CN109134655B - A kind of monoclonal antibody and its preparation method and application of anti-PDGFR α - Google Patents

Abstract

The invention belongs to biological medicine antibody arts, more specifically, the invention discloses the monoclonal antibodies and its preparation method and application of anti-PDGFR α a kind of.Anti- PDGFR alpha monoclonal antibodies of the invention have good bioactivity, compared with control antibodies, PDGFR α albumen can be more effectively combined, PDGFR α albumen is preferably closed in protein level and is combined with its receptor, and has more preferably inhibition ability of cell proliferation.The monoclonal antibody can also have the good prospect of the drugs such as preparation treatment tumour, anti-autoimmune disease individually or with other anti-tumor drug use in conjunction in immunotherapy of tumors and diagnosis and screening.

Description

A kind of monoclonal antibody and its preparation method and application of anti-PDGFR α

Technical field

The present invention relates to biomedicine fields, and in particular to a kind of monoclonal antibody of anti-PDGFR α and preparation method thereof and Using.

Background technique

Platelet derived growth factor (platelet-derived-growth-factor, PDGF) is that intra platelet free calcium goes out Most important growth factor, be present in a variety of tissues such as blood vessel, bone, in angiogenesis, embryonic stage primitive gut shape At the very crucial effect of performer during, cell division and proliferation, central nervous system and bone development etc..Current grinds Study carefully and show that the factor includes 5 kinds of dimer ligands, it may be assumed that PDGF-AA ,-AB ,-BB ,-CC and-DD.The PDGF ligand of 5 seed types Must in conjunction with the corresponding receptor on cell membrane its biological effect of rear competence exertion.

Pdgf receptor is made of Liang Zhong subunit, i.e. PDGFR α and PDGFR β.When the receptor and its ligand knot of cell surface After conjunction forms dimer, active cell intracellular domain tyrosine residue autophosphorylation, or promote to activate the junket of special target protein Histidine residue phosphorylation, thus signal is incoming intracellular, the biological function through tandem type enlarge-effect regulating cell.

The activation of PDGF and PDGFR signal path takes part in glioma, brain tumor, sarcoma, leukaemia, gastric cancer, ovary The generation of the diseases such as cancer.Effect of the angiogenesis in tumour is formed is extremely important.There is no new vessels to be formed, entity tumor is just It can not grow.Therefore, anti-tumor neovascularization generation is of great significance in terms of neoplasm growth and transfer.Some researches show that PDGFR receptor is overexpressed in NK/T cell lymphoma and mediated cell survival (Lu L, et al. Platelet-derived growth factor receptor alpha (PDGFRα) is overexpressed in NK/T-cell lymphoma and mediates cell survival. Biochem Biophys Res Commun. 2018;504(2):525- 531.).And numerous studies have demonstrated that PDGFR α and PDGFR β crucial making of promoting in human body during vascularization With, therefore the new drug initiative project that monoclonal antibodies drug is very attractive and prospect is developed using it as target.

Soft tissue sarcoma (STS) is a kind of rare and heterogeneous malignant tumour.According to the literature, the annual U.S. about occurs 12000 cases, death toll about 3900/annual, account about all adult cancer diagnosis less than 1%, children are pernicious swollen The 15% of tumor.In STS, 50 kinds of different histological subtypes are had more than, the most common STS includes between pleomorphism sarcoma, gastrointestinal tract Matter tumor, embryonal-cell lipoma, leiomyosarcoma, synovial sarcoma and malignant schwannoma etc..And the demography of soft tissue sarcoma, (Li Shu waits 687 soft tissue sarcoma's clinical pathology statistical analysis of single centre there are larger difference for Natural history of treated and clinical effectiveness [J] China Oncological Surgery magazine, 2015,7 (01): 6-8+13.).Although being directed to local lesion, soft tissue sarcoma at present, mainly It is treated in the way of operation excision and radiotherapy etc., but the effect of chemotherapy still has larger dispute；For metastatic STS, entirely Body treatment is main treatment method.For most of STS sufferers, first-line treatment scheme is usually to utilize anthracene nucleus medicament Or use it with another chemotherapy drugs in combination, new antibody class drug is developed, urgently at present to meet tumour sufferer Demand.

Resistance is passed through by the anti-PDGFR alpha monoclonal antibodies Olaratumab that Lilly Co., Eli. (Eli Lilly) is researched and developed The combination of disconnected PDGF and PDGFR α, inhibits the growth of tumour cell, and being approved for that treatment suffers from can not be by cancer hand The adult of the advanced stage soft tissue sarcoma of art or radiotherapy, is possibly used for treating other malignant tumours in the future.Olaratumab (or being IMC-3G3) is a kind of 1 monoclonal antibody of full humanized IgG, optionally in conjunction with PDGFR receptor.This combination Can prevent a variety of platelet derived growth factors (PDGF) ligand, including such as PDGF-AA, PDGF-BB, PDGF-AB and PDGF-CC is in conjunction with its receptor, to prevent activation (Davis EJ, the Chugh R. Spotlight on of receptor olaratumab in the treatment of soft-tissue sarcoma: design, development, and place in therapy. Drug Des Devel Ther. 2017;11:3579-3587.).Olaratumab can also hinder The only phosphorylation of the downstream signaling molecule of ligand induction, for example, the signaling molecules such as AKT and mitogen-activated protein kinase (MAPK) It is phosphorylated.Olaratumab in 2016 and the therapeutic scheme of adriamycin application obtain US and European approval, as controlling It the first-line drug of adult advanced stage soft tissue sarcoma patient is treated, is got nowhere and overall survival with improving, however the drug is not yet To the approval of China's Drug Administration.

