sequence shown in SEQ ID No.4 is the variable region of heavy chain of Amv6 antibody.
The VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.3, and the amino acid of its variable region of heavy chain is as shown in SEQ ID No.6.Aminoacid sequence shown in SEQ ID No.3 is the variable region of light chain of Amv6 antibody, aminoacid sequence shown in SEQ ID No.6 is the variable region of heavy chain after the mutational site stack found of the present invention, these mutational sites comprise: S31 sports D, and G53 sports P, and A88 sports P.
More preferably, these single-point orthomutations on light, heavy chain of the present invention can further superpose, and finally produce light chain and the heavy chain amino acid sequence as shown in SEQ ID NO.5 and SEQ ID NO.6.The heavy chain of itself and parental antibody is that SEQ ID NO.4, light chain are that after SEQ ID NO.3 combination, the thermostability of antibody further improves.
Its thermostability of antibody producing after SEQ ID NO.5 and SEQ ID NO.6 combination is the highest, compared with parental antibody, improves more than 7 ℃.The mutant antibody that thermostability improves, its external serum shelf-stability and long-term shelf-stability also improve thereupon.
The VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain can also be as shown in SEQ ID No.3, and the aminoacid sequence of its variable region of heavy chain is selected from any shown in SEQ ID No.2 or the sequence of a plurality of simple point mutations.Simple point mutation described herein refers to that antibody heavy chain variable region only has a mutational site with respect to parent variable region of heavy chain (shown in SEQ ID No.4).
The VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain is selected from any shown in SEQ ID No.1 or the sequence of a plurality of simple point mutations, and the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID No.4.Simple point mutation described herein refers to that antibody chain variable region only has a mutational site with respect to parent variable region of light chain (shown in SEQ ID No.3).
The VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain is selected from any shown in SEQ ID No.1 or the sequence of a plurality of simple point mutations, described a plurality of for being less than 7; The aminoacid sequence of its variable region of heavy chain is selected from the sequence of any shown in SEQ ID No.2 or two simple point mutations.
Further, the feature that its light chain directed mutants thermostability improves, has certain universality, is equally applicable to the anti-VEGF mutant of another strain antibody A mv4, take SEQ ID NO.8 and SEQ ID NO.4 as antibody light, that variable region of heavy chain forms.
The VEGF antibody that stability provided by the invention is high, the aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.8, and the amino acid of its variable region of heavy chain is as shown in SEQ ID No.4.Aminoacid sequence shown in SEQ ID No.8 is the aminoacid sequence of undergoing mutation after stack in the variable region of light chain of Amv4 antibody.
Further, antibody of the present invention, the aminoacid sequence of its variable region of light chain is selected from the sequence of any simple point mutation shown in SEQ ID No.7, and the amino acid of its variable region of heavy chain is as shown in SEQ ID No.4.
The invention provides the application that above-mentioned antibody be take in the disease therapeuticing medicine that VEGF is target in preparation.
The present invention also provides medicine or the detection reagent of above-mentioned antibody.
In the present invention, the variable region of light chain of parental antibody and weight chain variable region amino acid sequence are respectively as shown in SEQ ID No.3 and 4.Wherein on light chain, the amino acid whose orthomutation of a plurality of single-points can improve the thermostability of antibody, and these orthomutations comprise, L21 sports I, and V29 sports I, and G52 sports A, and S54 sports N, and T57 sports P, and A61 sports D, and G97 sports P; On heavy chain, also have the amino acid whose orthomutation of a plurality of single-points can improve the thermostability of antibody, these orthomutations comprise, S31 sports D, and G53 sports P, and A88 sports P.
First this research adopt ZDock method to carry out restricted stiff molecule to Amv6 with the interaction of VEGF and dock, and adopt on this basis Rosetta SnugDock method to re-start flexible part docking and mixture optimization to the structure of mixture, finally by Rosetta Antibody program, the molecular structure in Amv6 Fv district is carried out to detailed analysis, avoid region and the amino-acid residue of being combined with VEGF, mainly from affecting a plurality of angles of antibody structure, carry out the design of mutant, comprise the diversity ratio of antibody and standard construction, amino acid frequency statistical study, β-bend position and type, special acid as Pro and Gly present position whether reasonable, the thermokinetics feature of molecule local stability and molecule unfolding etc., and a plurality of angles such as weight chain interface stability, provide different designs, improved the accuracy of mutant design, design altogether 42 of mutant antibody, the mutational site that wherein stability is improved to effect is 18, more than rate of accuracy reached to 40%.
