CN109021104A - A kind of antibody of anti human platelet derivative growth factor beta receptor and its application - Google Patents

Abstract

The invention discloses a kind of antibody of anti human platelet derivative growth factor beta receptor and its applications.It includes heavy chain variable region, heavy chain variable region includes CDR1, CDR2 and/or CDR3；The amino acid sequence of CDR1 is as shown in SEQ ID NO.2, and the amino acid sequence of CDR2 is as shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is as shown in SEQ ID NO.4.The antibody can be combined with the extracellular region of people PDGFR β, and efficiently inhibit or block the combination of PDGFb and PDGFR β, to lower or cut off corresponding signal path；Its purposes includes but are not limited to, the signal path for inhibiting PDGFR beta mediated and the drug etc. for preparing the associated disease caused by PDGFR signal beta access.

Description

A kind of antibody of anti human platelet derivative growth factor beta receptor and its application

Technical field

The invention belongs to bioengineering fields, and in particular to a kind of antibody of anti human platelet derivative growth factor beta receptor And its application.

Background technique

Platelet derived growth factor (Platelet-derived growth factor, PDGF) is generally existing one The growth factor that kind is encoded by sis proto-oncogene, it is initially isolated from platelet alpha, it is mainly synthesized by megacaryocyte, in group Injury repair is knitted, is played the role of in angiogenesis and cell differentiation procedure very important.PDGF molecule is a glycoprotein two Aggressiveness can be divided into-AA ,-BB and-AB three types.PDGF and its receptor (Platelet-derived growth factor Receptor, PDGFR) there is relatively high expression in a variety of human tumors, such as cancer of pancreas, Small Cell Lung Cancer, gastric cancer and cream Gland cancer etc..PDGFR is a kind of receptor tyrosine kinase (Receptor tyrosine kinase, RTK) positioned at cell surface, Two hypotypes: PDGFR α and PDGFR β (Matsui et al.1989) can be divided into.PDGFR β can specificity and PDGF-BB Or-AB is activated in conjunction with (Herdaran et al.1991).After being activated, dimer can be formed between two PDGFR, And activate the signal in downstream to go to access by autophosphorylation, to realize the function of its regulating cell differential growth.PDGF letter Number transmitting is involved in a variety of human diseases, including disease relevant to pathology neovascularization, blood vessel and fibrosis Disease, tumour growth and eye disease.Accordingly, it has been suggested that the inhibitor of PDGF signal transmitting is used for a variety of Curing circumstances.For example, It was suggested that being used to treat a variety of diseases and illness for the inhibitor of PDGFR β.(Andrae et al. (2008) Genes Dev 22 (10):1276-1312).PDGFR beta inhibitor includes her horse of non-specific small molecule tyrosine kinase inhibitors such as methanesulfonic acid For Buddhist nun, Sunitinib malate and CP-673451 and anti-PDGFR β antibody (see, e.g. U.S. Patent number 7,060,271； 5,882,644；7,740,850；With U.S. Patent Application Publication No. 2011/0177074).Also it has proposed for anti-ligand to be adapted to Body (such as anti-PDGF-B) is used for treatment use.However, this field exists to new, high specific and strong PDGF signal Transmit the demand of inhibitor.

Current monoclonal antibody is mostly from mouse, therefore antibody has antigen as heterologous protein in human body Property, significantly limit its use on clinical treatment.In order to overcome this disadvantage, there has been proposed many solutions, Including people/people or people/murine hybridoma technology, chimeric antibody teclmology, display technique of bacteriophage.People/people or people/murine hybridoma technology Since its technical difficulty has dropped out market, be primarily now humanized antibody is prepared with external antibody engineering method, but this Often affinity is bad for the antibody that class method obtains, and easily assembles, and has high parent from humanization or human antibody is prepared in vivo And power, it is good water-soluble and the advantages of be not susceptible to aggregation, therefore people Quan Yuan is prepared from human antibody transgenic mice Antibody most researches and develops advantage, and the mainstream of antibody exploitation from now on.Heavy chain antibody is the antibody for removing two light chains, compared to biography System antibody, it more easily passes vascular wall and enters inside solid tumor, advantage is had more in terms of researching and developing antitumor monoclonal antibody medicine, separately The single domain antibody being derived outside from heavy chain antibody is the minimum antibody molecule found so far, in tumor imaging, treatment and Diagnosis aspect can be widely applied.Modular nature based on heavy chain antibody, further application is to prepare while being directed to The bispecific antibody of two different target antigens.

Summary of the invention

The technical problem to be solved by the present invention is to overcome existing anti human platelet derivative growth factor β in the prior art by Body (PDGFR β) antibody specificity is not high, affinity is low and prepares antibody using phage display screening, particularly heavy chain is anti- Body feature at high cost provides the antibody of anti-PDGFR β a kind of, especially monoclonal heavy chain antibody and its application.The antibody energy It is enough to be combined with the extracellular region of people PDGFR β, and can efficiently inhibit or block the combination of PDGFb and PDGFR β, to lower Or the corresponding signal path of cutting；Its purposes includes but are not limited to, inhibit the beta mediated signal path of PDGFR and prepare by The drug etc. for the associated disease that PDGFR signal beta access causes.

