CN104292330B - A kind of high human anti-VEGF antibody of stability and its application - Google Patents
A kind of high human anti-VEGF antibody of stability and its application Download PDFInfo
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Abstract
The invention provides a kind of high human anti-VEGF antibody of stability and its application.Verified by computer aided molecular design Binding experiment, heat endurance transformation is carried out to the light and weight chain variable region of one plant of anti-vegf function antibody, on the premise of affinity of antibody is not reduced, obtain the more preferable mutant antibodies of heat endurance, each mutational site is overlapped mutation, the mutant antibodies heat endurance of acquisition improves more than 7 DEG C compared with parental antibody highest.The high mutant antibody molecule of stability that the present invention is obtained, is conducive to the storage after the antibody patent medicine, while providing the experience of reference of being available for for the stability transformation of other antibody.
Description
It is on March 8th, 2013, Application No. 201310075262.X entitled " one kind the applying date that the application, which is,
The divisional application of the application for a patent for invention of the high human anti-VEGF antibody of stability and its application ".
Technical field
The present invention relates to protein engineering field, more particularly, to treatment employment source anti-vascular endothelial cell growth factor
(VEGF) the external heat endurance transformation of antibody, in particular it relates to for one plant of anti-vegf human antibody, pass through computer molecule
Analogue technique carries out structure mould to antibody and built, and carries out rite-directed mutagenesis to the amino acid for influenceing antibody structure stability, passes through reality
The evaluation that affinity and heat endurance are carried out to mutant is tested, screening obtains the mutant antibodies that heat endurance is significantly improved.
Background technology
Compared with other protein drugs, antibody drug have Stability Analysis of Structures, be easy to expression and purification, it is easily stored and in vivo half
Decline the phase it is long the features such as, this is also that it turns into one of major reason of the leading strength of field of biological pharmacy.The structural stability of antibody
Be influence its can turn into one of good key factor for clinical medicine molecule, good structural stability is to maintain antibody
With the basis of antigentic specificity binding ability.It is theoretical that antibody structure stability study is not only protein folding in biophysics
Important component, be also in therapeutic antibodies development process must in face of the problem of.Structural stability not only influences antibody
Expression, purifying and store, the affinity and specificity for also influenceing it to be combined in vivo with target antigen, so as to cause its internal
The increase of the loss or toxic and side effect of biological activity.
People are to the whole evolution of the concern of Antibody stability along with therapeutic antibody industry.To influence antibody knot
The factor of structure stability and its structural unstable mechanism is triggered also to carry out compared with in-depth study.Heat inactivation is antibody structure
Unstable most common form, evaluate anti-thermal inactivation ability of antibody be evaluate its structural stability important indicator.
The heat endurance of antibody inherently refers to, native state (Native state) and its unfolding state of antibody
The difference of free energy between (Unfolded state), i.e. folding free energy.In theory, antibody heat endurance is higher, its heat resistanceheat resistant
Deactivation is stronger, and this develops specific important meaning to antibody.First, the thermostability of antibody is higher, then its new polypeptide chain
The probability that misfolding (mis-folding) is produced when assembling in the cell is lower, so that amount of soluble expression is also higher.Therefore,
The stability of antibody is improved, production cost can be greatly reduced, so that medicine is easy to popularization.Secondly, the research of last decade
It is also shown that the heat endurance of antibody is related to its tolerance in vivo to various protease.Antibody heat endurance is higher,
What then its structure was folded is compacter, and then its internal protease cutting site is less susceptible to exposure outside, therefore in vivo more not
Easily it is easily degraded by proteases, so that its remaining active ingredient in same volume under removing speed in vivo is more, and this
Objectively make it that its blood concentration is higher in the case of dosage identical.Importantly, the stability of antibody is it
The guarantee of correct biological function is exercised, Antibody stability is higher, it keeps the time of bioactivity also longer in vivo.Thus
It can be seen that, higher heat endurance is that can one plant of therapeutic antibodies finally go on one of key factor that is clinical and putting on market.
In addition, the heat endurance of antibody is also vital for properties such as its shelf-life and storage conditions.It is thermally-stabilised higher,
Holding time then under the same conditions is also longer, and the requirement to Conservation environment is relatively low --- and this is certain
Storage and the logistics cost of antibody are also reduced in degree.Therefore, the properties such as affinity of antibody, specificity and expression quantity are being ensured
In the case of not being affected substantially, its heat endurance is improved to the full extent, and there is important reality for antibody drug research and development
Meaning and application value.