The relevant multinomial clinical test of Olaratumab is also in progress, including ongoing multinomial three phases clinic examination It tests and expands its effectiveness range with other kinds of clinical test to determine the biomarker of Olaratumab clinical Benefit, it is special It is not the effectiveness for pediatric population.For example, for the clinical research that gastrointestinal stromal tumor (GIST) indication is unfolded, it is existing Data shows that most of GIST are by activation KIT tyrosine kinase receptor mutation driving, and GIST is to small molecule kinase inhibitors With sensibility, such as Imatinib, Sutent and Rui Gefeini etc..In preclinical study, Olaratumab is different in sarcoma Induced growth inhibits in kind transplantation model, and causes in glioblastoma xenograft, total PDGFR transfer and phosphorylation water Pancake is low.Olaratumab can also promote the internalization or downward of cell surface PDGFR, it is thus possible to which having influences internal kinases Activity (Moroncini G, the et al. Developments in the management of structural domain growth driving mutation of advanced soft-tissue sarcoma - olaratumab in context. Onco Targets Ther. 2018;11:833-842.).In addition, KIT mutant GIST also PDGF-B expression Rs, can provide the target of antibody targeted therapy in turn Mark and the tumour potential treatment method resistant to small molecule kinase inhibitors.

However, finding that there are high risks for the drug in the process of clinical application of Olaratumab.In therapeutic process In, the side effect for generating most serious is neutrophilic granulocytopenia (neutrophil leucocyte white blood cell count(WBC) is low), about 55% patient's meeting The neutrophilic granulocytopenia that severity is 3 grades or 4 grades occurs；There are about the intermuscular bones that 3 grades or 4 grades can occur for 8% patient Bone pain, remaining common side effect include nausea, fatigue, catarrh, alopecia, vomiting, diarrhea, loss of appetite, abdominal pain, The symptoms such as neurotrosis and headache further include infusion correlated response and the harm to embryo-fetus.

Studies have shown that the infusion correlated response of patient, including tide should be monitored during giving the treatment of Olaratumab monoclonal antibody It is red, be short of breath, bronchial spasm, fever/feel cold, low blood pressure, fash and serious low blood pressure, anaphylactic shock or heart The symptoms such as all standing.In a clinical test, receive there are 70 trouble in 485 patients that at least one Olaratumab is treated There is infusion correlated response in person's (14%).In this 70 patients, 68 patients's (97%) produce 1-2 grades of infusion reactions, 3 grades or stronger of infusion reaction has occurred in 11 patients's (2.3%), or even the case where 1 death (0.2%) occurs (Penniman L, et al. Olaratumab (Lartruvo): An Innovative Treatment for Soft Tissue Sarcoma. P T. 2018;43 (5): 267-270.).Olaratumab monoclonal antibody also has certain embryo-fetus Harmfulness --- it is based on animal experiment and pharmacology, which may cause fetus injury, although not about it in gestation The definite data used, it is recommended that the women of child-bearing age are arranged during treatment and in three months using contraception after last time dosage It applies, to prevent to be pregnant.

And the price of Olaratumab monoclonal antibody is also higher, and by taking the U.S. as an example, the average sales price of Olaratumab can Up to 2889 dollars, if used with adriamycin, annual medical expense may be up to 100,000 dollars or more. (Zuluaga-Sanchez S, et al. Cost-Effectiveness of Olaratumab in Combination with Doxorubicin for Patients with Soft Tissue Sarcoma in the United States. Sarcoma. 2018;2018:6703963.).Although the current drug not yet lists at home, its country can be predicted Price also can be very high, it is difficult to be born by China patient.

Therefore, that there are types is more single, toxic side effect is higher and price for existing anti-PDGFR alpha monoclonal antibodies drug The disadvantages of expensive.Novel anti-PDGFR alpha monoclonal antibodies class drug is researched and developed, higher selectivity and affinity, lower are made it have Toxic side effect and more cheap drug cost, to provide the choosing of more monoclonal antibodies drugs for tumor patient It selects, has become urgent problem to be solved.

Summary of the invention

Technical problem to be solved by the invention is to provide a kind of anti-PDGFR alpha monoclonal antibodies, affinity is higher, malicious Side effect is relatively small.

Therefore, the first purpose of this invention is to provide a kind of amino acid sequence of anti-PDGFR alpha monoclonal antibodies.

Second object of the present invention is to provide the nucleic acid molecule for encoding the anti-PDGFR alpha monoclonal antibodies.

Third object of the present invention is to provide the expression vector comprising the nucleic acid molecule.

Fourth object of the present invention is to provide the host cell comprising the expression vector.

Of the invention the 5th is designed to provide a kind of preparation method of anti-PDGFR alpha monoclonal antibodies.

Of the invention the 6th is designed to provide the composition comprising the anti-PDGFR alpha monoclonal antibodies.

Of the invention the 7th is designed to provide the anti-PDGFR alpha monoclonal antibodies in preparation PDGFR alpha molecule retardance Application in drug.

To achieve the goals above, this invention takes following technical solutions:

One of the technical solution that the present invention takes are as follows: a kind of monoclonal antibody of anti-PDGFR α, the monoclonal antibody include Heavy chain variable region and light chain variable region, the amino acid sequence of the heavy chain variable region as shown in SEQ ID NO:7 in sequence table, The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:13 in sequence table.

The monoclonal antibody of anti-PDGFR α of the present invention is conventional monoclonal antibody comprising two heavy chain of antibody With two antibody light chains, including heavy chain variable region and light chain variable region.Each variable region includes complementary determining region (CDR region), i.e. tri- structural domains of CDR1, CDR2 and CDR3.Preferably, the weight of the monoclonal antibody of anti-PDGFR α of the present invention Chain variable region amino acid sequence is as shown in SEQ ID NO:7 in sequence table, preferably, its heavy chain variable region further includes CDR region, Middle CDR1 amino acid sequence is as shown in SEQ ID NO:8 in sequence table, SEQ ID NO:9 in CDR2 amino acid sequence such as sequence table Shown, CDR3 amino acid sequence is as shown in SEQ ID NO:10 in sequence table.The amino acid sequence of its light chain variable region such as sequence In table shown in SEQ ID NO:13, preferably, its light chain variable region further includes the area CDR ', wherein CDR1 ' amino acid sequence such as sequence In list shown in SEQ ID NO:14, CDR2 ' amino acid sequence is as shown in SEQ ID NO:15 in sequence table, CDR3 ' amino Acid sequence is as shown in SEQ ID NO:16 in sequence table.Preferably, the anti-PDGFR alpha monoclonal antibodies are source of people.

