CN107987163A

CN107987163A - Monoclonal antibody 9A and its application

Info

Publication number: CN107987163A
Application number: CN201711261508.7A
Authority: CN
Inventors: 高婵
Original assignee: Hangzhou Sumgen Biotech Co Ltd
Current assignee: Hangzhou Sumgen Biotech Co Ltd
Priority date: 2017-12-04
Filing date: 2017-12-04
Publication date: 2018-05-04
Anticipated expiration: 2037-12-04
Also published as: CN107987163B

Abstract

The invention discloses a kind of separated binding molecule, which is the monoclonal antibody or its antigen-binding fragment for AXL albumen.Experiment in vitro proves that the monoclonal antibody can be combined with AXL protein-specifics, there is stronger affine activity, and then can prevent and prevent disease caused by AXL protein overexpressions.On the one hand the achievement in research of the present invention provides new method for clinical diagnosis, being on the other hand overexpressed relevant disease with AXL for clinical treatment tumour etc. provides drug candidate.

Description

Monoclonal antibody 9A and its application

Technical field

The invention belongs to cellular immunology, biology field, is related to a kind of monoclonal antibody for AXL, in addition, The invention further relates to the preparation method and purposes of the antibody.

Background technology

Cancer has become a main cause for triggering human death.One data shows, the hair of global tumour Sick rate and the death rate are in the trend risen year by year：The morbidity number of global cancer in 2008 reaches 12,700,000, it is contemplated that will to the year two thousand thirty Reach 20,300,000；The death toll as caused by cancer in 2008 is 7,600,000, it is contemplated that will increase to 13,200,000 to the year two thousand thirty.2015 China Cancer statistics is shown, according to the pathogenesis of cancer number of 2000~2011 years and the data trend analysis of death toll, knot Fruit shows that estimated 2015 China's cancer neopathy number of cases are 429.2 ten thousand, and death toll is 281.4 ten thousand.As cancer is sent out The increase of sick rate and the death rate, cancer have become the first cause for promoting China's resident's disease death, become very important The problem of publilc health.

Chemotherapy has played important function as one of conventional cancer therapy means in treatment of cancer, but since it is special The opposite sex is poor, and clinic has larger toxic side effect, therefore medical staff and researcher are seeking more preferable therapeutic strategy.Molecule Targeted therapy has orientation, the advantage of positioning, it is possible to reduce patient medication dosage, it is secondary to reduce poison compared to traditional chemotherapy Reaction, improves therapeutic effect, has become the research hotspot of global field of cancer treatment.The research of last decade molecular targeted agents Quickly grow, substantial amounts of biological targeting cancer therapy drug obtains FDA's approval listing.Monoclonal antibody drug With its high specificity, safe, toxicity is low, clinical efficacy is good the advantages that become neoplasm targeted therapy priority research areas it One.

Receptor tyrosine kinase (receptor tyrosine kinase, RTK) is the transmembrane receptor of cell surface, is divided into Extracellular fragment, transmembrane region and Intracellular domain, wherein Intracellular domain contain kinase activity, are sent out in the signal transduction of Normal and malignant cells Wave important effect.1991, a receptor tyrosine kinase subfamily is found that in chronic myelocytic leukemia --- TAM Family.Into including Tyro-3, Axl and Mer, their ligand is 6 (growth of growth retardation specific gene for TAM families Arrest-specific 6, Gas6) encoded protein molecular.In recent years, substantial amounts of studies have shown that AXL is a variety of pernicious swollen All high expression in knurl, including lung cancer, breast cancer, colon cancer, stomach cancer, liver cancer and oophoroma etc..AXL can promote cells survival, Migration, invasion and attack and transfer, strengthen chemosensitivity.

Many research reports show in recent years, during targeting with chemotherapeutic drug therapy cancer patient, the super table of AXL Up to being important mechanisms causing targeted therapy and classic chemotherapy drug resistance to produce.At present in many tumours, such as lung Cancer, breast cancer, cancer of the esophagus, patients with gastrointestinal stromal tumors and acute myeloblastic leukemia etc., it was observed that being produced due to this mechanism Raw drug resistance.Show come more evidences, AXL kinases participates in the drug resistance as caused by different mechanisms in kinds of tumor cells Property, the overexpression of AXL kinases has become cancer patient and a major reason of drug resistance occurs.By to axl receptor junket ammonia The suppression of acid kinase can reduce rush survival-signal, the invasive ability of blocks tumor of tumour cell, increase targeted drug treatment With chemosensitivity degree.The presence of these advantages causes target spot strategy reasonables of the AXL as treatment of cancer.