At present, include for the classical several method of comparison that Antibody stability is transformed：The method of CDR transplanting, based on crystalline substance
The computer aided molecular design of body structure, the computer aided molecular design based on antibody homology modeling, based on protein folding
Molecular modification strategy under folded theoretical direction and the molecular modification strategy based on existing structure knowledge etc..In conventional research
In, for Antibody stability transformation, successfully report is a lot, and its method used is also not quite similar.By above-mentioned one kind or
The application of several method, the report for having successful case.Although the technical system of maturation has not yet been formed in these technical methods, this
A little researchs but for deeper into understanding antibody structure stability provide substantial amounts of material.
Because antibody includes two chains of light and heavy chain, therefore its structure is more complicated, and its integrally-built stability depends on
In：Several aspects such as interface stability and hinge area stability between light chain and the respective stability of heavy chain, light and weight chain, and it is light
The stability of heavy chain is subdivided into local stability and resistance to overturning.From the influence factor point of influence protein structure stability
Analysis, influence local stability sexual factor includes local structural entropy, folding and unfolding free energy, β-bend position and type, special ammonia
Base is sour as whether Pro and Gly present positions are reasonable, internal and surface amino groups are sour and its local environment is very harmonious and increase key
The hydrogen bond at position is conducive to constitutionally stable active force etc. with other, and the resistance to overturning of molecule is except depending on local stability
Property outside, it is also contemplated that the overall unfolding order of molecule and the equimolecular thermokinetics feature of unfolding energy barrier.These
All referring to the important idea and technical method for leading Antibody stability transformation.
Antibody molecule is the more special protein molecular of a class, and its structure is in addition to heavy chain CDR3 changes are huge, other 5 CDR
The structure change of area and framework region is smaller, and most of antibody has more similar standardized structural in the part in addition to HCDR3
Feature.This architectural feature of antibody, for by Computer-Aided Drug Design (Computer Aided Drug Design,
CADD) molecular modification for antibody provides possible.At present, antibody knot is improved by the method for computer aided molecular design
The successful case of structure stability is a lot.Intrinsically, CADD is that molecular simulation (molecular modeling) method exists
The integrated application of pharmaceutical field.With the progress of the perfect and technology of molecular simulation theory, molecule simulation method is just more and more
Ground be used for protein structure-functional relationship, protein and part be mutually distinguishable and the research work of drug design among.
Now, molecule simulation method has become the means that experimental study is difficult to substitute in some aspects.According to molecule simulation method
Theoretical foundation, can be roughly divided into three classes, i.e., based on Newtonian mechanics, based on quantum mechanics and Knowledge based engineering molecular simulation side
Method.Wherein the molecule simulation method based on Newtonian mechanics includes molecular dynamics simulation (molecular dynamics
Simulation), the various warps such as molecular docking (molecular docking) and Blast search (homology modeling)
Allusion quotation method.Its advantage is that theoretical foundation is sturdy, and calculating speed is very fast, and result of calculation is reliable in most cases, therefore
Become the main stream approach in current molecular simulation field.
Computer homology modeling techniques provide good platform support to understand the structure of antibody and compound, in recent years
Come, antibody molecule is designed or transformed by Computer homology modeling techniques, external affinity especially is carried out to antibody
Ripe reported success is more and more, it was demonstrated that the technology develops field in antibody has important effect, even starts in the world
The method for attempting to screen by computer simulation obtains required target antibody.However, because the antibody drug of China is developed
Start late, related technical staple is rare to carry out antibody using the design of computer molecular also in the initial stage of setting up
The report of molecule optimization, especially not yet finds to use it for the report of Antibody stability transformation.
Applicant always works on the R＆D work of original therapeutic antibodies, passes through the Large Copacity human antibody resource from structure
Storehouse technology obtains some original candidate antibodies kinds.In the research of VEGF target antigen therapeutic antibodies, applicant obtains
Obtained one plant has the antibody molecule suitable with bevacizumab neutralization activity in vitro, is expected to turn into brand-new anti-vegf candidate's medicine
Thing.But its structural stability is poor.Therefore need to obtain the mutant antibody molecule that stability is obviously improved, be that it is further opened
Hair anti vegf agents lays the foundation, while also being intended to improve the theoretic knowledge to antibody molecular physics structural stability and calculating
Machine ancillary drug MOLECULE DESIGN provides technology and theoretical branch in the deficiency and defect in the field for the transformation of other antibody drugs
Hold.
The content of the invention
It is an object of the invention to provide the anti-VEGF antibody and its active fragment that heat endurance is significantly improved.
Second object of the present invention is to provide the application of anti-VEGF antibody.