The anti-PDGFR alpha monoclonal antibodies can be the full length sequence of antibody, be also possible to anti-PDGFR α human antibody Partial Fragment, the segment are preferably Fab, Fab ', F (ab ')₂, Fv or scFv etc..Preferably, the anti-PDGFR α source of people Antibody is source of people.It is furthermore preferred that the anti-PDGFR α human antibody is IgG₁、IgG₂Or IgG₄Type antibody.

The present invention further provides the derivative of the anti-PDGFR α human antibody, the derivative is anti-for PDGFR α source of people The segment of body, antibody/antibody fragment-factor fusion protein, antibody/antibody fragment-chemical coupling thing；The anti-PDGFR α source of people The segment of antibody be Fab, Fab ', F (ab ')₂, Fv or scFv etc..

Preferably, antibody/antibody fragment-the factor fusion protein is specially antibody-factor fusion protein or antibody piece Section-factor fusion protein.Preferably, antibody/antibody fragment-the chemical coupling thing is specially antibody-chemical coupling thing or resists Body segment-chemical coupling thing.

Monoclonal antibody of the present invention can be prepared by this field routine techniques, including hybridoma technology, phage display technology Show technology, single lymphocyte gene clone technology etc., preferably passes through hybridoma technology from wild type or transgenic mice system Standby monoclonal antibody.

The two of the technical solution that the present invention takes are as follows: a kind of isolated nucleic acid molecule, the monoclonal antibody heavy Coding nucleotide sequence is as shown in SEQ ID NO:5 in sequence table, SEQ in the coding nucleotide sequence of light chain such as sequence table Shown in ID NO:11.

The preparation method of the nucleic acid molecule is this field customary preparation methods, is preferably comprised following preparation method: By gene clone technology such as PCR method etc., the nucleic acid molecule for encoding the monoclonal antibody is obtained, or by artificial Complete sequence synthetic method obtains the nucleic acid molecule of coding said monoclonal antibody.

As known to those skilled in the art, the nucleotide sequence for encoding the amino acid sequence of said monoclonal antibody can be appropriate Replacement is introduced, missing, changes, be inserted into or increase to provide the homologue or its conservative series of variation of a polynucleotide. The homologue of polynucleotide or its conservative series of variation in the present invention, can be by encoding the monoclonal antibody gene One or more bases are replaced, lack or increase within the scope of holding antibody activity to be made.

The three of the technical solution that the present invention takes are as follows: a kind of expression vector, the expression vector contain core as described above Thuja acid molecule.

Wherein the expression vector is the expression vector of this field routine, is referred to comprising regulating and controlling sequence appropriate, such as open Promoter sequences, terminator sequence, polyadenylation sequences, enhancer sequence, marker gene and/or sequence and other are appropriate The expression vector of sequence.The expression vector can be virus or plasmid, such as bacteriophage appropriate or phasmid, more technologies Details is referred to such as Sambrook, Molecular Cloning:A Laboratory Manual, the second edition, Cold Spring Harbor Laboratory Press, 1989.Known technology and scheme of many for nucleic-acid manipulation refer to Current Protocols in Molecular Biology, the second edition, Ausubel etc. write.Expression in the present invention carries Body refers to bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenopathy Poison, retrovirus or other carriers.

Preferably, the expression vector is selected from one of pHLX101, pEE14.4, pCHO 1.0 or pcDNA3.1 or more The combination of kind, is more preferably pcDNA3.1.Preferably, the expression vector includes the volume of the monoclonal antibody heavy and light chain Code nucleotide sequence, the coding nucleotide sequence of heavy chain variable region and light chain variable region, heavy chain CDR1, CDR2 and CDR3 with And light chain CDR1 ', CDR2 ' and the coding nucleotide sequence in the area CDR3 ' etc..

The four of the technical solution that the present invention takes are as follows: a kind of host cell, the host cell include table as described above Up to carrier.

Host cell of the present invention is the various host cells of this field routine, as long as being able to satisfy makes above-mentioned recombination table It is steadily voluntarily replicated up to carrier, and the entrained nucleotide can be by effective expression.Host cell in the present invention It can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as lactation Zooblast.The expression vector is preferably comprised: COS, CHO(Chinese hamster ovary, Chinese Hamster Ovary), HeLa cell line, myeloid cell series for example SP2/0 cell line, NS0, sf9, sf21, DH5 α, BL21 (DE3) orE.coliTG1、 One of B- cell or hybridoma of YB2/0 cell line etc. and conversion or a variety of combinations.More preferably it isE.coli TG1, BL21 cell (expression single-chain antibody or Fab antibody) or CHO-K1 cell (expression overall length IgG antibody).

Foregoing expression vectors are converted into host cell, currently preferred recombinant expression transformants can be obtained.Wherein The method for transformation is this field conventional transformation methods, preferably chemical transformation, heat shock method or electric robin.

The five of the technical solution that the present invention takes are as follows: the preparation method of the monoclonal antibody of anti-PDGFR α comprising following Step:

A) under expression condition, host cell as described above is cultivated, expresses the monoclonal antibody of anti-PDGFR α；

B) monoclonal antibody of separation and the anti-PDGFR α of purification step a) gained.

The Isolation and purification method of the cultural method of host cell of the present invention, the monoclonal of the anti-PDGFR α is Conventional method in that art, concrete operation method please refers to corresponding cell culture technology handbook and monoclonal antibody isolates and purifies Technical manual.

Host cell used in the present invention is the prior art, can be directly acquired by commercial sources, used in culture Culture medium be also various conventional mediums, those skilled in the art can rule of thumb select applicable culture medium, be suitable for place It is cultivated under conditions of chief cell growth.After host cell growth is to cell density appropriate, with suitable method (such as temperature Degree conversion or chemical induction) promoter that induces selection, cell is further cultured for a period of time.Recombination in the above methods is more Peptide can be expressed in cells, or on the cell membrane, or secreted outside the cell.If desired, can using its physics, it is chemical and Other characteristics are separated by various separation methods and purify the albumen of recombination.These methods are well known to those skilled in the art 's.The example of these methods includes but is not limited to: conventional renaturation process, with protein precipitant processing (salting-out method), from The heart permeates broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, efficient liquid phase Chromatograph the combination of (HPLC) and various other liquid chromatography technologies and these methods.