The present invention solve the technical solution of above-mentioned technical problem first is that: a kind of anti human platelet derivative growth factor β by The antibody of body, it includes heavy chain variable region, the heavy chain variable region includes CDR1, CDR2 and/or CDR3；The amino of the CDR1 Acid sequence as shown in SEQ ID NO.2, encode its nucleotide sequence preferably as shown in the 76th~99 of SEQ ID NO.5, The amino acid sequence of the CDR2 as shown in SEQ ID NO.3, encode its nucleotide sequence preferably such as the of SEQ ID NO.5 Shown in 151~174, the amino acid sequence of the CDR3 as shown in SEQ ID NO.4, encode its nucleotide sequence preferably such as Shown in the 289th~312 of SEQ ID NO.5.Preferably, the heavy chain variable region includes CDR1, CDR2 and CDR3；More preferably Ground, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1；Further more preferably, the weight chain variable is encoded The nucleotide sequence in area is as shown in SEQ ID NO.5.

The antibody of the invention may also include heavy chain constant region, and the heavy chain constant region is that this field is conventional, preferably For source of people or small source of mouse, more preferably human antibody heavy's constant region；The heavy chain variable region is that this field is conventional, preferably For source of people.

According to the present invention, the antibody refers to the antibody of this field routine, preferably monoclonal antibody, antibody overall length egg White, antigen-antibody binding domain protein matter segment, bispecific antibody, multi-specificity antibody, single-chain antibody (single chain Antibody fragment, scFv), single domain antibody (single-domain antibody, sdAb) or single domain antibodies (Signle-domain antibody)；The monoclonal antibody can be developed by number of ways and technology, including hybridization Tumor technology, display technique of bacteriophage, single lymphocyte gene clone technology etc., mainstream be by hybridoma technology from wild type or Transgenic mice prepares monoclonal antibody.

Antibody full-length proteins are the antibody full-length proteins of this field routine comprising heavy chain variable region, light chain variable region, again Chain constant region and constant region of light chain.Preferably, the heavy chain variable region and light chain variable region of antibody and small source of mouse heavy chain constant region and Mouse endogenous light chain constant region constitutes antibody full-length proteins.Alternatively, it is highly preferred that the heavy chain variable region and light chain variable region of antibody with Source of people heavy chain constant region and source of people constant region of light chain constitute human antibody full-length proteins.Most preferably, antibody full-length proteins are IgG1, IgG2, IgG3 or IgG4.

Single-chain antibody is the single-chain antibody of this field routine comprising heavy chain variable region, light chain variable region and 15~20 The small peptide of amino acid.

Antigen-antibody binding domain protein matter segment is the antigen-antibody binding domain protein matter segment of this field routine comprising The Fd section of light chain variable region, constant region of light chain and heavy chain constant region.Preferably, antigen-antibody binding domain protein matter segment is Fab With F (ab ')₂。

Single domain antibody is the single domain antibody of this field routine comprising heavy chain variable region and heavy chain constant region.

Single domain antibodies are the single domain antibodies of this field routine, only include heavy chain variable region.

Current antibody is the common antibody formation of two heavy chain two light chains, and heavy chain antibody is a kind of missing light chain Novel antibodies form, the method for preparing heavy chain antibody at present have immune camel to screen to obtain antibody, cost by phage display Higher, the later period, which still needs to progress humanization, can just obtain the heavy chain antibody of full source of people, domestic at present platelet-derived without full source of people The heavy chain antibody of growth factor receptors.Another difficult point of PDGFR β heavy chain antibody exploitation is due to transgenic mice and normal Mouse is had any different, traditional monoclonal antibody Development Techniques, it is intact apply to transgenic mice when, Bu Nengda To due effect.It is therefore, it is necessary to especially optimize for transgenic mice, i.e., repeatedly immune to obtain high serum titer, it establishes Play matched efficiently immune, fusion monoclonal antibody Development Techniques and sensitive quick screening, analytical technology and platform.

In a preferred embodiment of the present invention, the antibody be include source of people heavy chain variable region and small source of mouse heavy chain constant region Heavy chain antibody.

Wherein, the preparation method of antibody is the preparation method of this field routine.Preparation method is preferably: from recombinant expression Separation is obtained or is obtained by artificial sequence synthetic protein in the expression transformant of vascular endothelial growth factor antibody.From weight Group expresses separation in the expression transformant of the antibody and obtains preferably following method: by code for said proteins and having point mutation Cloned nucleic acid molecule into recombinant vector, gained recombinant vector is transformed into transformant, recombinant expression transformants are obtained, lead to Culture gained recombinant expression transformants are crossed, can isolate and purify and obtain the antibody.

The present invention solve the technical solution of above-mentioned technical problem second is that: a kind of nucleic acid, the coding anti-human blood are small The antibody of plate derivative growth factor beta receptor；Preferably, the amino acid sequence of the antibody of the nucleic acid encode includes such as SEQ ID Sequence shown in NO.1；More preferably, the nucleotide sequence of encoding said antibody includes the sequence as shown in SEQ ID NO.5.

As known to those skilled in the art, the base sequence of the amino acid sequence of encoding such antibodies can be suitably introduced into and replace It changes, lack, change, be inserted into or increase to provide the homologue of a polynucleotide.The homology of polynucleotide in the present invention Object can be by keeping being replaced within the scope of antibody activity, lack to the one or more bases for encoding the antibody sequence gene It loses or increases to be made.

The present invention solve the technical solution of above-mentioned technical problem third is that: a kind of recombinant expression comprising the nucleic acid carries Body.

Wherein recombinant expression carrier can be obtained by conventional method in that art, it may be assumed that be connected to nucleic acid molecules of the invention It is built-up on various expression vectors.Expression vector is the various carriers of this field routine, as long as it can hold load aforementioned nucleic acid Molecule.Carrier preferably includes: various plasmids, clay, bacteriophage or viral vectors etc..