The present invention resists with computer molecular simulateo is very poor to one plant of heat endurance and external shelf-stability
The variable region of VEGF function antibodies has carried out stability transformation in all directions, is screened, is finally obtained by affinity and stability
Multiple affinity are basically unchanged, but have to the Antibody stability light and heavy chain mutational site of positive acting, and are further confirmed
These mutational sites further can be mutated by being superimposed, and antibody heat endurance is further improved.Its final heat endurance compared with
Parental antibody improves more than 7 DEG C.
The antibody that the present invention is provided, various antibody formations are all contained in interior.Such as, anti-VEGF antibody can be whole antibody or anti-
Body fragment.And then, antibody can be labeled detection label, be fixed on solid phase or crosslinking heteromeric complexes (such as cytotoxicity thing
Matter).Antibody can diagnose or treat use.In diagnostic application, the present invention provides a kind of method for detecting vegf protein presence.
For this purposes, the present invention provides a kit, including antibody and the technology explanation for detection.
The human anti-VEGF antibody that the present invention is provided, the amino contained as shown in SEQ ID No.1 of its light chain variable district
Acid sequence, its weight chain variable district contains the amino acid sequence as shown in SEQ ID No.2；Or
Its light chain variable district contains the amino acid sequence as shown in SEQ ID No.7, and its weight chain variable district contains such as SEQ
Amino acid sequence shown in ID No.2.
Wherein, the amino acid sequence shown in SEQ ID No.1 is the human anti-VEGF antibody Amv6 that inventor researches and develops in advance
(amino acid sequence and encoding gene of its light and heavy chain variable region are shown in SEQ ID No.3,4 and SEQ ID No.9,11 respectively,
The correlated series of Amv6 antibody also can be found in Chinese patent ZL 201210533178.3) light chain variable district amino acid mutation
Sequence；Amino acid sequence shown in SEQ ID No.2 is the amino acid mutation sequence of the weight chain variable district of Amv6 antibody；SEQ ID
Amino acid sequence shown in No.7 is human anti-VEGF antibody Amv4 (its light chain variable district encoding gene that inventor researches and develops in advance
See SEQ ID No.10) light chain variable district amino acid mutation sequence.
Further, the high anti-VEGF antibody of the stability of the invention provided, the amino acid sequence such as SEQ of its light chain variable district
Shown in ID No.5, the amino acid of its weight chain variable district is as shown in SEQ ID No.4.Amino acid wherein shown in SEQ ID No.5
Sequence be present invention discover that mutational site superposition after light chain variable district, these mutational sites include：Wrap in these mutational sites
Include：L21 sports I, and V29 sports I, and G52 sports A, and S54 sports N, and T57 sports P, and A61 sports D, G97 mutation
For P；Amino acid sequence shown in SEQ ID No.4 is the weight chain variable district of Amv6 antibody.
The high anti-VEGF antibody of stability that the present invention is provided, the amino acid sequence such as SEQ ID of its light chain variable district
Shown in No.3, the amino acid of its weight chain variable district is as shown in SEQ ID No.6.Amino acid sequence shown in SEQ ID No.3 is
The light chain variable district of Amv6 antibody, the amino acid sequence shown in SEQ ID No.6 be present invention discover that mutational site superposition after
Weight chain variable district, these mutational sites include：S31 sports D, and G53 sports P, and A88 sports P.
It is highly preferred that these single-point orthomutations in the light and heavy chain of the present invention can be further superimposed, it is final to produce such as
Light chain and heavy chain amino acid sequence shown in SEQ ID NO.5 and SEQ ID NO.6.It is SEQ ID with the heavy chain of parental antibody
NO.4, light chain are that the heat endurance of antibody after SEQ ID NO.3 are combined further is improved.
Antibody its heat endurance highest produced by after SEQ ID NO.5 and SEQ ID NO.6 combinations, is carried compared with parental antibody
It is high more than 7 DEG C.The mutant antibodies that heat endurance is improved, its iii vivo serum shelf-stability and long-term shelf-stability are also therewith
Improve.
The high anti-VEGF antibody of stability that the present invention is provided, the amino acid sequence of its light chain variable district can also be such as SEQ
Shown in ID No.3, the amino acid sequence of its weight chain variable district is selected from any one or more simple point mutations shown in SEQ ID No.2
Sequence.Simple point mutation described herein refers to heavy chain of antibody variable region relative to parent's weight chain variable district (SEQ ID No.4 institutes
Show) only one of which mutational site.
The high anti-VEGF antibody of stability that the present invention is provided, the amino acid sequence of its light chain variable district is selected from SEQ ID
The sequence of any one or more simple point mutations shown in No.1, the amino acid sequence such as SEQ ID No.4 institutes of its weight chain variable district
Show.Simple point mutation described herein refers to antibody light chain variable region relative to parent's light chain variable district (shown in SEQ ID No.3)
Only one of which mutational site.