The present invention screens the gene order for obtaining purpose antibody from the cell strain of Colony Culture, to construct eukaryon table Up to carrier, the activity of antibody can be rebuild after expression, obtain anti-PDGFR alpha monoclonal antibodies.

The six of the technical solution that the present invention takes are as follows: a kind of composition, it includes a effective amount of anti-PDGFR α as described above Monoclonal antibody and pharmaceutically acceptable carrier.

Monoclonal antibody of the invention can by any of mode compounding pharmaceutical composition in this field come using.This Kind composition is using the monoclonal antibody as active constituent, in addition one or more pharmaceutically acceptable carriers, diluent, filling Agent, bonding agent and other excipient, this depends on administration mode and designed dosage form.Known to the branch art personnel of this field Treat inert inorganic or organic carrier include but is not limited to lactose, cornstarch or derivatives thereof, talcum, vegetable oil, Wax, fat, more antelope based compounds such as polyethylene glycol, water, sucrose, ethyl alcohol, glycerol, such, various preservatives, lubrication Agent, dispersing agent, corrigent.Moisturizer, antioxidant, sweetener, colorant, stabilizer, salt, buffer are such to be added Entering wherein, these substances are used to help the stability of formula as needed or help to improve activity or its biological effectiveness, Inhibitor can be used in such a composition, can be the form of its original chemical itself, or optionally use its drug Learn the form of acceptable salt, monoclonal antibody of the invention can be administered alone, or with various combination medicine-feedings, and with it is other Combining form is administered healing potion together.The composition so prepared may be selected well known by persons skilled in the art as needed What mode appropriate is administered gained monoclonal antibody.

Anti- PDGFR alpha monoclonal antibodies provided by the invention can form drug with pharmaceutically acceptable carrier together For preparation compositions to more stably play curative effect, these preparations can guarantee anti-PDGFR alpha monoclonal antibodies disclosed by the invention Amino acid core sequence conformation integrality, while the polyfunctional group for going back protected protein matter prevents its degradation (including but unlimited In cohesion, deamination or oxidation).Under normal conditions, it for liquid preparation, can usually be saved under the conditions of 2 °C -8 °C at least steady It is 1 year fixed, for lyophilized preparation, stablize in 30 °C of holdings at least six months.The anti-PDGFR alpha monoclonal antibodies preparation can be system The preparations, preferably water needle or lyophilized preparation such as medicine field is commonly suspended, water needle, freeze-drying,

For the water needle or lyophilized preparation of the monoclonal antibody of anti-PDGFR α disclosed by the invention, pharmaceutically acceptable Carrier is preferably comprised but is not limited to: one or a combination set of surfactant, solution stabilizer, isotonic regulator and buffer. Wherein surfactant is preferably comprised but is not limited to: nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80)；Poloxamer(such as poloxamer 188)；Triton；Lauryl sodium sulfate (SDS)；Laurel sulfuric acid Sodium；Myristyl, sub- oil base or octadecyl sarcosine and Pluronics and Monaquat^tmDeng additional amount should make to resist The granulating trend of PDGFR alpha monoclonal antibodies is minimum.Solution stabilizer one of is preferably comprised but is not limited to be exemplified below or it Combination: carbohydrate, for example, reducing sugar and nonreducing sugar；Amino acids, for example, monosodium glutamate or histidine；Alcohols, example Such as: the additional amount of trihydroxylic alcohol, advanced sugar alcohol, propylene glycol, polyethylene glycol, solution stabilizer should make the preparation eventually formed exist Those skilled in the art thinks to reach stable time interior holding stable state.Isotonic regulator is preferably comprised but is not limited to One or a combination set of sodium chloride, mannitol.Buffer is preferably comprised but is not limited to: Tris, histidine buffering liquid, citric acid One or a combination set of salt, phosphate buffer.

The seven of the technical solution that the present invention takes are as follows: the monoclonal antibody of anti-PDGFR α as described above is at PDGFR α points of preparation Purposes on sub- blocking medicine.

The application is more preferably to prepare the purposes on PDGFR alpha molecule blocking medicine, preferably, the preparation PDGFR α Purposes on molecule blocking medicine is specially the purposes prepared on oncotherapy or diagnosing tumor class drug.

Drug of the present invention is preferably antitumor, treatment autoimmune disease, treatment infectious diseases and/or The drug of resisting transplant rejection reaction is more preferably anti-tumor drug, treatment autoimmune disease drug, it is therefore preferable to antitumor Drug.The monoclonal antibody of anti-PDGFR α of the present invention can be used alone or be used in combination with other anti-tumor drugs.Institute Stating other anti-tumor drugs is this field conventional anticancer drugs, including antibody class drug or small molecule, anti-tumor drug.It is described Antibody class drug is this field conventional antibody class drug, is preferably comprised anti-PD-1 monoclonal antibody, her monoclonal antibody etc..It is described small Molecule antineoplastic medicament is this field conventional medicine, including taxol, 5-Fu pyrimidine etc..

Wherein the targeted tumour of the anti-tumor drug is preferably comprised but is not limited to: lung cancer, liver cancer, oophoroma, palace Neck cancer, cutaneum carcinoma, colon cancer, glioma, bladder cancer, breast cancer, kidney, cancer of the esophagus, gastric cancer, oral squamous cell carcinoma, One of urothelial cell cancer, cancer of pancreas and/or H/N tumors are a variety of.

The so-called anti-tumor drug of the present invention, refers to the drug for inhibiting and/or treating tumour, may include with tumour The delay of related symptoms development and/or the reduction of these severity of symptom, further comprise already present tumour with disease The mitigation of shape and the appearance for preventing other symptoms further include the transfer etc. for reducing or preventing tumour.

Monoclonal antibody of anti-PDGFR α and combinations thereof is when to animal administration including people in the present invention, administration Age and weight of the dosage because of patient, disease traits and seriousness and administration route and it is different, can refer to zoopery knot Fruit and various situations, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 1-1800mg/ days.