The present invention fully uses computer molecular simulateo, from affecting all angles of antibody protein stability, consider, employing is based on experience, based on structure with based on several different methods such as statistical study, the weight chain of antagonism VEGF antibody A mv6 has carried out respectively the design of tens of plant mutant bodies, build and screening, 11 the light chain orthomutation sites and 7 the heavy chain orthomutation sites that thermostability are improved to effect have finally been obtained, further to the sudden change that superposes of these sites, 7 stack sudden changes on light chain have been realized, 3 stack sudden changes on heavy chain, the thermostability of stack mutant is further improved, final mutant antibody thermostability has improved more than 7 ℃ compared with parental antibody, for improving structural stability and the physico-chemical property of this function antibody, provide better selection.
Accompanying drawing explanation
Figure 1 shows that pABG1 carrier information schematic diagram;
Figure 2 shows that pAB κ-Amv6L and pABG1-Amv6H carrier information schematic diagram;
Figure 3 shows that the analysis of Amv6 serum shelf-stability;
Figure 4 shows that the long-term shelf-stability of Amv6 accelerates interpretation;
Figure 5 shows that the interpretation of result of Amv6 thermal stability determination;
The Fab that Figure 6 shows that the Amv6 of prediction is combined mixture model diagram with VEGF;
Figure 7 shows that the comparison of light chain simple point mutation body and Amv6 thermostability;
Figure 8 shows that the comparison of heavy chain simple point mutation body and Amv6 thermostability;
Figure 9 shows that the changing conditions of light chain multi-point joint mutant thermostability;
1: acrivastine; 2:amv6-L97P; 3:Amv6-L52G-L57P-L97P;
4：Amv6-L21I-L52G-L57P-L61D；5：Amv6-L21I-L29I-L52G-L57P-L61D；
6：Amv6-L21I-L29I-L52G-L57P-L61D-L97P；
7：Amv6-L21I-L29I-L52G-L54N-L57P-L61D-L97P；
Figure 10 shows that the comparison of heavy chain multi-point joint mutant and Amv6 thermostability;
Figure 11 shows that light, heavy chain multi-point joint mutant thermostability changing conditions;
1: acrivastine; 2:Amv6-L21I-L52G-L57P-L61D;
3：Amv6-L21I-L52G-L57P-L61D-L97P；
4：Amv6-L21I-L29I-L52G-L54N-L57P-L61D-L97P；
5：Amv6-L21I-L52G-L57P-L61D-H31D-H53P-H88P；
6：Amv6-L21I-L52G-L57P-L61D-L97P-H31D-H53P-H88P；
7：Amv6-L21I-L29I-L52G-L54N-L57P-L61D-L97P-H31D-H53P-H88P；
8：Amv6-H31D-H53P-H88P
Figure 12 shows that thermostability improves the impact of the long-term shelf-stability of antagonist;
Figure 13 shows that Amv4 light chain single-point and the mutant of stack sudden change and the comparison of Amv4 thermostability.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
Embodiment 1 VEGF antibody Amv6 stability study
One, materials and methods
1, material: mammalian cell HEK293T cell is purchased from invitrogen company, serum free medium Freestyle
tM293 express substratum (article No.: 12338-026) with transfection medium Opti-MEMI(article No.: 31985-070) be Gibco company product, transfection reagent 293fectin
tMreagent(article No.: 12347-019) Ye Wei Invitrogen company product.High-fidelity DNA polymerase primeStar archaeal dna polymerase (Takara company product), primer is synthetic to be completed by the raw work in Shanghai.HRP-goat anti-human igg antibody is purchased from biological company limited of Zhong Shan Golden Bridge.96 hole ELISA are Cornning company product.Purification media ProteinA1ml prepacked column is Invitrogen company product.Restructuring VEGF165 albumen sterling is that HEK293T transient expression purifying obtains.Amv6 antibody weight chain expression vector pAB κ-Amv6L and pABG1-Amv6H preserve for this chamber, and carrier information is shown in Fig. 1 and 2.Control antibodies acrivastine is Roche Holding Ag's product.