The high anti-VEGF antibody of stability that the present invention is provided, the amino acid sequence of its light chain variable district is selected from SEQ ID
The sequence of any one or more simple point mutations shown in No.1, the multiple is less than 7；The amino acid sequence of its weight chain variable district
The sequence of any one or two simple point mutations shown in column selection from SEQ ID No.2.
Further, the characteristics of its light chain directed mutants heat endurance is improved, it is same suitable with certain universality
For another plant of anti-vegf mutant antibodies Amv4, i.e., using SEQ ID NO.8 and SEQ ID NO.4 as the variable district's groups of light and heavy chain
Into antibody.
The high anti-VEGF antibody of stability that the present invention is provided, the amino acid sequence such as SEQ ID of its light chain variable district
Shown in No.8, the amino acid of its weight chain variable district is as shown in SEQ ID No.4.Amino acid sequence shown in SEQ ID No.8 is
The light chain variable district of Amv4 antibody undergo mutation superposition after amino acid sequence.
Further, antibody of the invention, the amino acid sequence of its light chain variable district is selected from appointing shown in SEQ ID No.7
The sequence of one simple point mutation, the amino acid of its weight chain variable district is as shown in SEQ ID No.4.
The invention provides application of the above-mentioned antibody in preparing using VEGF as the disease therapeuticing medicine of target.
Present invention also offers the medicine of above-mentioned antibody or detection reagent.
In the present invention, the light chain variable district of parental antibody and heavy chain variable amino acid sequence are respectively such as SEQ ID
Shown in No.3 and 4.The orthomutation of multiple single-point amino acid can improve the heat endurance of antibody wherein on light chain, these orientations
Mutation includes, and L21 sports I, and V29 sports I, and G52 sports A, and S54 sports N, and T57 sports P, and A61 sports D,
G97 sports P；Also the orthomutation for having multiple single-point amino acid on heavy chain can improve the heat endurance of antibody, these orientations
Mutation includes, and S31 sports D, and G53 sports P, and A88 sports P.
Interaction of this research first using ZDock methods to Amv6 and VEGF has carried out restricted rigid molecule pair
Connect, and re-started the local docking of flexibility to the structure of compound using Rosetta SnugDock methods on this basis
With compound optimization, detailed analysis finally is carried out to the molecular structure in Amv6 Fv areas with Rosetta Antibody programs,
Region and the amino acid residue combined with VEGF is avoided, mainly multiple angles from influence antibody structure carry out setting for mutant
Meter, includes comparison in difference, amino acid frequency statistical analysis, β-bend position and type, the special ammonia of antibody and normal structure
Whether base acid such as Pro and Gly present positions are reasonable, the thermokinetics feature of molecule local stability and molecule unfolding, with
And multiple angles such as light and weight chain interface stability, different designs are provided, the accuracy of mutant design is improved, sets altogether
Mutant antibodies 42 are counted, wherein the mutational site for having improvement result to stability is 18, rate of accuracy reached to more than 40%.
The present invention fully uses computer molecular simulateo, is carried out from all angles of influence antibody protein stability comprehensive
Close and consider, using based on experience, based on structure and based on a variety of methods such as statistical analysis, to anti-vegf antibody A mv6 light and weight chain
The design of tens of plant mutant bodies has been carried out respectively, has been built with screening, and is finally obtained have improvement result to heat endurance 11
These sites are further overlapped mutation, realize light chain by light chain orthomutation site and 7 heavy chain orthomutation sites
On 7 superposition mutation, 3 superposition mutation on heavy chain, the heat endurance of superposition mutant further improves, final mutation
Body antibody heat endurance improves more than 7 DEG C compared with parental antibody, to improve the structural stability and physicochemical property of the function antibody
There is provided more preferable selection.
Brief description of the drawings
Fig. 1 show pABG1 carrier information schematic diagrames；
Fig. 2 show pAB κ-Amv6L and pABG1-Amv6H carrier information schematic diagrames；
Fig. 3 show the analysis of Amv6 serum shelf-stability；
Fig. 4 show the long-term shelf-stability Acceleration study interpretations of result of Amv6；
Fig. 5 show Amv6 thermal stability determination interpretations of result；
Fig. 6 show the Amv6 of prediction Fab and VEGF combination complex model figure；
Fig. 7 show the comparison of light chain single-point mutants and Amv6 heat endurances；
Fig. 8 show the comparison of heavy chain single point mutation body and Amv6 heat endurances；
Fig. 9 show the situation of change of light chain multi-point joint mutant heat endurance；
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 show the comparison of heavy chain multi-point joint mutant and Amv6 heat endurances；
Figure 11 show light and heavy chain multi-point joint mutant heat endurance situation of change；
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
The heat endurance that Figure 12 show improves the influence to the long-term shelf-stability of antibody；
Figure 13 show mutant and the comparison of Amv4 heat endurances of Amv4 light chains single-point and superposition mutation.