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: anti-PDGFR alpha monoclonal antibodies provided by the invention have good biology Activity has affinity more higher than comparison medicine, more preferably inhibits ligand-receptor binding ability than control drug and inhibits thin The ability of born of the same parents' proliferation.The monoclonal antibody can individually or with other anti-tumor drug use in conjunction in immunotherapy of tumors and In diagnosis and screening, treatment tumour, infectious diseases, autoimmune disease and anti-immunity repulsion etc. can be efficiently applied to In the preparation of drug.

Detailed description of the invention

Fig. 1 is the affinity capacity experimental result of anti-PDGFR Alpha antibodies.

Fig. 2 is the ligand-receptor combination inhibition assay result of anti-PDGFR Alpha antibodies.

Fig. 3 is the experimental result that anti-PDGFR Alpha antibodies inhibit U-118MG cell Proliferation.

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.Room temperature as described in the examples be this field routine room temperature, generally 10 ~ 30 DEG C.

Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text In addition explicitly point out, singular "one", " one " and " this " include plural form.

When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment, Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real The existing present invention.

Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc. Molecular Cloning:A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989 And Third Edition, 2001；Ausubel etc., Current Protocols In Molecular Biology, John Wiley & Sons, New York, 1987 And Periodic Updates；The Series Methods In Enzymology, Academic Press, San Diego；Wolffe, Chromatin Structure And Function, Third Edition, Academic Press, San Diego, 1998；Methods In Enzymology, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With Methods In Molecular Biology, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

Embodiment 1 expresses people PDGFR α extracellular region recombinant protein

It synthesizes people PDGFR α-AP fusion (being synthesized by Nanjing Genscript Biotechnology Co., Ltd.), wherein PDGFR α Gene order is that SEQ ID NO:1, AP gene order is SEQ ID NO:2, and two sections of sequence connections are that PDGFR α-AP merges base Cause, design forward and reverse primer are respectively as follows:

5 '-TAACGGGGCCGGCAAGCCGCCACCATGGGGACTTCCCATCCGG-3 ' (SEQ ID NO:3) and 5 '-T CACTACGTATCAGTGGTGGTGGTGGTGGTGGCCTG-3 ' (SEQ ID NO:4) obtains coding PDGFR α-AP for expanding The gene of fusion protein introduces restriction endonuclease sites Ngo MIV and SnaBI in the upstream and downstream of gene respectively, and PCR condition is 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 60s, 35 circulations, 72 DEG C of extension 10min, archaeal dna polymerase are TAKARA Products.

PCR product recycles (QIAquick Gel Extraction Kit is Omega bio-tek Products) after purification through Ago-Gel, is added Restriction enzyme Ngo MIV and SnaBI carry out digestion, and through DNA reclaim reagent, (Omega bio-tek company produces digestion products Product) after purification with to answer macro Chinese continuous heavy rain biotechnology share by Shanghai with the carrier pHLX101(of identical digestion with restriction enzyme limited Company's building, is attached reaction referring to Chinese patent CN201210211812.1).Connection product is transformed into DH5 α large intestine bar Bacterium is coated on the 2YT agar medium containing 50 μ g/ml carbenicillins.The positive colony of acquisition is being contained into 50 μ g/ml It is cultivated in the 2YT fluid nutrient medium of carbenicillin, after Invitrogen company sequence verification, examination is largely extracted with plasmid Agent box (Omega bio-tek Products) extracts positive colony plasmid.

Using the method for polyethyleneimine (Polyethylenimine, PEI) by plasmid DNA transfection into 293F cell (buying from Invitrogen company), after transfecting 6 hours, cell is centrifuged, replaces 293 fresh freestyle Medium culture solution (GIBCO Products) continues shaking table culture.Cell can be observed after 24 and 48 hours, after transfection Start daily to cell count within 3rd day, expression time is usually 7-10 days, the harvest cell training when viable cell density is lower than 50% Nutrient solution supernatant.

The building and expression of the anti-PDGFR α human antibody carrier for expression of eukaryon of embodiment 2

Heavy chain of antibody (SEQ ID NO:5) and light chain (SEQ ID NO:11) gene are had by Nanjing Jin Sirui biotechnology The synthesis of limit company.The DNA sequences encoding of above-mentioned heavy chain and light chain is expanded using PCR reaction, in heavy chain of antibody and light chain Gene both ends introduce restriction enzyme site appropriate, wherein the end of antibody heavy chain gene 5 ' and 3 ' ends introduce respectively Ngo MIV and SnaBI restriction enzyme site, forward primer sequence are as follows:

5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ ID NO:17)

Reverse primer sequences are as follows:

5 '-GCTCTACGTATCATTTACCTGGAGACAGGGAGAGGC-3 ' (SEQ ID NO:18), antibody light chain can Become area's gene 5 ' end and 3 ' ends and introduce Ngo MIV and SnaBI, forward primer sequence respectively are as follows:

5 '-ACCTGCCGGCAAGCCGCCACCATGGGTTGGAGCCTCATCTTGCTCTTC-3 ' (SEQ ID NO:17)

Reverse primer sequences are as follows:

5 '-GCATCTTACGTATTATTAACACTCTCCCCTGTTGAAG -3 ' (SEQ ID NO:19).It is anti-using PCR It answers, condition is 95 DEG C of 10s, 60 DEG C of 10s, 72 DEG C of 10s, and 35 circulations, 72 DEG C of extension 10min, archaeal dna polymerase is TAKARA company Product.After PCR amplification, by PCR product through agarose gel electrophoresis recovery purifying.Restriction enzyme is added in light-chain variable region gene Enzyme Ngo MIV and SnaB I carries out digestion, and heavy chain variable region gene is added restriction enzyme Ngo MIV and SnaBI and carries out enzyme Cut, purified after digestion through DNA purification kit, and with the carrier pHLX101 of identical digestion with restriction enzyme.Connection produces Object is transformed into DH5 α Escherichia coli, is coated on the 2YT agar medium containing 50 μ g/ml carbenicillins.The positive of acquisition It is cloned in the 2YT fluid nutrient medium containing 50 μ g/ml carbenicillins and cultivates, by Invitrogen company sequence verification Afterwards, positive colony plasmid is extracted with a large amount of extraction agent boxes of plasmid.