The present invention solve above-mentioned technical problem technical solution fourth is that: a kind of weight comprising the recombinant expression carrier Group expression transformant.

Wherein, the preparation method of above-mentioned recombinant expression transformants is the preparation method of this field routine, preferably: will be upper State recombinant expression carrier convert into host cell be made.Above-mentioned host cell is the various host cells of this field routine, As long as being able to satisfy replicates above-mentioned recombinant expression carrier steadily voluntarily, and the entrained nucleic acid can be by effective expression It can.It preferably, is to state host cell as E.coliTG1 or BL21 cell (expression single-chain antibody or Fab antibody) or CHO-K1 Cell (expression overall length IgG antibody).Aforementioned recombinant expression plasmid is converted into host cell, can be obtained currently preferred heavy Group expression transformant.Wherein above-mentioned method for transformation be this field conventional transformation methods, it is therefore preferable to chemical transformation, heat shock method or Electric robin.

The present invention solve above-mentioned technical problem technical solution fifth is that: a kind of anti human platelet derivative growth factor The preparation method of the antibody of beta receptor comprising following steps: the recombinant expression transformants are cultivated, are obtained from culture The antibody of anti human platelet derivative growth factor beta receptor.

The present invention solve above-mentioned technical problem technical solution sixth is that: the anti human platelet derivative growth factor β by Drug of the antibody of body in preparation and platelet derived growth factor and/or platelet derived growth factor BB related disease In application；Preferably, the drug is anti-tumor drug.

The present invention solve above-mentioned technical problem technical solution seventh is that: a kind of pharmaceutical composition, active constituent include Above-mentioned antibody.Preferably, above-mentioned pharmaceutical composition is for preventing or treating antitumor medicine composition.

The administration route of the above-mentioned pharmaceutical composition of the present invention is preferably drug administration by injection or oral administration.It is above-mentioned to be administered to Medicine is preferably comprised the approach such as intravenous injection, intramuscular injection, intraperitoneal injection, intracutaneous injection or subcutaneous injection.Above-mentioned medicine group The various dosage forms that object is this field routine, the preferably form of solid, semisolid or liquid are closed, can be aqueous solution, non-aqueous Solution or suspension are more preferably tablet, capsule, granule, injection or infusion agent etc..

Preferably, present invention pharmaceutical composition above-mentioned further includes one or more pharmaceutical carriers.Pharmaceutical carrier above-mentioned For this field conventional pharmaceutical carrier, pharmaceutical carrier above-mentioned can be any appropriate physiology or pharmaceutically acceptable drug Auxiliary material.Excipient substance above-mentioned is the excipient substance of this field routine, it preferably includes pharmaceutically acceptable excipient, filling Agent or diluent etc..It is highly preferred that above-mentioned pharmaceutical composition include 0.01~99.99% above-mentioned antibody and 0.01~ 99.99% pharmaceutical carrier, wherein percentage is the mass percent for accounting for described pharmaceutical composition.

Preferably, the amount of application of above-mentioned pharmaceutical composition is effective quantity, and above-mentioned effective quantity is that can alleviate or postpone disease The amount of disease, degeneration or damaging disease progression.Above-mentioned effective quantity can be measured with individual primary, and will be based partially on wait control The considerations for the treatment of symptom and sought result.Those skilled in the art can be by using above-mentioned factors such as individual primaries and using not Effective quantity is determined more than conventional experiment.

The present invention solve above-mentioned technical problem technical solution eighth is that: above-mentioned antibody preparation prevention or treatment blood platelet Application in derivative growth factor or the drug of the relevant disease of its receptor.The disease is preferably tumour, vascular diseases and fibre Dimensionization disease, the tumour are preferably cancer of pancreas, Small Cell Lung Cancer, gastric cancer and breast cancer.

The present invention solve above-mentioned technical problem technical solution ninth is that: aforementioned pharmaceutical compositions preparation prevention or treatment Application in platelet derived growth factor or the drug of the relevant disease of its receptor.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that:

The present invention utilizes heavy chain antibody transgenic mice integrated use panimmunity method, has developed a high-affinity With the antibody of the anti human platelet derivative growth factor beta receptor of high biological activity, especially monoclonal heavy chain antibody.The antibody Show excellent property, can be combined with the extracellular region of people PDGFR β, and can efficiently inhibit or block PDGFb with The combination of PDGFR β, to lower or cut off corresponding signal path.The purposes of the antibody, includes but are not limited to, and inhibits The drug etc. for the associated disease that PDGFR beta mediated signal path and preparation is caused by PDGFR signal beta access.

Detailed description of the invention

Fig. 1 is the serum titer of 5 HCAb mouse.

Fig. 2A is heavy chain antibody ELISA result in conjunction with immunogene；Fig. 2 B is heavy chain antibody and other the non-phases for having hFc Close the ELISA binding curve of albumen.

Fig. 3 is the heavy chain antibody with flow cytomery gradient dilution and the cell line binding curve for being overexpressed target spot.