Embodiment
Following examples further illustrate present disclosure, but should not be construed as limiting the invention.Without departing substantially from
In the case of spirit and essence of the invention, the modification or replacement made to the inventive method, step or condition belong to the present invention
Scope.
If not specializing, the conventional meanses that technological means used is well known to those skilled in the art in embodiment.
The anti-VEGF antibody Amv6 stability studies of embodiment 1
First, materials and methods
1st, material：Mammalian cell HEK293T cells are purchased from invitrogen companies, serum free medium
Freestyle TM293 expression culture medium (article No.s：12338-026) with transfection medium Opti-MEMI (article No.s：31985-070) it is
Gibco Products, transfection reagent 293fectinTMReagent (article No.s：12347-019) also produced for Invitrogen companies
Product.High-fidelity DNA polymerase primeStar archaeal dna polymerases (Takara Products), primer synthesis is completed by Shanghai life work.
HRP- goat anti-human igg antibodies are purchased from biological Co., Ltd of Zhong Shan Golden Bridge.96 hole ELISA are Cornning Products.Purifying is situated between
Matter ProteinA 1ml prepacked columns are Invitrogen Products.It is HEK293T transient expressions to recombinate VEGF165 albumen sterling
Purifying is obtained.Amv6 antibody weight chain expression vector pAB κ-Amv6L and pABG1-Amv6H preserves for this room, and carrier information is shown in figure
1 and 2.Control antibodies Acrivastine is Roche Holding Ag's product.
2nd, method
2.1 recombinant protein antigen VEGF165 preparation：
By VEGF165 genes (given by BIO ENGINEERING INST MILITARY doctor Shi Minglei, specific source and
The visible Shi Minglei thesis for the doctorate of sequence：Construction expression and the identification of VEGF soluble recepters are recombinated, the Chinese People's Liberation Army is military
The Academy of Medical Sciences, 2008) it is cloned into eucaryon transient expression vector pABG1 (Fig. 2) using common molecular cloning process.It is specific and
Speech, carrier is cloned into using restriction enzyme site EcoR I and BamH I, builds recombinant expression plasmid, and utilize HEK293T cells pair
Recombinant plasmid is expressed, and is purified using 6 the histidine-tagged of His of the C-terminal introducing in VEGF165, obtains albumen
Packing freezes after sterling, protein quantification.
The expression and purification of 2.2Amv6 antibody
By recombinant plasmid pAB κ-Amv6L and pABG1-Amv6H with mol ratio 1：1 transfection HEK293T cells, are resisted entirely
The transient expression of body, cells and supernatant is collected after 3 days, and expression supernatant is purified through ProteinA affinity columns, pure
Antibody samples desalination is in pH7.4 PBS after change.And using spectrophotometric determination its 280nM absorbance, itself divided by
It is the concentration of antibody after extinction coefficient 1.35.Purified antibodies are put in 4 DEG C of preservations.
The serum shelf-stability analysis of 2.3 antibody
Test antibodies are independently sub-packed in the sterile PBS buffer containing 50% serum, and concentration is 5ug/ml, 50ul/ pipes point
Loaded on sterile PCR pipe, 37 DEG C are positioned over after sealing, a sample is collected in different time points, freezes in -20 DEG C, finally leads to
Cross the reactivity that ELISA method analyzes different sample point samples and VEGF.That is, VEGF is coated with 50ul in 96 holes with 1ug/ML concentration
Plate, 4 DEG C of coatings are stayed overnight, secondary daily skimmed milk power Seal treatment, and detection antibody is diluted to 50ng/ML detection with confining liquid
After concentration, 96 orifice plates added after closing, 37 DEG C of effect 1h, washed with PBST and added after 5 times the HRP- that skimmed milk power is closed in advance
After goat anti-human igg, 37 DEG C of effect 30min, PBST is washed 5 times, adds the colour developing of substrate nitrite ion, and in after 5min with 2M's
H2SO4 terminating reactions are simultaneously compared with the sample of 4 DEG C of placements, evaluate the stability that antibody is placed in 37 DEG C of serum.
The long-term shelf-stability (accelerated test) of 2.4 antibody
Antibody is independently sub-packed in sterile pH7.0 PBS, concentration is 5 μ g/ml, 50 μ l/ pipes, after sealing
37 DEG C are put in, 1 part of sample is collected in different time points, is frozen in -20 DEG C, different sample points are analyzed finally by ELISA method
The reactivity of sample and VEGF.Specific ELISA information is shown in 2.3.