Using the Neon system of Invitrogen company, by the plasmid DNA transfection of linearisation to Chinese hamster ovary celI, (purchase is certainly Invitrogen company).Chinese hamster ovary celI after transfection is diluted colonized culture, is containing 50 μm of ol methionine imino group generations It is sieved in 94113 culture mediums (IrvineScientific Products) of sulfone (methionine sulfoximine, MSX) Choosing, to obtain monoclonal cell system, is named as K25.

By the monoclonal cell system K25 of acquisition in containing 94113 culture mediums of 50 μm of ol methionine imino groups for sulfone into Row shaking flask culture, expression time are usually 7-14 days, harvest cell culture supernatant when viable cell density is lower than 50%.It utilizes Goat anti-human igg Fd(Meridian Products) and horseradish peroxidase-labeled goat-anti people's constant region of light chain carry out it is double The content of antibody in Sandwich ELISA detection expression supernatant, using untransfected supernatant as negative control, the IgG sterling conduct of people Standard items.Experimental result shows that the destination protein for expressing acquisition can be identified there is human IgG antibody's feature by two antibody, and And the human antibody expression quantity of building is in 200 ~ 500 μ g/ml, expression with higher.Using Protein A affinity chromatography Column isolates and purifies purpose antibody K25 from cells and supernatant.

Control antibodies Olaratumab antibody base according to described in patent (referring to Chinese patent CN101613409B) Cause is synthesized by Nanjing Genscript Biotechnology Co., Ltd., and heavy chain and light chain gene are building up to pHLX101 expression vector respectively In.The Neon system for recycling Invitrogen company, by the plasmid DNA transfection of linearisation to Chinese hamster ovary celI.CHO after transfection Cell is diluted colonized culture, containing 50 μm of ol methionine imino groups for sulfone (methionine sulfoximine, MSX it is screened in 94113 culture mediums (IrvineScientific Products)), to obtain monoclonal cell system.Most Control antibodies Olaratumab is obtained by expression and purifying.

The identification of the anti-PDGFR α human antibody affinity of embodiment 3

By goat anti-human igg Fd(Meridian Products) it is coated with into ELISA Plate (30 μ L of working volume) by 2 μ g/ml, 4 DEG C stand overnight.It is washed 3 times with the conventional phosphoric acid salt buffer (PBST) of the Tween 20 containing 0.05%.With 5% degreasing ox Milk room temperature is closed 1 hour.It is washed 3 times with PBST, antibody K25 and Olaratumab that embodiment 2 prepares is configured to 6 respectively μ g/ml concentration, and 3 times of gradient dilutions are carried out, totally 8 gradients, every hole are added 30 μ l, are stored at room temperature 1 hour.It is washed 3 times with PBST, PDGFR α-AP cell expression the supernatant prepared in 30 μ l embodiments 1 is added, is stored at room temperature 1 hour.It is washed 6 times, is added with PBST PNPP colour developing, is read at 405nm with microplate reader.The affinity capacity experimental result of anti-PDGFR Alpha antibodies is as shown in Figure 1, from knot Fruit, which can be seen that the monoclonal antibody K25 that present invention screening obtains, has apparent affinity, affinity to PDGFR α receptor It is significantly improved than control antibodies Olaratumab.

4 ligands of embodiment-antibody, which combines, inhibits Function Identification

By PDGF-AA(Peprotech Products) it is coated with into ELISA Plate (30 μ L of working volume) by 1 μ g/ml, 4 DEG C It stands overnight.It is washed 3 times with PBST.It is closed 1 hour with the PBS room temperature of the BSA containing 1%.It is washed 3 times with PBST.It prepared by embodiment 2 The antibody K25 and Olaratumab of acquisition are configured to 5 μ g/ml concentration, and carry out 3 times of gradient dilutions, totally 8 gradients.It again will be real PDGFR α-AP cell expression the supernatant being prepared in example 1 is applied, is diluted with PBST by 1:10, is then pressed with the antibody diluted 1:1 mixing, is stored at room temperature 1.5 hours, is then added in the ELISA Plate closed, be stored at room temperature 1 hour.It is washed 6 times with PBST.With PNPP colour developing, is read at 405nm with microplate reader.Ligand-receptor combination inhibition assay result such as Fig. 2 institute of anti-PDGFR Alpha antibodies Show, from the results, it was seen that the present invention, which screens gained monoclonal antibody K25, can effectively inhibit PDGFR α receptor and its ligand The combination of PDGF-AA, inhibiting effect more preferably than control antibodies Olaratumab, with the increase of MAb concentration, more Early reach minimum.

Inhibition Function Identification of 5 antibody of embodiment for vascular endothelial cell proliferation

U-118MG cell (ATCC, HTB-15) is cultivated with the DMEM culture medium added with 10% FBS, by every hole 4000 A cell/100 μ l are inoculated into overnight incubation in 96 well culture plates.Supernatant carefully is sucked, the DMEM with 200 μ l without FBS is cultivated Base is washed 2 times, and the DMEM culture medium that 100 μ l are free of FBS is added in every hole, carries out hungry culture 2 days to cell.Carefully suck Clearly.Antibody K25 and Olaratumab that embodiment 2 prepares are configured to 20 μ g/ml concentration, and carry out 3 times of gradient dilutions, Totally 8 gradients are added in 96 well culture plates by every 75 μ l of hole, tissue culture plate are placed in CO₂In incubator, 37 DEG C, 5% CO₂, it is incubated for 30min.PDGF-AA is configured to 400ng/ml, is added in 96 well culture plates by every 75 μ l of hole, is i.e. PDGF-AA is whole Concentration is 200ng/ml, and tissue culture plate is placed in CO₂In incubator, 37 DEG C, 5% CO₂, cultivate 4 days.15 μ l are added in every hole, Cell culture is placed in CO by the MTT of 5mg/ml₂In incubator, 37 DEG C, 5% CO₂, it is incubated for 3 h.With microplate reader at 565nm Reading.Anti- PDGFR Alpha antibodies inhibit the experimental result of U-118MG cell Proliferation as shown in figure 3, can be seen that from result, and the present invention sieves Choosing gained monoclonal antibody K25 has the ability for significantly inhibiting cell Proliferation to U-118MG cell, and rejection ability is more anti-than control Body Olaratumab is stronger, with the increase of antibody concentration, can more effectively inhibit U-118MG cell Proliferation.