AXL targeted drugs under study for action mainly include small molecule tyrosine kinase inhibitors and anti-AXL monoclonals at present Antibody etc..

At present, it has been reported that a variety of micromolecular inhibitors for AXL paths, some of them have come into clinical examination Test the stage, such as R428, s49076, LY2801653 and MP-470, SKI-606, MGCD265, ASP2215, XL184 etc.. R428 (BGB324) is first using AXL as therapy target and earliest into the kinase inhibitor of clinical experimental stage.R428 can To suppress the phosphorylation of AXL and Akt, downstream signaling pathway is blocked.R428 can dose-dependently suppress high invasion and attack breast cancer The growth of cell MDA-MB-231 and 4T1.In 4T1 breast cancer mouse models, after cutting off the mammary gland of mouse, treated through R428, Metastasis of cancer is can inhibit, extends survival time of mice；In addition, R428 collaboration cis-platinums can suppress hepatoma Metastasis；R428, which has had been enter into, to be faced 2 phase conceptual phases of bed.And the research for targeting the antibody of AXL just starts to walk, the stage of preclinical study is currently under.

In conclusion AXL is the potential target spot for the treatment of of cancer and medicament research and development.Object of this investigation is to screen targeting The functional monoclonal antibody of AXL.

The content of the invention

It is an object of the invention to provide a kind of binding molecule for AXL containing unique complementary determining region, the knot Close molecule has significant affinity interaction to AXL albumen.

To achieve these goals, present invention employs following technical solution：

The present invention provides a kind of separated binding molecule, the binding molecule includes：

(1)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 1:Heavy chain CDR2, SEQ ID NO shown in 2:3 institutes The heavy chain CDR3 shown；And/or

(2)SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 4:Light chain CDR2, SEQ ID NO shown in 5:6 institutes The light chain CDR3 shown.

As one aspect of the present invention, binding molecule of the invention includes：

(1) heavy chain variable region, the heavy chain variable region have SEQ ID NO:Amino acid sequence shown in 7；And/or

(2) light chain variable region, the light chain variable region have SEQ ID NO:Amino acid sequence shown in 8.

Further, the binding molecule includes：

(1) heavy chain, the heavy chain have SEQ ID NO:Amino acid sequence shown in 13；And/or

(2) light chain, the light chain have SEQ ID NO:Amino acid sequence shown in 11.

The binding molecule of the present invention can be complete immunoglobulin molecules, can also be antigen-binding fragment, including But it is not limited to Fab, F (ab '), F (ab ') 2, Fv, dAb, Fd, complementary determining region (CDR) fragment, single-chain antibody (scFv), divalence Single-chain antibody, single chain variable fragment phage antibody, double specific duplex antibody, three chain antibodies, four chain antibodies and at least containing being enough to assign With (more) peptide or its fragment of the fragment of AXL binding domain-immunoglobulins.

The binding molecule of the present invention can also specifically bind one or more fragments of AXL albumen.For treatment and/or in advance For the method for the anti-disease induced by AXL protein overexpressions, the binding molecule can preferably specifically bind cell surface egg White matter.

As another aspect of the present invention, the function that binding molecule of the invention further includes foregoing binding molecule becomes Body.If become physical efficiency and parent binding molecule competition specific binding AXL or its protein fragments, then it is assumed that the Variant molecules are these The functional variety of invention binding molecule.In other words, the functional variety remains to combine AXL albumen or its protein fragments.

Functional variety include but not limited to primary structural sequence it is substantially similar but containing for example in parent binding molecule The derivative of undiscovered external or internal chemistry and/or biochemical modification.This modification includes acetylation, phthalein, nucleosides The sour either covalent attachment of the covalent attachment of nucleotide derivative, lipid or lipid derivate, crosslinking, disulfide formation, sugar Base, hydroxylating, methylate, aoxidize, the processing of Pegylation, proteolysis, phosphorylation etc..In other words, parental generation, which combines, divides Modification in the amino acid and/or nucleotide sequence of son is not significantly affected or changed by described nucleotide sequence coded or contain There is the binding characteristic of the binding molecule of the amino acid sequence, i.e., described binding molecule remains to identify and combines its target position.