2, method
The preparation of 2.1 recombinant protein antigen VEGF165:
VEGF165 gene (is so kind as to give by doctor Shi Minglei of BIO ENGINEERING INST MILITARY, concrete source and the visible Shi Minglei Ph D dissertation of sequence: construction expression and the evaluation of restructuring VEGF soluble receptors, Academy of Military Medicine, PLA, 2008) utilize conventional molecular cloning method to be cloned into eucaryon transient expression carrier pABG1(Fig. 2).Particularly, utilize restriction enzyme site EcoR I and BamH I to be cloned into carrier, build recombinant expression plasmid, and utilize HEK293T cell to express recombinant plasmid, the histidine-tagged purifying that carries out of 6 His that utilization is introduced at the C of VEGF165 end, obtain albumen sterling, after protein quantification, packing is frozen.
The expression and purification of 2.2Amv6 antibody
By recombinant plasmid pAB κ-Amv6L and pABG1-Amv6H with mol ratio 1:1 transfection HEK293T cell, carry out the transient expression of whole antibody, collecting cell culture supernatant after 3 days, carries out purifying through ProteinA affinity column to expressing supernatant, and after purifying, the desalination of antibody sample is in the PBS of pH7.4.And utilizing its absorbance at 280nM of spectrophotometric determination, it is the concentration of antibody after divided by optical extinction coefficient 1.35.After purifying, antibody is put in 4 ℃ of preservations.
2.3 the serum shelf-stability of antibody is analyzed
Antibody to be measured is independently sub-packed in containing in the aseptic PBS damping fluid of 50% serum, concentration is 5ug/ml, 50ul/ pipe is sub-packed in aseptic PCR pipe, after sealing, be positioned over 37 ℃, in different time points, collect a duplicate samples, frozen in-20 ℃, finally by the reactivity of the different sampling spot samples of ELISA methods analyst and VEGF.; VEGF is coated with 50ul in 96 orifice plates with 1ug/ML concentration, 4 ℃ of coated spending the night, inferior daily skim-milk sealing treatment; detection antibody is diluted to the detectable level of 50ng/ML with confining liquid; add 96 orifice plates after sealing, after 37 ℃ of effect 1h, with PBST, wash the HRP-goat anti-human igg who adds the pre-sealing of skim-milk after 5 times; after 37 ℃ of effect 30min; PBST washing 5 times, adds the colour developing of substrate nitrite ion, and after 5min, uses the H of 2M
2sO4 termination reaction also compares with the sample of 4 ℃ of placements, evaluates the stability that antibody is placed in 37 ℃ of serum.
The long-term shelf-stability (accelerated test) of 2.4 antibody
Antibody is independently sub-packed in the PBS damping fluid of aseptic pH7.0, concentration is 5 μ g/ml, 50 μ l/ pipes, after sealing, be put in 37 ℃, in different time points, collect 1 duplicate samples, frozen in-20 ℃, finally by the reactivity of the different sampling spot samples of ELISA methods analyst and VEGF.Concrete ELISA information is shown in 2.3.
The thermal stability analysis of 2.5 antibody
(1) sample preparation: antibody to be measured is diluted to the working concentration of 5ug/ml with PBS, divides and be filled to PCR pipe, 50ul/ pipe; (2) heating: also continue 2 hours with grads PCR programmed heating sample.Temperature range is rule of thumb generally selected 45～70 ℃, and thermograde is generally no more than 2 ℃.(3) ELISA detects: the reactivity of antibody sample and VEGF after adopting conventional ELISA methods analyst to process, and concrete ELISA information is shown in 2.3; (4) data analysis: do temperature-sample ELISA colour developing value correlation curve, analyze different samples after heat treated with the variation of VEGF antigen binding capacity.
Two, result
The serum shelf-stability of 1.Amv6
Amv6 and control antibodies acrivastine are processed to the combination activity of rear elisa assay Amv6 and VEGF by method 2.3, result shows (Fig. 3), Amv6 places unstable in serum, and the activity presenting is progressively lost trend, and control antibodies acrivastine stable existence in serum.