Fig. 4 detects heavy chain antibody and block on human brain perivascular cell for protein immunoblot method to be drawn by ligand PDGFb The phosphorylated CREB level up-regulation risen.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

The preparation of embodiment 1PDGFR β antibody

(1) preparation (source of people PDGFR β ECD-hFc albumen) of immunogene

1, original structure design is immunized

PDGFR β is receptor tyrosine kinase, and extracellular domain includes 5 immunoglobulin-like rings.HFc is that have height The protein macromolecule for spending immunogenicity is used for the preparation of immunogene as carrier protein, is crosslinking in other antigens, enhances compound Immunogenicity.The product of PDGFR β extracellular region (ECD) Yu human constant region (hFc) is devised herein, and the C-terminal of product has His Label.Number is PDGFR β ECD-hFc-His.Specifying information is shown in Table 1.

1 immunogene structural information of table

2, the expression and purifying of immunogene

Nucleotide sequence containing amino acid sequence in the coding above-mentioned table 1 of PDGFR β ECD (wherein, is encoded into source of people PDGFR The number that the nucleotides sequence of β albumen is listed in Genebank is NC_000005.10) be cloned into human IgG Fc segment (hFc) with And 6 × His (hFc and His label passes through pcr clone to pCpC carrier) pCpC carrier (be purchased from Invitrogen, V044-50) and by established standard molecular biology method plasmid is prepared.Specific method referring to Sambrook, J., Fritsch,E.F.,and Maniatis,T.(1989).Molecular Cloning:A Laboratory Manual, Second Edition(Plainview,New York:Cold Spring Harbor Laboratory Press).By the matter Grain transient transfection method infection HEK293 cell (being purchased from ATCC, the U.S.), it is affine that amplification takes supernatant to be loaded to albumin A after 2 weeks Chromatographic column (10ml MabSelect SuRe is purchased from GE healthcare, article No. 17-5438), while being detected with ultraviolet (UV) Instrument monitors the variation of ultraviolet absorption value (A280nm).The affine layer of albumin A is cleaned with PBS phosphate buffer (pH 7.4) after loading Analysis column returns to baseline until ultraviolet absorption value, is then eluted with 50mM sodium citrate buffer solution (pH 3.0), collects from albumin A parent With the PDGFR β ECD with hFc eluted on chromatographic column.It was dialysed with PBS phosphate buffer (pH 7.4) in 4 DEG C of refrigerators Night.Albumen after dialysis is sub-packed in -80 DEG C of preservations after 0.22 micron is sterile filtered, that is, obtains the immunogene hPDGFR β of purifying ECD-hFc-his。

(2) preparation of hybridoma

1, immunogen immune mouse

Utilize immunogen immune heavy chain antibody transgenic mice (the HCAb mouse, purchased from Holland harbour public affairs of above-mentioned preparation Department), mouse is raised under the conditions of SPF (no-special pathogen, Specific pathogen Free).When initial immunity, it is immunized 25 μ l are injected intraperitoneally in original after being emulsified with Freund's complete adjuvant, i.e. every mouse injects 50 μ g immunogenes.When booster immunization, immunogene 25 μ l are injected intraperitoneally after being emulsified with incomplete Freund's adjuvant, i.e. every mouse injects 50 μ g immunogenes.Initial immunity and first time It is spaced 2 weeks between booster immunization, is spaced 3 weeks between booster immunization every time later.Generally can be booster immunization 1~7 time, preferably 4~6 times.

2, indirect elisa method detects immune serum potency

ELISA is the abbreviation of enzyme linked immunoadsorbent assay, is developed after immunofluorescence and radioimmunoassay technique A kind of immunoenzyme technics come, for the technology since the beginning of the seventies comes out, development is very rapid, has been widely used for biology at present Learn many fields with medical science.Cheek is taken a blood sample after each booster immunization 1 week in the present invention, detects serum with indirect ELISA The antibody titer and specificity of middle immunogene.

Concrete operations are as follows:

The hPDGFR β ECD-hFc-His being prepared in 1 μ g/mL " preparation of (one) immunogene " is taken to be dissolved in phosphate Buffer is coated in ELISA Plate, and 100 holes μ L/, 4 DEG C overnight.Next day (contains 0.1% (V/V) Tween- using PBS buffer solution 20) three times, with 0.5% gelatin confining liquid, 200 hole μ L/, 37 DEG C are closed 1 hour board-washing, three times using PBS buffer solution board-washing, small Cheek blood sampling in 7 days after mouse is immune in second, mouse immune serum is used to be diluted containing 2% newborn bovine serum 10mM PBS buffer solution, is added Enter ELISA Plate, 100 37 DEG C of the holes μ L/ 1 hour, 1:10 is added in board-washing afterwards three times⁴Dilution horseradish peroxidase-labeled goat-anti is small again Mouse IgG, 100 37 DEG C of the holes μ L/ 1 hour, TMB colour developing is added in 100 holes μ L/ after board-washing, and room temperature is protected from light 20min, adds 50 μ L/ hole 2M HCl terminates reaction, surveys 450nm absorption value, is used as negative control using mice serum before immune, must be compared with measured value and control value >= 2.1 be the positive potency to judge immune serum.

As a result as shown in Fig. 1 and table 2.Fig. 1 display gained serum titer is high；Table 2 illustrates, is immunized through PDGFR β extracellular region The Post-immunisation serum of mouse has different degrees of combination to immunogene, antigen-antibody reaction is presented, wherein highest dilution exists 100000 or so.Wherein blank control is 1% (w/w) BSA, and wherein batch refers to the 7th day after the 1st booster immunization mice serum, Data in table are OD450nm value.For 9396, serum is can be with the antigen that is coated on elisa plate when 1:100 dilutes Very strong combination occurs, shows as OD value and is up to 3.3, OD value gradually decreases after 10 times of dilutions.