The thermal stability analysis of 2.5 antibody
(1) sample preparation：Test antibodies are diluted to 5ug/ml working concentration, packing to PCR pipe, 50ul/ with PBS
Pipe；(2) heat:Sample is heated with grads PCR program and continues 2 hours.Temperature range is rule of thumb typically chosen 45~70 DEG C,
Thermograde is usually no more than 2 DEG C.(3) ELISA is detected：Antibody samples and VEGF after being analyzed and processed using conventional ELISA method
Reactivity, specific ELISA information is shown in 2.3；(4) data analysis：Temperature-sample ELISA colour developing value correlation curves are done, analysis is not
With the change of sample after a heating treatment with VEGF antigen binding capacities.
2nd, result
1.Amv6 serum shelf-stability
Elisa assay Amv6 and VEGF combination after Amv6 and control antibodies Acrivastine are handled by method 2.3
Activity, as a result shows (Fig. 3), and Amv6 places unstable in serum, is presented active loss trend progressively, and control antibodies Ah
Cut down STING stable existence in serum.
2.Amv6 shelf-stability
Elisa assay Amv6 and VEGF combination are lived after Amv6 control antibodies Acrivastine is handled by method 2.4
Property, as a result show (Fig. 4), Amv6 places unstable for a long time at 37 DEG C, be presented active loss trend progressively, and control antibodies Ah
Cut down STING stability preferable.
3.Amv6 heat endurance
Elisa assay Amv6 and VEGF reaction after Amv6 and control antibodies Acrivastine are handled by method 2.4
Property, as a result show (Fig. 5), Amv6 heat endurances are far below control antibodies Acrivastine, stable about 58 DEG C of its half inactivation.
The above results are pointed out, and Amv6 molecule vitro stabilities are poor, show as 37 DEG C long in pH7.0 PBS and serum
Phase places unstable, and the heat endurance of antibody is to evaluate the important indicator of stability of molecule, the prompting of thermal stability analysis result
Amv6 heat endurances are poor.
Influence of the Amv6 light chain mutants of embodiment 2 to heat endurance
First, method
1.Amv6 variable region structure moulds are built
Antibody is the functional protein that a class is widely studied, and passes through the summary and analysis of the structural information to antibody, mesh
It is preceding to have summarized a set of effective structure prediction and screening scheme, than the variable region that accurately mould builds out antibody.
In the present invention, partial code in Rosetta Antibody is modified and parallelization processing has been carried out, be allowed to be adapted to meter
Calculate environment.In real work, mould is carried out to Amv6 variable region using the Rosetta Antibody softwares of localization and built, mould
Symbiosis is built into 5000 models.Marking sequence is carried out to these models, marking is selected and comes preceding 10 model and carry out careful point
Analysis, chooses the optimal model of bulk properties and is used as final model.Using this method, can very accurate mould build out except heavy chain
All CDR regions beyond CDR3, for the heavy chain CDR3 areas of shorter (within 12 residues), can also provide relatively accurate mould
Type.
2.Amv6-VEGF binding patterns are predicted
The purpose of the present invention is that to greatly improve its steady under the premise of not influenceing between Amv6 and VEGF affinity and be specific
It is qualitative, it is therefore necessary to which that mould builds out Amv6 and VEGF composite structure.By the Alanine-scanning (Ala-scan) of early stage and
Substantial amounts of rite-directed mutagenesis tests (number of patent application：201210533178.3) Amv6 and VEGF, are more clearly obtained
Combination interface information, therefore Amv6-VEGF compound can be predicted by restricted docking.First, utilize
ZDock Amv6 variable regions and VEGF are carried out it is restricted dock, and cluster analysis is carried out to result, therefrom chooses marking and cluster
The optimal result of property is used as possible binding pattern；Then, the rigidity obtained to ZDock using Rosetta SnugDock is right
Binding fruit re-starts local docking and optimized, and complex model is chosen according to marking and experiment information.Finally confirm with most suitable
Complex model is illustrated in figure 6 according to progress mutant design.