The functional variety can have conserved sequence modification, including 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, addition and missing.These modifications can To be imported by the known standard technique in this area, such as the mutagenesis of directed mutagenesis and random PCR mediation, and can include natural And alpha-non-natural amino acid.

Conserved amino acid substitution includes wherein amino acid residue by another amino with similar structure or chemical property The substitution of sour residue substitutions.The family of amino acid residue with similar side chain oneself through limiting in the art.These families wrap Include the amino acid (such as lysine, arginine, histidine) with basic side chain, acidic side chains (such as aspartic acid, Glutamic acid), without charge polarity side chain amino acid (such as asparagus fern phthalein amine, paddy ammonia phthalein amine, serine, threonine, tyrosine, half skin Propylhomoserin, tryptophan), nonpolar side chains (such as glycine, alanine, valine, leucine, isoleucine, dried meat ammonia Acid, phenylalanine, methionine), branched side chains (such as threonine, valine, isoleucine) and aromatic side chain Amino acid (such as tyrosine, phenylalanine, tryptophan).It will be appreciated that can also use except above-mentioned family it Outer other amino acid residue families mode classifications.In addition, variation can have a non-conservative 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, for example, amino acid by Another radical amino acid replacement with different structure or chemical property.Similar small variation may also comprise amino acid deletions or Person is inserted into, or both.It is can be found that using computer program well known in the art and determines which amino acid residue can be by Substitution, insertion or the guidance lacked without eliminating immunologic competence.

In addition, functional variety can include truncation of the amino acid sequence at amino terminal either carboxyl terminal or this both ends Body.The functional variety of the present invention can be affine with identical or different, higher or lower combination compared with parent binding molecule Property, but remain to combine AXL albumen or its fragment.

Also comprising the modification to hypervariable region, hypervariable region includes the amino acid residue from CDR and comes from the functional variety The amino acid residue of hypervariable loop.Functional variety within the scope of the present invention has at least about with parent binding molecule described herein 50% to about 99%, be preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, even more Preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, especially at least about 95% to About 99%, and the especially at least amino acid sequence homology of about 97% to about 99%.

Computerized algorithm well known by persons skilled in the art such as Gap or Bestfit can be used for most preferably arrays of amino acid Sequence is to be contrasted and clearly similar or identical amino acid residue.Functional variety can be known by using this area Common molecular biology method changes parent binding molecule or one part and obtains, the described method includes but be not limited to fallibility Mutagenesis, direct mutagenesis and the heavy chain and/or light chain reorganization method that PCR, oligonucleotides instruct.

The functional variety of the present invention has affine activity for AXL.The affine activity can compared with parent binding molecule With identical or higher or lower.Hereafter, when using term " binding molecule ", it is also covered by the function of the binding molecule and becomes Body.

Present invention also offers the nucleic acid molecules of foregoing binding molecule.

The foregoing binding molecule of nucleic acid molecule encoding.

Further, the nucleic acid molecules include SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:At least one nucleotide sequence in nucleotide sequence shown in 12, or including with SEQ ID NO:9、SEQ ID NO:10、 SEQ ID NO:14、SEQ ID NO:At least one nucleotide sequence in nucleotide sequence shown in 12 is at least more than 80% Nucleotide sequence.

Preferably, the nucleic acid molecules include SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:At least one nucleotide sequence in nucleotide sequence shown in 12.

Specific, concrete, SEQ ID NO:The corresponding nucleotide sequence of amino acid sequence shown in 7 is SEQ ID NO:9；SEQ ID NO:The corresponding nucleotide sequence of amino acid sequence shown in 8 is SEQ ID NO:10；SEQ ID NO:Amino shown in 13 The corresponding nucleotide sequence of acid sequence is SEQ ID NO:14；SEQ ID NO:The corresponding nucleotide of amino acid sequence shown in 11 Sequence is SEQ ID NO:12.

It will be appreciated by persons skilled in the art that the functional variety of these nucleic acid molecules is also the part of the present invention.Function becomes Body is such nucleotide sequence, by using standard genetic code can by its directly translation with provide with from parent nucleic acid molecules The identical amino acid sequence of the sequence of middle translation.

Once obtain related sequence information, it is possible to obtain related sequence in large quantity with recombination method.This is usual It is to be cloned into carrier, then is transferred to cell, it is then isolated related from the host cell after propagation by conventional method Sequence.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.It is in general, logical After first synthesizing multiple small fragments, the very long fragment of sequence can be obtained by being then attached again.