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 table

<110>Shanghai Fu Honghan continuous heavy rain Biotechnology Ltd.

Shanghai Fu Honghan continuous heavy rain Biology Pharmacy Co., Ltd

<120>a kind of monoclonal antibody and its preparation method and application of anti-PDGFR α

<141> 2018-11-28

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1560

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggggactt cccatccggc gttcctggtc ttaggctgtc ttctcacagg gctgagccta 60

atcctctgcc agctttcatt accctctatc cttccaaatg aaaatgaaaa ggttgtgcag 120

ctgaattcat ccttttctct gagatgcttt ggggagagtg aagtgagctg gcagtacccc 180

atgtctgaag aagagagctc cgatgtggaa atcagaaatg aagaaaacaa cagcggcctt 240

tttgtgacgg tcttggaagt gagcagtgcc tcggcggccc acacagggtt gtacacttgc 300

tattacaacc acactcagac agaagagaat gagcttgaag gcaggcacat ttacatctat 360

gtgccagacc cagatgtagc ctttgtacct ctaggaatga cggattattt agtcatcgtg 420

gaggatgatg attctgccat tataccttgt cgcacaactg atcccgagac tcctgtaacc 480

ttacacaaca gtgagggggt ggtacctgcc tcctacgaca gcagacaggg ctttaatggg 540

accttcactg tagggcccta tatctgtgag gccaccgtca aaggaaagaa gttccagacc 600

atcccattta atgtttatgc tttaaaagca acatcagagc tggatctaga aatggaagct 660

cttaaaaccg tgtataagtc aggggaaacg attgtggtca cctgtgctgt ttttaacaat 720

gaggtggttg accttcaatg gacttaccct ggagaagtga aaggcaaagg catcacaatg 780

ctggaagaaa tcaaagtccc atccatcaaa ttggtgtaca ctttgacggt ccccgaggcc 840

acggtgaaag acagtggaga ttacgaatgt gctgcccgcc aggctaccag ggaggtcaaa 900

gaaatgaaga aagtcactat ttctgtccat gagaaaggtt tcattgaaat caaacccacc 960

ttcagccagt tggaagctgt caacctgcat gaagtcaaac attttgttgt agaggtgcgg 1020

gcctacccac ctcccaggat atcctggctg aaaaacaatc tgactctgat tgaaaatctc 1080

actgagatca ccactgatgt ggaaaagatt caggaaataa ggtatcgaag caaattaaag 1140

ctgatccgtg ctaaggaaga agacagtggc cattatacta ttgtagctca aaatgaagat 1200

gctgtgaaga gctatacttt tgaactgtta actcaagttc cttcatccat tctggacttg 1260

gtcgatgatc accatggctc aactggggga cagacggtga ggtgcacagc tgaaggcacg 1320

ccgcttcctg atattgagtg gatgatatgc aaagatatta agaaatgtaa taatgaaact 1380

tcctggacta ttttggccaa caatgtctca aacatcatca cggagatcca ctcccgagac 1440

aggagtaccg tggagggccg tgtgactttc gccaaagtgg aggagaccat cgccgtgcga 1500

tgcctggcta agaatctcct tggagctgag aaccgagagc tgaagctggt ggctcccacc 1560

<210> 2

<211> 1521

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

cgaattcctt acgtaagatc ttccggaatc atcccagttg aggaggagaa cccggacttc 60

tggaaccgcg aggcagccga ggccctgggt gccgccaaga agctgcagcc tgcacagaca 120

gccgccaaga acctcatcat cttcctgggc gatgggatgg gggtgtctac ggtgacagct 180

gccaggatcc taaaagggca gaagaaggac aaactggggc ctgagatacc cctggccatg 240

gaccgcttcc catatgtggc tctgtccaag acatacaatg tagacaaaca tgtgccagac 300

agtggagcca cagccacggc ctacctgtgc ggggtcaagg gcaacttcca gaccattggc 360

ttgagtgcag ccgcccgctt taaccagtgc aacacgacac gcggcaacga ggtcatctcc 420

gtgatgaatc gggccaagaa agcagggaag tcagtgggag tggtaaccac cacacgagtg 480

cagcacgcct cgccagccgg cacctacgcc cacacggtga accgcaactg gtactcggac 540

gccgacgtgc ctgcctcggc ccgccaggag gggtgccagg acatcgctac gcagctcatc 600

tccaacatgg acattgacgt gatcctaggt ggaggccgaa agtacatgtt tcccatggga 660

accccagacc ctgagtaccc agatgactac agccaaggtg ggaccaggct ggacgggaag 720

aatctggtgc aggaatggct ggcgaagcgc cagggtgccc ggtatgtgtg gaaccgcact 780

gagctcatgc aggcttccct ggacccgtct gtgacccatc tcatgggtct ctttgagcct 840

ggagacatga aatacgagat ccaccgagac tccacactgg acccctccct gatggagatg 900

acagaggctg ccctgcgcct gctgagcagg aacccccgcg gcttcttcct cttcgtggag 960

ggtggtcgca tcgaccatgg tcatcatgaa agcagggctt accgggcact gactgagacg 1020

atcatgttcg acgacgccat tgagagggcg ggccagctca ccagcgagga ggacacgctg 1080

agcctcgtca ctgccgacca ctcccacgtc ttctccttcg gaggctaccc cctgcgaggg 1140

agctccatct tcgggctggc ccctggcaag gcccgggaca ggaaggccta cacggtcctc 1200

ctatacggaa acggtccagg ctatgtgctc aaggacggcg cccggccgga tgttaccgag 1260

agcgagagcg ggagccccga gtatcggcag cagtcagcag tgcccctgga cgaagagacc 1320

cacgcaggcg aggacgtggc ggtgttcgcg cgcggcccgc aggcgcacct ggttcacggc 1380

gtgcaggagc agaccttcat agcgcacgtc atggccttcg ccgcctgcct ggagccctac 1440

accgcctgcg acctggcgcc ccccgccggc accaccgacg ccgcgcaccc gggttcaggc 1500

caccaccacc accaccactg a 1521

<210> 3

<211> 43

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

taacggggcc ggcaagccgc caccatgggg acttcccatc cgg 43

<210> 4

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tcactacgta tcagtggtgg tggtggtggt ggcctg 36