2 ELISA of table detects PDGFR β extracellular region immunized mice serum antibody titer

3, cell fusion

By PDGFR β made from selected every mouse last time 100 above-mentioned (one) parts of μ g of immune intraperitoneal injection Extracellular region protein (mouse being immunoreacted for immunogene) puts to death mouse after 5 days, collect splenocyte.NH is added₄OH is extremely Final concentration 1% (w/w) cracks the red blood cell adulterated in splenocyte, obtains splenocyte suspension.With 1000 turns of DMEM basal medium It is eccentric cleaning cell 3 times per minute, then mixed by number of viable cells 4:1 ratio and murine myeloma cell SP20 (being purchased from ATCC) It closes, is merged using PEG method.

4, hybridoma is identified

Fused cell be diluted to containing 20% fetal calf serum, 1 × HAT DMEM culture medium in, the percentage be matter Measure percentage.Then 1 × 10 is pressed⁵/ 200 every holes μ l are added in 96 porocyte culture plates, are put into 5%CO₂, in 37 DEG C of incubators, The percentage is percent by volume.Respectively with envelope antigen PDGFR β extracellular region protein-hFc-his and hFc after 14 days ELISA (microwell plate protein assay) screen selects cell fusion plate supernatant, and feminine gender ratio positive in ELISA is greater than to 10 positive Clonal expansion is containing 10% (w/w) HT fetal calf serum to 24 orifice plates, and DMEM (invitrogen) is at 37 DEG C, 5% (v/v) CO₂Item Expand culture under part.Culture takes the culture solution for expanding culture in 24 orifice plates to be centrifuged after 3 days, supernatant is collected, to supernatant Antibody subtype analysis is carried out, determines that the combination activity to PDGFR β positive cell (please divide in conjunction with active detection method with FACS Not referring to embodiment 3 (one) and embodiment 3 (two)).According to 24 orifice plate the selection results, the miscellaneous of value > 50 MFI in FACS experiment is selected Friendship oncocyte is qualified positive colony, and qualified hybridoma is selected to be carried out with limiting dilution assay in 96 orifice plates Subclone (is purchased from invitrogen) 37 DEG C in the DMEM culture medium containing 10% (w/w) FBS, 5% (v/v) CO₂Under the conditions of train It supports.Preliminary screening is carried out with ELISA within 10 days after subclone, select single positive monoclonal amplification and continue to cultivate to 24 orifice plates.3 days Determine that antigen binding is positive and assesses bioactivity (evaluation criteria FACS with PDGFR beta receptor ligand binding assay with FACS afterwards Value > 50 MFI in experiment).

5, hybrid tumor cell amplification

According to 24 orifice plate sample detections as a result, picking out optimal clone, and cultivated in the DMEM containing 10% (w/w) FBS In base (be purchased from invitrogen) at 37 DEG C, 5% (v/v) CO₂Under the conditions of the optimal clone is expanded culture, liquid nitrogen freeze It deposits up to hybridoma of the present invention, and can be used for subsequent antibody producing and purifying.

The production and purifying of 2 antibody of embodiment

The antibody concentration that hybridoma generates is lower, and only about 1-10 μ g/mL, concentration change greatly.And in culture medium Fetal calf serum ingredient contained by multiple protein and culture medium caused by cell culture has not many biological activity assays With the interference of degree, it is therefore desirable to carry out small-scale (1-5mg) antibody producing purifying.

The resulting hybridoma of embodiment 1 is inoculated into T-75 Tissue Culture Flask and with production medium (Hybridoma Serum free medium is purchased from Invitrogen company) domestication 3 generations of passage.It is good to its growth conditions, inoculating cell training Support rolling bottle.500mL production medium is added in the culture rolling bottle of each 2L, inoculating cell density is 1.0 × 10⁵/mL.Cover tightly bottle Lid, rolling bottle is placed on the Rotary Machine in 37 DEG C of incubators, 3 revs/min of revolving speed.After continuous rotation culture 14 days, cell is collected Culture solution, filtering removal cell, and clarified with 0.45 μm of membrane filtration to culture supernatant.Clear culture supernatant can horse On carry out purifying or -30 DEG C freeze.

Monoclonal antibody in the culture supernatant (300mL) of clear hybridoma (is purchased from GE with 2mL albumin A column Healthcare it) purifies.Albumin A column first uses equilibration buffer (PBS phosphate buffer, pH7.2) to balance, then by clear training Feeding supernatant is loaded to albumin A column, and coutroi velocity was at 3mL/ minutes.Albumin A column is cleaned with equilibration buffer after loading, is put down The volume of weighing apparatus buffer is 4 times of albumin A column bed volumes.With eluent (the sweet ammonia salt acid buffer of 0.1M, pH2.5) elution of bound PDGFR β antibody on Protein G column monitors elution profile (A280 ultraviolet absorption peak) with UV detector.Collect the anti-of elution Body, is added in 10%1.0M Tris-HCl buffer and pH, the percentage are percent by volume, uses PBS phosphoric acid immediately after Buffer dialysed overnight changes liquid for second day 1 time and continues dialysis 3 hours.PDGFR β antibody after collecting dialysis, with 0.22 μm Filter is sterile filtered, Preservation in sterile condition to get purifying heavy chain PDGFR β antibody.

The PDGFR β antibody of purifying is carried out protein concentration (A280/1.4), purity etc. to test and analyze, as a result such as 3 institute of table Show.