3. mutant design
In the present invention, 3 kinds of mutant design sides such as empirical method, local structural entropy method and structured analysis method are mainly employed
Method is to improve Amv6 stability.Empirical method refers to that the experience using statistical information and under design accumulation in the past carries out protein and changed
The method made, such as be substituted for the high frequency amino acid residue such as Gly, Ser and Pro by the amino acid residue of corner.Local knot
Structure entropy (Local structure entropy, LSE) method is to carry out statistical analysis according to the structure in PDB result databases
And a kind of method transformed according to result of calculation protein.(it is usually that 4 amino acid are residual by calculating certain length
Base) peptide fragment, in the frequency of occurrence of a certain position, can be inferred to the LSE of a specific amino acid sequence according to formula, and LSE is lower
Illustrate that this possesses the structure of the sequence more stable.Structured analysis method is actually to albumen according to the comprehensive analysis to protein structure
The method that matter stability is transformed, typically all to be beaten based on physics and semiempirical scoring functions the mutant of design
Point, and select the preferable mutant progress experimental verification of marking.Specific design is the results detailed in result.
4. the vector construction of mutant antibodies, protein expression and purifying
The method that mutant light and heavy chain construction of recombinant vector is mutated using plasmid Fast Fixed-point is carried out, reference literature [Wang Rong
It is great, Chen Ruichuan, Liu Runzhong.The optimization method of quick point mutation.Xiamen University's journal (natural science edition), 2008, Vol47, sup
2,282-285].It is specific to this experiment：Respectively using Amv6 light and heavy chains recombinant vector as plasmid template, each mutational site is set
A pair of mutant primers (specific primer information is omited, and design principle is with reference to above-mentioned bibliography) are counted, weight chain gene is pinpointed
Mutation, using the paired primer containing mutational site, using the plasmid of parental antibody as template, carries out Standard PCR (DNA high-fidelities
Polymerase and dNTP etc. are Takara Products), PCR primer is directly handled with Dpn I enzymes, and PCR reagent is used afterwards
Box is reclaimed, and the mutant gene products of recovery are converted into E. coli competent, the recombinant vector after being mutated, through sequencing
Confirm the correct rear transient expression for mutant；The transient expression of mutant and purifying are carried out by the 2.2 of embodiment 1.
5.ForteBio QKe system measurement mutant antibodies affinity
VEGF 165 is utilized into biotin reagent box (GE Products, article No.：) biotinylation is carried out, will be biotinylated
VEGF is diluted to 100nM concentration with PBS, is coated in streptavidin sensor surface (ForteBIO, a division of
Pall Life Sciences, article No.:18-5020), the time is 20min, and 5min is cleaned to sensor using HEPES EP, will
Mutant antibodies and parental antibody Amv6 to be measured is put in detection hole with 50nM concentration, and makes it affine with chain after cleaning
The VEGF of plain sensor surface is combined, and binding time is 10min, to be combined to reach compound after equilibrium state in HEPES EP
Dissociated, Dissociation time is 20min.Affinity analysis is carried out using ForteBio software kits.
6. mutant antibodies thermal stability analysis
Method is 2.5 identical with embodiment 1.
3rd, result
With Amv6 light-chain variable sequences (SEQ ID NO.3) for object, light chain simple point mutation 28 is designed altogether, to each
Single-point mutants are carried out after expression and purification and protein quantification, and relative affinity is carried out using Fortebio protein-interactings system
Measure, evaluate the difference of its affinity and parental antibody Amv6, statistics is as shown in table 1.Affinity is kept not substantially
The mutant antibodies of change carry out further Evaluation of Thermal Stability, and statistics is shown in Table 1.Wherein, there are 11 simple point mutation body heats
Stability increases compared with parental antibody, and partial results are as shown in Figure 7.Wherein, heat endurance improve most notably L97P this
Individual site.
Influence of the Amv6 light chain single-point orthomutations of table 1 to Amv6 affinity and heat endurance
Note：Affinity change represents that mutant affinity situation of change is that is, affine equal to mutant using Amv6 as control
Power/Amv6 affinity；1 represents essentially unchangedization；0.5 represents slightly about 1 times of raising；1.5th, 2 and 3 represent to be slightly different respectively
Degree is reduced.This research thinks mutant of the affinity change between 0.5~2 times it is believed that affinity is basically unchanged.It is hot steady
It is qualitative：↑ represent that heat endurance improves；↓ represent heat endurance reduction；- represent that heat endurance is basically unchanged.
Influence of the Amv6 heavy chain mutants of embodiment 3 to heat endurance
With Amv6 weight chain variabl area sequences (SEQ ID NO.4) for object, heavy chain single point mutation 17 is designed altogether, to each
Single-point mutants are carried out after expression and purification and protein quantification, and relative affinity is carried out using Fortebio protein-interactings system
Measure, evaluate the difference of its affinity and parental antibody Amv6, statistics is as shown in table 2.Affinity is kept not substantially
The mutant antibodies of change carry out further Evaluation of Thermal Stability, and method is with reference to embodiment 2, and statistics is shown in Table 2.Wherein, have 6
Individual single-point mutants heat endurance increases compared with parental antibody, and partial results are as shown in Figure 8.