At present, it is already possible to completely by chemical synthesis come obtain the binding molecule of the coding present invention (or its fragment, or its Derivative) nucleotide sequence.Then the nucleotide sequence can be introduced to various existing DNA moleculars as known in the art (or such as Carrier) and cell in.In addition, it can will be also mutated by chemical synthesis in the sequence of binding molecule incorporated in the present invention.

Present invention also offers a kind of expression vector for including foregoing nucleic acid molecules, except foregoing nucleic acid Outside molecule, expression vector further includes the expression regulation sequence being operatively connected with the sequence of nucleic acid molecules.

These expression vectors can be used for converting appropriate host cell, allow it to marking protein.

Present invention also offers a kind of host containing foregoing nucleic acid molecules or foregoing expression vector is thin Born of the same parents.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；It is or high Deng eukaryotic, such as mammalian cell.Representative example has:Escherichia coli, streptomyces；The bacterium of salmonella typhimurium Cell：Fungal cell's such as yeast；Plant cell；The insect cell of drosophila S2 or Sf9；CHO, COS, 293 cell or Bowes are black Zooblast of plain oncocyte etc..

In specific embodiments of the present invention, the host cell is CHO.

Converted with recombinant DNA, transfection host cell can be carried out with routine techniques well known to those skilled in the art.Some are adopted Conversion, transfection method include but is not limited to：Calcium phosphate precipitation, conventional mechanical methods for example microinjection, electroporation, Liposome packaging etc..

The transformant of acquisition can use conventional method culture, with the binding molecule of the expression present invention.According to host used Cell, culture medium used may be selected from various conventional mediums in culture.Trained under conditions of suitable for host cell growth Support.After host cell growth is to appropriate cell density, is induced and selected with suitable method (such as temperature transition or chemical induction) The promoter selected, a period of time is further cultured for by cell.

The binding molecule of the present invention is preferably produced using mammalian cell, and mammalian cell usually requires Cultivated in culture medium containing serum.After needing the adaptation process to cell progress serum-free, cell can be allowed in serum-free Normally grown in culture medium.

The present invention provides a kind of pharmaceutical composition including foregoing binding molecule, detection product or diagnosis production Product.

Further, described pharmaceutical composition further includes pharmaceutically acceptable carrier.

Term " pharmaceutically acceptable " used herein refer to when biomolecule ontology and composition suitably give animal or During people, unfavorable, allergy or other adverse reactions that they will not be produced." pharmaceutically acceptable carrier " used herein should When with the present invention binding molecule it is compatible, can the blended effect without composition is greatly lowered under normal conditions Fruit.

Can be carbohydrate as the specific example of some of pharmaceutically acceptable carrier or its component materials, such as lactose, Portugal Grape sugar and sucrose；Starch, such as cornstarch and potato starch；Cellulose and its derivates, as sodium carboxymethylcellulose, ethyl are fine Dimension element and methylcellulose；Tragacanth powder；Malt；Gelatin；Talcum；Kollag, such as stearic acid and magnesium stearate；Sulphur Sour calcium；Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil；Polyalcohol, such as propane diols, sweet Oil, D-sorbite, mannitol and polyethylene glycol；Alginic acid；Emulsifying agent, such as Tween；Wetting agent, such as NaLS； Toner；Flavor enhancement；Tablet agent, stabilizer；Antioxidant；Preservative；Apirogen water；Isotonic salting liquid；And phosphate buffer Deng.

Various formulations can be made as needed for the composition of the present invention, and can be by doctor according to patient category, age, weight Substantially the factor such as disease condition, administering mode determines that the dosage beneficial to patient is administered.Administering mode can for example be adopted With injection or other therapeutic modalities.

Present invention also offers a kind of immunoconjugates, the immunoconjugates include at least one combination described herein point Son and the molecule for further including the detectable part/material of at least one label.

The mark of the immunoconjugates of the present invention can be therapeutic agent, but they can also be detectable part/thing Matter.Mark suitable for treating and/or preventing can be toxin either its funtion part, antibiotic, enzyme, enhancing phagocytosis or Other binding molecules of immunostimulation.