The PDGFR β antibody test analysis that table 3 purifies

Clone number	Antibody purity	Protein concentration (mg/mL)
			39E10E12B3	> 90%	1.2

The calibrating of 3 antibody of embodiment

(1) enzyme-linked immunosorbent assay (ELISA) detects antigen antibody binding sites

The PDGFR β antibody of the resulting purifying of embodiment 2 is carried out and hPDGFR β-hFc-his albumen made from embodiment 1 It is combined reaction respectively.

The immunogene for the purifying that embodiment 1 obtains is diluted to 5.0 μ g/mL of final concentration with PBS, then with the 100 every holes μ l It is added to 96 hole elisa plates.4 DEG C of overnight incubations are sealed with plastic foil, and second day with board-washing liquid [PBS+0.01% (v/v) Tween20] board-washing 2 times, confining liquid [PBS+0.01% (v/v) Tween20+1% (w/w) BSA] room temperature is added and closes 2 hours. Confining liquid is outwelled, the 100 every hole μ l of PDGFR β antibody of the resulting purifying of embodiment 2 is added.After 37 DEG C are incubated for 2 hours, board-washing is used Liquid [PBS+0.01% (v/v) Tween20] board-washing 3 times.The secondary antibody that HRP (horseradish peroxidase) label is added (is purchased from Sigma), 37 DEG C be incubated for 2 hours after, with board-washing liquid [PBS+0.01% (v/v) Tween20] board-washing 3 times.Tmb substrate 100 is added The 100 every hole μ l terminate liquid (1.0N HCl) is added after incubation at room temperature 30 minutes in the every hole μ l.With ELISA plate reading machine (SpectraMax 384plus is purchased from Molecular Device) reads A450nm numerical value, as a result as shown in Fig. 2 and table 4, Middle IgG control is mouse IgG, and the data in table are OD450nm value.The result of table 4 and Fig. 2 explanation: ELISA wrapper sheet antigen, 100nM heavy chain antibody of the present invention carries out 1:10 gradient dilution as initial concentration, is the OD value after reacting, i.e. Fig. 2A in table 4； Due to there is hFc label protein in immunogene, elisa plate has been coated with other bands albumen of hFc in Fig. 2 B, these antibody are not With they occur antigen-antibody reaction, illustrate this heavy chain antibody specifically with antigen binding rather than with label protein knot It closes.

The combination of 4 ELISA of table detection PDGFR β antibody and PDGFR β extracellular domain

(2) combination of Flow cytometry experiments (FACS) detection antibody and PDGFR β expression cell

(in detail by the nucleotide sequence containing coding source of people PDGFR β full length amino acid (being detailed in Uniprot P09619) sequence See Genebank ID:NC_000005.10) it imports HEK293 cell strain and must contain HEK293 stable cell line (this of people PDGFR β Place is known as HEK293-hPDGFR β stable cell line), then expand culture in T-75 Tissue Culture Flask to 90% convergence degree, inhales Culture medium to the greatest extent is washed 2 times with PBS buffer solution (Phosphate buffer saline) (being purchased from Invitrogen), then uses nothing (Versene solution: the being purchased from Life technology company) processing of enzyme cell dissociation buffer and collection cell.It is slow with PBS Fliud flushing is washed cell 2 times, and cell is diluted to 2 × 10 with PBS buffer solution after progress cell count⁶It is small to be added 2% for cell/mL Cow's serum confining liquid, the percentage are mass percent, are incubated at room temperature 15 minutes, then use PBS buffer solution centrifuge washing 2 It is secondary.The cell of collection FACS buffer solution (PBS+2%FBS, the percentage are mass percent) is suspended into 3 × 10⁶Carefully Born of the same parents/mL are added in 96 hole FACS reaction plates by every 100 μ l of hole, and the PDGFR β antibody that the resulting purifying of embodiment 2 is added is to be measured The every 100 μ l of hole of sample, 4 DEG C are incubated for 1 hour.With FACS buffer solution centrifuge washing 2 times, every 100 μ l fluorescence (Alexa of hole is added 488) secondary antibody (being purchased from Invitrogen) marked, 4 DEG C are incubated for 1 hour.With FACS buffer solution centrifuge washing 3 times, every hole is added 100 μ l fixers [4% (v/v) paraformaldehyde] suspension cell is used FACS buffer solution centrifuge washing 2 times after ten minutes.With 100 μ LFACS buffer suspension cell, with FACS (FACS Calibur is purchased from BD company) detection and analysis result.As a result such as Fig. 3 and Shown in table 5, table 5 illustrate, test antibodies in combination with cell surface PDGFR β.Wherein IgG control is mouse IgG (mIgG), in table Data by survey cell mass average fluorescent strength value (MFI).Fig. 3 explanation, with the antibody incubation cell of gradient dilution, is used in combination The secondary antibody (be purchased from Life technologies) of alxa-488 label is incubated for again, can measure 39E10E12B3 and The cell line for being overexpressed PDGFR β has good combination, and negative control mIgG does not obtain fluorescence signal.