The shelf-stability of 2.Amv6
The combination that Amv6 control antibodies acrivastine is processed to rear elisa assay Amv6 and VEGF by method 2.4 is active, result shows (Fig. 4), Amv6 places unstable at 37 ℃ long-term, the activity presenting is progressively lost trend, and control antibodies acrivastine stability is better.
The thermostability of 3.Amv6
Amv6 and control antibodies acrivastine are processed to the reactivity of rear elisa assay Amv6 and VEGF by method 2.4, result shows (Fig. 5), and Amv6 thermostability is far below control antibodies acrivastine, and its half inactivation is stable is about 58 ℃.
The above results prompting, Amv6 molecule vitro stability is poor, in the PBS showing as at pH7.0 and serum, place unstablely for 37 ℃ long-term, and the thermostability of antibody is to evaluate the important indicator of stability of molecule, and thermal stability analysis results suggest Amv6 thermostability is poor.
The impact of embodiment 2Amv6 light chain sudden change on thermostability
One, method
1.Amv6 variable region structure mould is built
Antibody is the functional protein that a class is widely studied, and summary and the analysis of the structural information by antagonist, summarized a set of effective structure prediction and screening scheme at present, and mould is built out the variable region of antibody more exactly.In the present invention, partial code in Rosetta Antibody has been carried out revising and having carried out parallelization processing, made it to be applicable to computing environment.In real work, adopt localized Rosetta Antibody software to carry out mould to the variable region of Amv6 and build, mould is built symbiosis and is become 5000 models.To the sequence of giving a mark of these models, selecting marking and coming front 10 model and carry out careful analysis, choose the model of bulk properties optimum as final model.Utilize the method, mould is built out all CDR district except heavy chain CDR3 very accurately, and the heavy chain CDR3 district for shorter (in 12 residues), also can provide relatively accurate model.
The prediction of 2.Amv6-VEGF binding pattern
The object of the invention is under avidity and specific prerequisite, significantly to improve its stability not affecting between Amv6 and VEGF, be therefore necessary that mould builds out the composite structure of Amv6 and VEGF.By the Alanine-scanning (Ala-scan) in early stage and a large amount of rite-directed mutagenesis experiment (number of patent applications: 201210533178.3), more clearly acquisition the bonding interface information of Amv6 and VEGF, therefore can to the mixture of Amv6-VEGF, predict by restricted docking.First, utilize ZDock to carry out restricted docking to Amv6 variable region with VEGF, and result is carried out to cluster analysis, therefrom choose the result of marking and cluster character optimum as possible binding pattern; Then, utilize the rigidity docking result that Rosetta SnugDock obtains ZDock re-start local docking and optimize, according to marking and experiment information, choose mixture model.Final confirmation is illustrated in figure 6 according to carrying out mutant design with the suitableeest mixture model.
3. mutant design
In the present invention, mainly adopted 3 kinds of mutant method of design such as empirical method, local structural entropy method and structured analysis method to improve the stability of Amv6.Empirical method refers to the method for utilizing the experience under Statistical information and design accumulation in the past to carry out protein transformation, such as the amino-acid residue of nook being replaced to the high frequency amino-acid residues such as Gly, Ser and Pro.Local structural entropy (Local structure entropy, LSE) method is according to a kind of method that the structure in PDB result database is carried out statistical study and according to calculation result, protein transformed.By calculating certain length (being generally 4 amino-acid residues) peptide section at the frequency of occurrence of a certain position, can infer according to formula the LSE of a specific aminoacid sequence, this structure that has this sequence of the lower explanation of LSE is more stable.Structured analysis method is actually according to the method that the comprehensive analysis of protein structure is transformed protein stability, general all based on physics and semiempirical scoring functions, the mutant of design is given a mark, and select the good mutant of marking and carry out experimental verification.Specific design is the results detailed in result.