Being used for immunoconjugates diagnosticability comprising detectable substance such as evaluation object, whether oneself is through suffering from AXL mistakes The disease of induced expression or a part of generation or progress for monitoring AXL and being overexpressed the disease induced as clinical trial program The effect of for example to determine designated treatment scheme.However, they can be used for other detections and/or analysis and/or diagnosis mesh 's.Detectable part/material includes but not limited to enzyme, prothetic group, fluorescent material, luminescent material, bioluminescent material, radioactivity Material, positron emitting metal and on-radiation paramagnetic metal ion.

In order to which the mark for detecting and/or analyzing and/or diagnostic purpose is used to mark binding molecule is specific dependent on using Detection/analysis/diagnostic techniques and/or method such as immunohistochemical staining (tissue) sample, flow cytometry, laser Scan cytometry detection, fluorescence immunoassay, enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), life Thing measure (such as phagocytosis measure), Western blotting application etc..For detection/analysis/diagnostic techniques known in the art And/or method suitably mark is well known to those skilled in the art.

Produced except through direct or indirect (such as passing through connector) is conjugated and chemistry outside immunoconjugates, it is described immune Conjugate can be produced as fusion protein, and the fusion protein includes the binding molecule of the present invention and suitable mark.Melt Hop protein can be produced by means known in the art, such as by building nucleic acid molecules and then expressing the nucleic acid molecules And recombinate and produce, the nucleic acid molecules include the nucleotide sequence and coding appropriate flags of in-frame encoding binding molecules Nucleotide sequence.

Further, the detection product or the diagnostic products include but not limited to detection reagent, kit, chip or examination Paper.Every the detection product for being capable of detecting when AXL protein expression levels or diagnostic products including binding molecule noted earlier are equal It is included within the scope of the present invention.

Present invention also offers a kind of method of the detection AXL protein levels of non-diagnostic purpose, it is characterised in that the side Method includes the following steps：

(1) sample containing AXL is extracted；

(2) sample that step (1) obtains is contacted with foregoing binding molecule；

(3) immune response of sample and binding molecule is detected.

The present invention also provides given it is a kind of using foregoing host cell produce the present invention binding molecule method, The described method includes cultivate foregoing host cell under suitable conditions and recycle the binding molecule.

Present invention also offers a kind of binding molecule produced by the above method.The binding molecule is antibody or it is anti- Former binding fragment.

Present invention also offers application of the foregoing binding molecule in detection product or diagnostic products are prepared；It is described Detection product is the product for detecting AXL expressing quantities, and the diagnostic products are disease of the diagnosis using AXL protein overexpressions as characterization The product of disease；Preferably, the disease using AXL protein overexpressions as characterization includes tumour, fibrosis or hepatic sclerosis.

The detection product or diagnostic products include foregoing binding molecule；The detection product includes but not limited to Detection reagent, kit, chip or test paper.It is every to be capable of detecting when AXL protein expression water including foregoing binding molecule Flat detection product or diagnostic products are included within the scope of the present invention.

Present invention also offers foregoing binding molecule to prepare prevention or treat what is induced by AXL protein overexpressions Application in the pharmaceutical preparation of disease；Preferably, the disease induced by AXL protein overexpressions includes tumour, or tumour is resistance to Pharmacological property.

The tumour includes but not limited to, breast cancer, colon cancer, prostate cancer, lung cancer, stomach cancer, oophoroma, endometrium Cancer, kidney, hepatocellular carcinoma, thyroid cancer, the cancer of the esophagus, chronic myelocytic leukemia (CML), acute myelocytic leukemia (AML), Osteosarcoma, melanoma, Head and neck squamous cell carcinoma.

The drug resistance of tumor induced by AXL protein overexpressions includes lung cancer, breast cancer, cancer of the esophagus, Gastrointestinal Stromal The drug resistance produced in the tumour such as cytoma and acute myeloblastic leukemia due to AXL high expression.

The antibody of disclosure of the invention can include one or more glycosylation sites in heavy chain and light chain variable region, such as originally Known in field, one or more glycosylation sites present in variable region can cause the antibody immunogenicity of enhancing, Or change the pharmacokinetics of antibody due to changing antigen binding.

The antibody of the present invention can be designed as comprising changing in Fc regions, typically change antibody one or more Functional characteristic, such as serum half-life, the cytotoxicity that complement combines, Fc acceptors combine, and/or antigen relies on.It is in addition, of the invention Antibody can be modified by sulphation and (e.g., one or more chemical groups can be connected to antibody), or be modified to change it Glycosylation, so as to change the one or more functions characteristic of antibody again.