Influence of the Amv6 heavy chain single-point orthomutations of table 2 to Amv6 affinity and heat endurance
| Mutational site | Affinity changes | Heat endurance | Mutational site | Affinity changes | Heat endurance |
| 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：Affinity change represents that mutant affinity situation of change is that is, affine equal to mutant using Amv6 as control
Power/Amv6 affinity；1 represents essentially unchangedization；0.5 represents slightly about 1 times of raising；1.5th, 2 and 3 represent to be slightly different respectively
Degree is reduced.This research thinks mutant of the affinity change between 0.5~2 times it is believed that affinity is basically unchanged.It is hot steady
It is qualitative：↑ represent that heat endurance improves；↓ represent heat endurance reduction；- represent that heat endurance is basically unchanged.
Influence of the Amv6 light and heavy chains of the embodiment 4 joint mutation to heat endurance
The result provided according to fact Example 2 and 3, respectively to significantly improving 11 light chain simple point mutations of antibody heat endurance
Mutation is overlapped with 6 heavy chain single point mutations, is obtained after light chain and heavy chain recombinant vector after superposition mutation, further by it
Transient expression and purifying are carried out with heavy chain and light chain recombinant vector cotransfection 293T cells, obtains prominent containing multiple mutational sites
Variant antibodies.Affinity comparative analysis, method ginseng are carried out to itself and parental antibody using Fortebio protein-interactings system
According to embodiment 2.As a result show (table 3), have its affinity after 7 light chain mutant sites and 3 heavy chain mutant site superpositions closeer
This antibody is without significant change.Further Evaluation of Thermal Stability is carried out to it, as a result shown, light chain the 21st, 29,52,
54,57,61 and 97 amino acids can be with simultaneous mutations, and heat endurance is further enhanced with the increase in mutational site
(Fig. 9), improving most obvious L97P compared with simple point mutation further significantly improves；Heavy chain the 31st, 53 and 88 amino acids
Can be with simultaneous mutation, and heat endurance is further enhanced (Figure 10) with the increase in mutational site；Also, the multiple spot of light chain is dashed forward
Become the mutant formed after being combined with the multipoint mutation of heavy chain, the more independent mutated light chain of its heat endurance or heavy chain substantially increase
By force (Figure 11).Wherein, the mutant that whole 7 mutational site superpositions of light chain and 4 heavy chain mutant site superpositions are produced, its heat is steady
Qualitative to reach that Tm values reach more than 66 DEG C, the Tm values (about 58.5 DEG C) compared with parental antibody Amv6 improve more than 7 DEG C.
Influence of the Amv6 light and heavy chain combination mutations of table 3 to affinity of antibody
The Amv4 light chain mutants of embodiment 5 resist the influence of body heat stability
It is different in CDR3 region sequences from Amv6 in another strain to verify this universality for improving Antibody stability method,
But belong on the light chain Amv4L (SEQ ID NO.8, its encoding gene is as shown in SEQ ID NO.10) of Kappa3 families and verify
These light chain mutant sites resist the influence of body heat stability.The light chain combined with heavy chain SEQ ID NO.4 to be formed one plant resist
VEGF specific antibodies Amv4 (SEQ ID NO.8 and SEQ ID NO.4 combinations).By L21I, L29I, L52G, L57P and L61D
Several mutational sites carry out single-point and combinatorial mutagenesis on SEQ ID NO.8, and its affinity and heat endurance are analyzed
And evaluation.Affinity measurement result shows (Figure 13).In Figure 13 Amv4-IIGPD refer to L21I, L29I, L52G, L57P and
The mutant that L61D simultaneous mutations are formed, the mutation in above-mentioned site has no significant effect to affinity, further evaluates its thermally-stabilised
Property result show that the heat that the single-point and combinatorial mutagenesis (SEQ ID No.7) in these sites can equally significantly improve Amv4 is steady
It is qualitative.The stability that the prompting light chain of the invention provided, weight mutational site improve anti-VEGF antibody has certain universality.
Claims (1)
1. a kind of human anti-VEGF antibody, it is characterised in that the amino acid sequence of its light chain variable district such as SEQ ID No.3 institutes
Show, the amino acid of its weight chain variable district is as shown in SEQ ID No.6.
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| WO2011153243A2 (en) * | 2010-06-02 | 2011-12-08 | Genentech, Inc. | Anti-angiogenesis therapy for treating gastric cancer |
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| WO2011153243A2 (en) * | 2010-06-02 | 2011-12-08 | Genentech, Inc. | Anti-angiogenesis therapy for treating gastric cancer |
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