<210> 5

<211> 1404

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt ccactccgag 60

gtgcagctgg tgcagtctgg ggctgaggtg aagaagcctg gggcctcagt gaaggtttcc 120

tgcaaggcat ctggatacac cttcaccagc tactatatgc actgggtgcg acaggcccct 180

ggacaagggc ttgagtggat gggaataatc aaccctagtg gtggtagcac aagctacgca 240

cagaagttcc agggcagagt caccatgacc agggacacgt ccacgagcac agtctacatg 300

gagctgagca gcctgagatc tggcgacacg gccgtgtatt actgtgcgag agcccggagt 360

ggtcatagtt ttgactactg gggccaggga accctggtca ccgtctcaag cgctagcacc 420

aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgaccgtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc cctgctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660

aacgtgaatc acaagcccag caacaccaag gtggacaaga aagcagagcc caaatcttgt 720

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accatcagtc 780

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 1320

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380

ctctccctgt ctccaggtaa atga 1404

<210> 6

<211> 345

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggagctga gcagcctgag atctggcgac acggccgtgt attactgtgc gagagcccgg 300

agtggtcata gttttgacta ctggggccag ggaaccctgg tcacc 345

<210> 7

<211> 115

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 7

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

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

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Gly Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ala Arg Ser Gly His Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr

115

<210> 8

<211> 10

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 8

Gly Tyr Thr Phe Thr Ser Tyr Tyr Met His

1 5 10

<210> 9

<211> 10

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 9

Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser

1 5 10

<210> 10

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 10

Ala Arg Ser Gly His Ser Phe Asp Tyr

1 5

<210> 11

<211> 702

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

atgggttgga gcctcatctt gctcttcctt gtcgctgttg ctacgcgtgt ccactccgac 60

atccagatga cccagtctcc atcctccctg tctgcatctg taggagacag agtcaccatc 120

acttgccggg caagtcagag cattagcagc tatttaaatt ggtatcagca gaaaccaggg 180

aaagccccta agctcctgat ctatgctgca tccagtttgc aaagtggggt cccatcaagg 240

ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagtct gcaacctgaa 300

gattttgcaa cttactactg tcaacagagt tacagtaccc ctccgacgtt cggccaaggg 360

accaaggtgg aaatcaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 420

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480

agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540

agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600

agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660

agctcgcccg tcacaaagag cttcaacagg ggagagtgtt aa 702

<210> 12

<211> 324

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 12

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct 240

gaagattttg caacttacta ctgtcaacag agttacagta cccctccgac gttcggccaa 300

gggaccaagg tggaaatcaa acga 324

<210> 13

<211> 108

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 13

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105

<210> 14

<211> 11

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 14

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 15

<211> 7

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 15

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 16

<211> 9

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 16

Gln Gln Ser Tyr Ser Thr Pro Pro Thr

1 5

<210> 17

<211> 48

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 17

acctgccggc aagccgccac catgggttgg agcctcatct tgctcttc 48

<210> 18

<211> 36

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 18

gctctacgta tcatttacct ggagacaggg agaggc 36

<210> 19

<211> 37

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 19

gcatcttacg tattattaac actctcccct gttgaag 37

Claims (10)

1. a kind of monoclonal antibody of anti-PDGFR α, which is characterized in that the monoclonal antibody includes heavy chain variable region and light chain can Become area, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:7 in sequence table, the ammonia of the light chain variable region Base acid sequence is as shown in SEQ ID NO:13 in sequence table.

2. the monoclonal antibody of anti-PDGFR α as described in claim 1, which is characterized in that the heavy chain variable region of the monoclonal antibody Further include 3 CDR regions, wherein CDR1 amino acid sequence is as shown in SEQ ID NO:8, CDR2 amino acid sequence such as SEQ ID NO: Shown in 9, CDR3 amino acid sequence is as shown in SEQ ID NO:10.

3. the monoclonal antibody of anti-PDGFR α as described in claim 1, which is characterized in that the light chain variable region of the monoclonal antibody It further include 3 areas CDR ', wherein CDR1 ' amino acid sequence is as shown in SEQ ID NO:14, CDR2 ' amino acid sequence such as SEQ ID Shown in NO:15, CDR3 ' amino acid sequence is as shown in SEQ ID NO:16.

4. a kind of nucleic acid molecules for encoding the monoclonal antibody of anti-PDGFR α as described in claim 1, which is characterized in that heavy chain Coding nucleotide sequence is as shown in SEQ ID NO:5, and the coding nucleotide sequence of light chain is as shown in SEQ ID NO:11.

5. nucleic acid molecule as claimed in claim 4, which is characterized in that monoclonal antibody heavy described in coding claim 1 The nucleotide sequence of variable region encodes the nucleotide sequence such as SEQ ID NO of its light chain variable region as shown in SEQ ID NO:6: Shown in 12.

6. a kind of expression vector, which is characterized in that the expression vector contains the list for encoding anti-PDGFR α as described in claim 1 The nucleic acid molecule of clonal antibody.

7. a kind of host cell, which is characterized in that the host cell contains expression vector as claimed in claim 6.

8. a kind of preparation method of the monoclonal antibody of anti-PDGFR α as described in claim 1, which is characterized in that the preparation side Method the following steps are included:

A) under expression condition, host cell as claimed in claim 7 is supported, expresses the monoclonal antibody of anti-PDGFR α；

B) monoclonal antibody of separation and the anti-PDGFR α of purification step a) gained.

9. a kind of composition, which is characterized in that the composition includes the list of a effective amount of anti-PDGFR α as described in claim 1 Clonal antibody and pharmaceutically acceptable carrier.

10. the monoclonal antibody of anti-PDGFR α as described in claim 1 is preparing the application in anti-glioma drug.