The association reaction of 5 FACS of table detection PDGFR β antibody and HEK293-hPDGFR β

PDGFR caused by 4 Western blotting of embodiment experiment detection PDGFR β antibody blocking PDGFb beta mediated downstream letter Number access

For being analyzed with Western blotting, with lysis buffer (1% (w/v) SDS, 1mM Tris (pH 7.4), 2mM vanadium Sour sodium, 2mM EGTA, 2mM EDTA, I mM phenylmethylsulfonyl fluoride and mM sodium fluoride) processing HBVP (human brain perivascular cell) Dissolved matter is obtained, and dissolved matter is boiled, at 4 DEG C with 10000g centrifugation 5 minutes, removes insoluble precipitating.By supernatant and SDS Sample buffer mixing, boils 10 minutes.SDS-PAGE and Western blotting are carried out according to the method for this field extensive utilization, is made Sample is as follows: 12%SDS- polyacrylamide gel, pvdf membrane (Millipore#IPVH00010, the U.S.), is used as and is used for phosphorus It is acidified the anti-phosphorylated CREB (Cell Signaling technology, the U.S.) and anti-ERK antibody of the first antibody of ERK (Cell Signaling technology, the U.S.)；Sheep anti-mouse igg antibody (the Santa being coupled with the HRP for being used as secondary antibody Cruze B1technology, the U.S.).See Fig. 4.Fig. 4 explanation: heavy chain antibody 39E10E12B3 can be blocked to be caused by PDGFb HBVP in ERK phosphorylation, and pERK phosphorylation is various kinds of cell functional parameter, is such as proliferated.

5 heavy chain variable amino acid sequencing of embodiment

Total serum IgE separation: subclone in the amplification of the rapid hybridoma of embodiment 1 (two) step 5 is cultivated on resulting Clear liquid was examined after antigen binding (i.e. after the calibrating of embodiment 3~5 and determination of activity), is passed through centrifugation and is collected 5 × 10⁷It is a Hybridoma is added 1mL Trizol and mixes and be transferred in 1.5mL centrifuge tube, is stored at room temperature 5 minutes；Add 0.2mL chloroform, Oscillation 15 seconds, in 4 DEG C after standing 2 minutes, 12000g is centrifuged 5 minutes, and supernatant is taken to be transferred in new 1.5mL centrifuge tube；It is added 0.5mL isopropanol mixes gently liquid in pipe, is stored at room temperature after ten minutes in 4 DEG C, and 12000g is centrifuged 15 minutes, abandons supernatant； 75% ethyl alcohol of 1mL (percentage is percent by volume) is added, gently washing precipitating, 4 DEG C, 12000g centrifugation is abandoned after five minutes Supernatant dries sediment, and the processed H of DEPC is added₂O dissolution (55 DEG C of water-baths promote solvent 10 minutes) is to get total serum IgE.

Reverse transcription and PCR: taking 1 μ g total serum IgE, configures 20 μ l systems, reacts 60 minutes after reverse transcriptase is added in 42 DEG C, in 7 DEG C of reaction termination in 10 minutes reactions.Configure 50 μ lPCR systems, including 1 μ lcDNA, every kind of primer 2 5pmol, 1 μ l DNA polymerization Enzyme and the buffer system of matching, 250 μm of oldNTPs；PCR program is set, 95 DEG C of initial denaturation 3 minutes, 95 DEG C of denaturation 30 seconds, is moved back Fire 55 DEG C 30 seconds, extend 72 DEG C 35 seconds, 35 circulation after again additionally in 72 DEG C extend 5 minutes, obtain PCR product.Wherein reverse transcription Kit used is PrimeScript RT Master Mix, is purchased from Takara, article No. RR036；Kit used in PCR Surpass fidelity enzyme including Q5, is purchased from NEB, article No. M0492.

Clone and sequencing: taking 5 μ l PCR products to carry out agarose gel electrophoresis detection, will test positive sample and is returned using column Kits are received, wherein QIAquick Gel Extraction Kit isGel&PCRClean-up is purchased from MACHEREY-NAGEL, Article No. 740609.It is attached reaction: sample 50ng, carrier T 50ng, 0.5 μ l of ligase, buffer 1 μ l, 10 μ of reaction system L obtains connection product in 16 DEG C of reaction half an hour, wherein the kit connected is T4DNA ligase, is purchased from NEB, article No. M0402； Take 5 μ l connection products that competent cell (the Ecos 101competent cells, purchased from Yeastern, article No. of 100 μ l is added FYE607 in), ice bath 5 minutes, then in 42 DEG C water-bath heat shock 1 minute, put back to and 650 μ l antibiotic-frees be added after 1 minute on ice SOC culture medium, in being recovered 30 minutes on 37 DEG C of shaking tables with the speed of 200RPM, it is solid that 200 μ l of taking-up are coated on antibiotic LB It is incubated overnight on body culture medium in 37 DEG C of incubators；Next day uses primer M13F (core on carrier T (being purchased from Takara, article No. 6011) Nucleotide sequence: GTAAAACGACGGCCAGT) and M13R (nucleotide sequence: CAGGAAACAGCTATGAC) 30 μ l PCR bodies of configuration System carries out bacterium colony PCR, dips bacterium colony pressure-vaccum in PCR reaction system with pipettor gun head, and 0.5 μ l point is sucked out in another piece To save bacterial strain on the LB solid culture ware of the ampicillin containing 100nM；PCR after reaction, it is solidifying to take out 5 μ l progress agarose Gel electrophoresis detection, positive sample is sequenced and is analyzed [referring to Kabat, " Sequences of Proteins of Immunological Interest,"National Institutes of Health,Bethesda,Md.(1991)].It surveys Sequence result is as shown in table 6~7.