4. the vector construction of mutant antibody, protein expression and purifying
Mutant is light, heavy chain construction of recombinant vector adopts the method for plasmid Fast Fixed-point sudden change to carry out, reference literature [Wang Ronghao, Chen Ruichuan, Liu Runzhong.The optimization method of Quick-Point sudden change.Xiamen University's journal (natural science edition), 2008, Vol47, sup2,282-285].Specific to this experiment, be: light with Amv6 respectively, heavy chain recombinant vectors is plasmid template, a pair of mutant primer is designed in each mutational site, and (concrete primer information slightly, principle of design is with reference to above-mentioned reference), weight chain gene is carried out to rite-directed mutagenesis, the paired primer that employing contains mutational site, the plasmid of parental antibody of take is template, carry out conventional PCR(DNA exo+ polymerase and dNTP etc. and be Takara company product), PCR product is directly processed with Dpn I enzyme, with PCR test kit, reclaim afterwards, the mutator gene product reclaiming is transformed to intestinal bacteria competence, obtain the recombinant vectors after sudden change, through order-checking confirm correct after for the transient expression of mutant, the transient expression of mutant and purifying are undertaken by 2.2 of embodiment 1.
5.ForteBio QKe systems measurement mutant affinity of antibody
VEGF165 is utilized to vitamin H test kit (GE company product, article No. :) carry out biotinylation, biotinylated VEGF is diluted to the concentration of 100nM with PBS, be coated in streptavidin sensor surface (ForteBIO, a division of Pall Life Sciences, article No.: 18-5020), time is 20min, use HEPES EP to clean 5min to sensor, mutant antibody to be measured and parental antibody Amv6 are all put in detection hole with the concentration of 50nM, and the VEGF of its streptavidin sensor surface after cleaning is combined, binding time is 10min, until in conjunction with after reaching equilibrium state, mixture being dissociated in HEPES EP, Dissociation time is 20min.Adopt ForteBio software package to carry out avidity analysis.
6. mutant antibody thermal stability analysis
2.5 identical with embodiment 1 of method.
Three, result
The Amv6 light chain variable region sequence (SEQ ID NO.3) of take is object, design altogether 28 of light chain simple point mutations, each simple point mutation body is carried out after expression and purification and protein quantification, adopt Fortebio protein-interacting system to carry out the mensuration of relative affinity, the difference of evaluating its avidity and parental antibody Amv6, statistics is as shown in table 1.The mutant antibody that avidity is remained unchanged substantially carries out further Evaluation of Thermal Stability, and statistics is in Table 1.Wherein, have 11 simple point mutation body heat stability to increase compared with parental antibody, partial results as shown in Figure 7.Wherein, thermostability improvement is apparent that this site of L97P most.
The impact of table 1Amv6 light chain single-point orthomutation on Amv6 avidity and thermostability
Note: avidity changes expression and take Amv6 as contrast, and mutant avidity changing conditions, equals mutant avidity/Amv6 avidity; 1 represents basic no variation; 0.5 raising of 1 times that represents slightly to have an appointment; 1.5,2 and 3 represent respectively to reduce slightly in various degree.This research thinks that the mutant that avidity changes between 0.5～2 times can think that avidity is substantially constant.Thermostability: ↑ expression thermostability is improved; ↓ represent that thermostability reduces;-represent that thermostability is substantially constant.
The impact of embodiment 3Amv6 heavy chain sudden change on thermostability
The Amv6 weight chain variabl area sequence (SEQ ID NO.4) of take is object, design altogether 17 of heavy chain simple point mutations, each simple point mutation body is carried out after expression and purification and protein quantification, adopt Fortebio protein-interacting system to carry out the mensuration of relative affinity, the difference of evaluating its avidity and parental antibody Amv6, statistics is as shown in table 2.The mutant antibody that avidity is remained unchanged substantially carries out further Evaluation of Thermal Stability, and method is with reference to embodiment 2, and statistics is in Table 2.Wherein, have 6 simple point mutation body heat stability to increase compared with parental antibody, partial results as shown in Figure 8.