Another modification that the antibody of the present invention can be designed is Pegylation.Antibody can by Pegylation from And for example, biology (such as serum) half-life period of increase antibody.In order to make antibody Pegylation, the antibody or its fragment are usual Under conditions of being suitable for one or more polyethylene glycol (PEG) group and being connected to the antibody or antibody fragment, reacted with PEG, Such as the active ester or aldehyde derivatives of polyethylene glycol.Preferably, which is (or similar by the PEG molecules with activity Reactive water-soluble polymer) carry out acylation reaction or alkylated reaction and realize.

The binding molecule of the present invention can be used alone or be applied in the mixture of the binding molecule comprising the present invention. In other words, the binding molecule can with combination application, such as include two or more kind the present invention binding molecule, its change The pharmaceutical composition of body or fragment.For example, it can be combined in a therapeutic scheme with different but complementary activity binding molecules In to reach desired prevention, treatment or diagnostic effect, but or can also will be with identical active binding molecule combination In a therapeutic scheme with reach it is desired prevention, treatment or diagnostic effect.

The binding molecule of the present invention can also be answered with other with identical or complementary function Drug combination, joint Effect can be the sum of binding molecule and other drugs function, can also be far longer than binding molecule and other drugs function The sum of, such case shows to generate synergistic effect between the binding molecule and other drugs.

Term " monoclonal antibody " used herein refers to the antibody obtained from a kind of substantially uniform colony, except a small number of possible Outside the existing mutation naturally occurred, the single antibody included in the colony is identical.Modifier " monoclonal " only represents anti- The characteristic of body, is obtained from substantially uniform antibody population, this cannot be construed to need to be produced with any specific process anti- Body.

Some parts of variable region are different in sequence in " variable " the expression antibody of term used herein, it is formed Combinations and specificity of the various specific antibodies to its specific antigen.Changeability, which is concentrated in light chain and heavy chain variable region, to be known as mutually Mend in three fragments determined in area (CDR) or hypervariable region.Four FR areas are respectively contained in the variable region of native heavy and light chain (more conservative part in variable region), they are generally in beta sheet configuration, are connected by three CDR for forming connection ring, can shape Into part β-pleated sheet structure.CDR in every chain is by FR areas firmly against together form together and with the CDR of another chain The antigen-binding site of antibody.Constant region does not participate in the combination of antibody and antigen directly, but they show different effects Function, such as participates in the cytotoxicity dependent on antibody.

As used herein, " AXL " and " AXL albumen " is interchangeably used.

As used herein, term " carrier " refers to expression vector and non-express carrier, and including virus and non-virus carrier, It includes the outer carrier (e.g., multicopy plasmid) of chromosome and integration vector (it is designed as may be incorporated into host chromosome).

" sample " as described herein covers several samples type, including the blood of biological origin and other body fluid samples Product, solid tissue sample such as tissue biopsy sample either tissue culture or derived from cell therein or its offspring.Should Term is additionally included in the sample handled after acquisition by any mode, such as some with agent treatment, dissolving or enrichment Component such as protein or polynucleotides.The term covers the various clinical samples derived from any species, also includes culture Cell, cell conditioned medium and cell lysates.

As used herein, the protein that term " separated " refers to isolate from its natural surroundings is (that is, from least one Separated in its natural adjoint other component).

The advantages of the present invention：

The present invention, using conventional antibody production techniques, is obtained efficient using AXL albuminous cells extracellular portion as antigen The monoclonal antibody of the anti-AXL of valency, high specific and high-affinity.Since the antibody titer is high, specificity is good, affinity is strong, The disease that induction is overexpressed by AXL can be used to prevent and treat directly as anti-AXL classes medicine.

Brief description of the drawings

Fig. 1 shows the physical map of carrier for expression of eukaryon pCMV-163；

Fig. 2 shows the combination situation using ELISA detection 9A antibody and antigen A XL-Fc；

Fig. 3 shows the combination situation using ELISA detection 9A antibody and antigen A XL；

Fig. 4 shows the combination situation using flow cytometer showed technology for detection 9A antibody and A549 cell surfaces AXL.

Embodiment

The present invention is further illustrated below by embodiment.It should be understood that the embodiment of the present invention is to be used to illustrate The present invention rather than limitation of the present invention.The simple modifications that essence carries out the present invention according to the present invention belong to the present invention Claimed scope.