6 PDGFR β antibody protein amino acid sequence of table number

Table 7 PDGFR β antibody gene (DNA) sequence number

Clone number	Heavy chain protein variable region
		39E10E12B3	5

Wherein, the number in table 6 and 7 is sequence number in sequence table, such as the heavy chain protein variable region of 39E10E12B3 The amino acid sequence that amino acid sequence is CDR1 in the heavy chain protein variable region of SEQ ID No.1,39E10E12B3 is SEQ ID The amino acid sequence of No.2, CDR2 are that the amino acid sequence of SEQ ID No.3, CDR3 are SEQ ID No.4；Coding The nucleotides sequence of the heavy chain protein variable region of 39E10E12B3 is classified as SEQ ID No.5.

Wherein, the nucleotides sequence for encoding CDR1 in the heavy chain protein variable region of 39E10E12B3 is classified as sequence table SEQ ID The 76th to the 99th in No.5；

The nucleotides sequence for encoding CDR2 in the heavy chain protein variable region of 39E10E12B3 is classified as in sequence table SEQ ID No.5 The 151st to the 174th；

The nucleotides sequence for encoding CDR3 in the heavy chain protein variable region of 39E10E12B3 is classified as in sequence table SEQ ID No.5 The 289th to the 312nd.

It should be understood that those skilled in the art can make the present invention various after having read above content of the invention Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.

SEQUENCE LISTING

<110>Shanghai Ruizhi Chemical Study Co., Ltd.；Triumphant favour sagacity biotechnology (Shanghai) Co., Ltd.

<120>a kind of antibody of anti human platelet derivative growth factor beta receptor and its application

<130> P1710588C

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 115

<212> PRT

<213> Homo sapiens

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

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

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Val Thr Gly Asp Val Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 2

<211> 8

<212> PRT

<213> Homo sapiens

<400> 2

Gly Phe Thr Phe Ser Ser Tyr Gly

1 5

<210> 3

<211> 8

<212> PRT

<213> Homo sapiens

<400> 3

Ile Trp Tyr Asp Gly Ser Asn Lys

1 5

<210> 4

<211> 8

<212> PRT

<213> Homo sapiens

<400> 4

Ala Val Thr Gly Asp Val Asp Tyr

1 5

<210> 5

<211> 345

<212> DNA

<213> Homo sapiens

<400> 5

gaggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc ggtaactggg 300

gatgttgact actggggcca gggaaccctg gtcaccgtct cctca 345

Claims (10)

1. a kind of antibody of anti human platelet derivative growth factor beta receptor, it includes heavy chain variable regions, which is characterized in that described Heavy chain variable region includes CDR1, CDR2 and/or CDR3；The amino acid sequence of the CDR1 as shown in SEQ ID NO.2, encode it Nucleotide sequence preferably as shown in the 76th~99 of SEQ ID NO.5, the amino acid sequence of the CDR2 such as SEQ ID Shown in NO.3, its nucleotide sequence is encoded preferably as shown in the 151st~174 of SEQ ID NO.5, the amino of the CDR3 Acid sequence as shown in SEQ ID NO.4, encode its nucleotide sequence preferably such as the 289th~312 institute of SEQ ID NO.5 Show.

2. antibody as described in claim 1, which is characterized in that the heavy chain variable region includes CDR1, CDR2 and CDR3；Preferably Ground, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.1；More preferably, the core of the heavy chain variable region is encoded Nucleotide sequence is as shown in SEQ ID NO.5.

3. antibody as claimed in claim 1 or 2, which is characterized in that the antibody further includes heavy chain constant region；Preferably, institute Stating heavy chain constant region is source of people or small source of mouse；And/or the heavy chain variable region is source of people.

4. antibody as claimed in any one of claims 1 to 3, which is characterized in that the antibody is monoclonal antibody, antibody overall length Albumen, antigen-antibody binding domain protein matter segment, bispecific antibody, multi-specificity antibody, single-chain antibody, single domain antibody or list Domain antibodies；Preferably heavy chain antibody.

5. a kind of nucleic acid, which is characterized in that it is encoded such as the derivative growth of the described in any item anti human platelets of Claims 1 to 4 The antibody of factor beta receptor；Preferably, the amino acid sequence of the antibody of the nucleic acid encode includes as shown in SEQ ID NO.1 Sequence；More preferably, the nucleotide sequence of encoding said antibody includes the sequence as shown in SEQ ID NO.5.

6. a kind of recombinant expression carrier comprising nucleic acid as claimed in claim 5.

7. a kind of recombinant expression transformants comprising recombinant expression carrier as claimed in claim 6.

8. a kind of preparation side of the antibody of the anti human platelet derivative growth factor beta receptor as described in any one of Claims 1 to 4 Method, which is characterized in that it includes the following steps: to cultivate recombinant expression transformants as claimed in claim 7, obtain from culture Obtain the antibody of anti human platelet derivative growth factor beta receptor.

9. a kind of pharmaceutical composition, which is characterized in that it includes if the described in any item antibody of Claims 1 to 4 are as activity Ingredient and pharmaceutically acceptable carrier；Preferably, the pharmaceutical composition includes 0.01~99.99% such as claim 1 Antibody described in any one of~4 and 0.01~99.99% pharmaceutical carrier, the percentage is to account for described pharmaceutical composition Mass percent.

10. a kind of antibody as described in any one of claims 1 to 4 or a kind of pharmaceutical composition as claimed in claim 9 Application of the object in the drug of preparation prevention or treatment disease relevant to human vascular endothelial growth factor or its receptor；Preferably Ground, the disease are tumour, vascular diseases or fibrotic disease；More preferably, the tumour is cancer of pancreas, cellule lung Cancer, gastric cancer or breast cancer.