The impact of table 2Amv6 heavy chain single-point orthomutation on Amv6 avidity and thermostability
| Mutational site | Avidity changes | Thermostability | Mutational site | Avidity changes | Thermostability |
| A23T | 1 | ↑ | D62P | 3 | ↑ |
| T28P | 0.5 | — | S63P | 1 | ↓ |
| T28D | 1.5 | ↑ | T69K | 1 | ↓ |
| S31D | 1.5 | ↑ | N77K | 1.5 | ↓ |
| S35L | 0.5 | ↓ | N77G | 1.5 | ↓ |
| T52S | 1.5 | - | Y80S | 0.7 | ↓ |
| G53P | 1 | ↑ | A88P | 0.5 | ↑ |
| G55D | 1 | ↓ | V93I | 1 | ↓ |
| E57S | 1.5 | ↓ | ? | ? | ? |
Note: avidity changes expression and take Amv6 as contrast, and mutant avidity changing conditions, equals mutant avidity/Amv6 avidity; 1 represents basic no variation; 0.5 raising of 1 times that represents slightly to have an appointment; 1.5,2 and 3 represent respectively to reduce slightly in various degree.This research thinks that the mutant that avidity changes between 0.5～2 times can think that avidity is substantially constant.Thermostability: ↑ expression thermostability is improved; ↓ represent that thermostability reduces;-represent that thermostability is substantially constant.
Embodiment 4Amv6 is light, heavy chain is combined the impact of sudden change on thermostability
The result providing according to fact Example 2 and 3, respectively to the sudden change that superposes of 11 light chain simple point mutations of obvious raising antibody thermostability and 6 heavy chain simple point mutations, obtain after the light chain and heavy chain recombinant vectors after stack sudden change, further itself and heavy chain and light chain recombinant vectors cotransfection 293T cell are carried out to transient expression and purifying, obtain the mutant antibody that contains a plurality of mutational sites.Adopt Fortebio protein-interacting system to carry out avidity comparative analysis to itself and parental antibody, method is with reference to embodiment 2.Result shows (table 3), have after 7 light chain mutational sites and the stack of 3 heavy chain mutational sites its avidity compared with parental antibody without considerable change.It is carried out to further Evaluation of Thermal Stability, result shows, the 21st, 29,52,54,57,61 of light chains and 97 amino acids can simultaneous mutations, and thermostability further strengthens (Fig. 9) with the increase in mutational site, compared with simple point mutation, improve the most obvious L97P and further significantly improve; The 31st, 53 of heavy chains and 88 amino acids can simultaneous mutations, and thermostability further strengthens (Figure 10) with the increase in mutational site; And by formed mutant after the multipoint mutation combination of the multipoint mutation of light chain and heavy chain, suddenly change more separately light chain or heavy chain of its thermostability all obviously strengthens (Figure 11).Wherein, all 7 mutational sites of light chain superpose and 4 mutant that the stack of heavy chain mutational site produces, and its thermostability reaches Tm value and reaches more than 66 ℃, compared with the Tm value of parental antibody Amv6 (being about 58.5 ℃), improves more than 7 ℃.
Table 3Amv6 is light, the impact of heavy chain combinatorial mutagenesis antagonist avidity
The impact of embodiment 5Amv4 light chain sudden change antagonist thermostability
For verifying the universality of this raising antibody stability approach, different at CDR3 region sequence from Amv6 in another strain, but the light chain Amv4L(SEQ ID NO.8 that belongs to Kappa3 family, its encoding gene is as shown in SEQ ID NO.10) on verified the impact of these light chain mutational site antagonist thermostabilitys.This light chain and heavy chain SEQ ID NO.4 are combined to form the anti-VEGF specific antibody of strain Amv4 (SEQ ID NO.8 and SEQ ID NO.4 combination).Single-point and combinatorial mutagenesis are carried out in the several mutational sites of L21I, L29I, L52G, L57P and L61D on SEQ ID NO.8, and its avidity and thermostability are analyzed and evaluated.Avidity measurement result shows (Figure 13).In Figure 13, Amv4-IIGPD refers to the mutant that L21I, L29I, L52G, L57P and L61D simultaneous mutation are formed, the sudden change in above-mentioned site has no significant effect avidity, further the result of its thermostability of evaluation shows, the single-point in these sites and combinatorial mutagenesis (SEQ ID No.7) can obviously improve the thermostability of Amv4 equally.The stability of pointing out light chain provided by the invention, weight mutational site to improve VEGF antibody has certain universality.
Claims (1)
1. a people source VEGF antibody, is characterized in that, the aminoacid sequence of its variable region of light chain is as shown in SEQ ID No.5, and the amino acid of its variable region of heavy chain is as shown in SEQ ID No.4.
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| CN103012589A (en) * | 2012-12-11 | 2013-04-03 | 上海赛伦生物技术有限公司 | Human anti-vascular endothelial cell growth factor antibody and application thereof |
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