1 antibody screening of embodiment

In order to avoid the skewed popularity of individual immunity background, ensure the diversity of antibody library as far as possible, utilize lymphocyte point Chaotropic separates 100 adult healthy people (men and women is fifty-fifty) peripheral bloods and 10 neonate (men and women is fifty-fifty) cord blood lymphocytes cells, 2 × 10 are collected altogether⁹A cell.Trizol methods extract total serum IgE, reverse transcription cDNA, conventional PCR method amplification different antibodies hypotype Variable region gene.According to antibody library carrier pDF (Military Medical Science Institute's proceeding, 2008,32 (4)：305-308,358.) letter Breath, introduces restriction enzyme site BssH II, Nhe I and (sequence is with Loxp511 sequences：SGGSTITSYNVYYTKLSSSGT (SEQ ID NO：15) connection peptide), ScFv (single-chain antibodies are spliced into by overlapping PCR method：VL-Linker (contains Loxp511 sequences)-VH, upstream and downstream introduces BssH II, Nhe I sites respectively) form (be shown in by specific method《Biological libraries skill Art》, the chief editor such as Shao Ningsheng, military medicine Science Press, the first edition in 2011).After electrophoretic separation, the scFv obtained is used BssH II, Nhe I digestion rear clones enter in the pDF carriers of same digestion processing, Electroporation Transformation Escherichia coli XL1-Blue (Agilent Technology).After SB nutrient solutions expand culture, add 1 × 10¹³Pfu helper virus VCSM13 (BioVector NTCC Inc.) infection, primary phage antibody library is obtained, measure potency is 8 × 10¹²cfu/mL.(infection multiplicity MOI in proportion >200) primary antibody storehouse and BS1365 bacterium [genotype are mixed：F’kan recA1endA1gryA96thi21Δ LacU169supE44hsdR17 (λ imm434X12cre)] (BioVector NTCC Inc.), the Cre expressed by BS165 bacterium (specific method refers to for recombinase-mediated loxp/loxp511 restructuring：Hum Antibodies.1999；9(1):67-77；J Biomol Screen.2014Feb 4；19(6):839-846.), large capacity recombinant antibodies storehouse is obtained.

Using mammalian cell-Chinese hamster ovary cell CHO-K1 (CCL-61^TM) expression people's AXL eggs (people AXL albumen extracellular fragment-Fc sections of human IgG1 fusion protein, is named as AXL-Fc, and the wherein sequence information of people AXL refers to NP_ in vain 068713, the sequence reference AEO21920.1 of human IgG1's Fc fragments) it is target spot, screen target antibody.5% skimmed milk power closes AXL- After the coated immunotubes of Fc (Maxisorp immunotubes, Thermo Nunc), above-mentioned phage antibody library is added, 37 DEG C incubate Educate 2h；Uncombined bacteriophage is discarded, TBS-T wash liquids 5 times, fully wash away non-specific adsorption bacteriophage；Add 1mL elutions Buffer solution (0.1mol/L glycine-HCl, pH 2.2) wash-out bacteriophage is simultaneously neutralized with 40 μ L 2mol/L Tris solution；Add Logarithmic phase XL1-Blue bacterium, SB culture mediums (SB nutrient solutions：Tryptone 30g, yeast extract 20g, MOPS 10g, is dissolved in In 950mL deionized waters, sodium hydroxide tune pH value to 7.0, is settled to 1L, autoclaving) and helper phage VCSM13 progress Amplification enrichment；Process 3-4 wheels are repeated, the freshly prepared logarithmic phase XL1-Blue bacterium of the phage-infect eluted are applied into training Plate is supported, after 37 DEG C are incubated overnight, random picking monoclonal to 96 hole depth orifice plates (Corning), phage- is carried out after expanding culture (specific method refers to ELISA：Military Medical Science Institute's proceeding, 2008,32 (4)：305-308,358.), detection and the knot of antigen Close characteristic；165 clones are identified altogether, wherein in the clone of 48 specific binding AXL-Fc, it is best that 9A combines activity.

Send 9A clones to sequencing, the variable region gene obtained is through http://www.abysis.org/ on-line analyses, are obtained In the 9A clones obtained, weight chain variabl area sequence such as SEQ ID NO:Shown in 7, its CDR1, CDR2, CDR3 region amino acid sequence is successively For SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3；Light-chain variable sequence is SEQ ID NO:8, its CDR1, CDR2, CDR3 region amino acid sequence are respectively SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6.Encoding the heavy chain can Become the nucleotide sequence such as SEQ ID NO in area:Shown in 9；The nucleotide sequence of coding light chain variable region such as SEQ ID NO:Shown in 10.

2 antibody expression of embodiment purifies

9A clone's cloning of V_H gene of acquisition is entered into the carrier for expression of eukaryon pCMV- containing human IgG constant region gene In 163, build intact antibody carrier, its physical map as shown in Figure 1 (each composition of carrier for expression of eukaryon pCMV-163 into Divide and be known in the art component, formed according to the restructuring of shown order).Whole antibody is known as 9A antibody (light-chain amino acid sequences For SEQ ID NO:11, nucleotides sequence is classified as SEQ ID NO:12；Heavy chain amino acid sequence is SEQ ID NO:13, nucleotides sequence Arrange SEQ ID NO:14).By the carrier for expression of eukaryon of acquisition, by using ExpiCHOTM Expression System reagents Box (Thermo Fisher Scientific, #A29133), by 9A eukaryotic expression vector transfections into CHO-S cells, passes through Goat anti-human igg (the GOAT Anti that ELISA experiments are marked using goat anti-human igg (KPL, #01-10-06) and horseradish enzyme Human (HRP), Thermo Fisher Scientific, #31412) carry out antibody in double sandwich-ELISA method detection supernatant Content, using untransfected supernatant as negative control, human IgG sterling is as standard items), antibody expression amount in culture supernatant is detected, Screening obtains the higher monoclonal cell strain of expression quantity.Enough supernatants are collected, utilize conventional Protein A affinity purification targets Antibody.

3 antibody binding activity of embodiment is analyzed

Target antigen AXL-Fc is coated with elisa plate bar, 1 μ g/ml, 4 DEG C overnight；After PBST washings, 10% tire ox is added Serum, when 37 DEG C of closings 1 are small；The 9A antibody of various concentrations is added, when 37 DEG C of reactions 1 are small；After PBST washings, horseradish peroxide is added The goat-anti human Fab secondary antibody (Goat Anti-human IgG (Fab') 2-HRP, Abcam) of compound enzyme mark, 37 DEG C are reacted 30 points Clock；PBST repeats board-washing 5 times, and residual drop is patted dry as far as possible on blotting paper；100 μ l TMB (eBioscience) are added per hole, Room temperature (20 ± 5 DEG C) avoid light place 1.5min；100 μ l 2N H are added per hole₂SO₄Terminate liquid terminates substrate reactions, microplate reader OD values, analysis antibody and target antigen AXL-Fc binding abilities are read at 450nm.9A antibody energy specific recognition target antigens AXL- Fc；Identification activity is in significant dose dependent, and the results are shown in Figure 2.

4 antibody specificity of embodiment identifies target antigen

By Milk (milk) (Beijing Bo Maide Bioisystech Co., Ltd), BSA (BOVOGEN), (Beijing justice sticks up god to CD19 State Bioisystech Co., Ltd), TROP2 (Sino Biological Inc.), BCMA (Yi Qiao Divine Land, Beijing biology Technology Co., Ltd.), CD47 (Beijing Mai Gepoer bio tech ltd), (Yi Qiao Divine Land, Beijing biotechnology has CD38 Limit company), each albumen and AXL (ACRO Biosystems) such as Gas6 (R＆D) be coated with elisa plate bar respectively, 1 μ g/ml, 4 DEG C Overnight；After PBST washings, 10% hyclone is added, when 37 DEG C of closings 1 are small；9A antibody is added, when 37 DEG C of reactions 1 are small； PBST washing after, add horseradish peroxidase-labeled goat anti-human igg's secondary antibody (Goat Anti-human IgG-HRP, Thermo Fisher Scientific), react at room temperature 30 minutes；PBST repeats board-washing 5 times, is patted dry as far as possible on blotting paper residual Stay drop；100 μ l TMB (eBioscience), room temperature (20 ± 5 DEG C) avoid light place 1.5min are added per hole；100 are added per hole μl 2N H₂SO₄Terminate liquid terminates substrate reactions, reads OD values at microplate reader 450nm, analyzes antibody and protein binding capacity.

The results show that 9A antibody can specific recognition target antigen AXL, but with Milk (milk), BSA, CD19, TROP2, The albumen such as BCMA, CD47, CD38, Gas6 are without significant association reaction；The results are shown in Figure 3.

5 antibody of embodiment identifies cell surface antigen

Utilize the combination of flow cytometer showed technology for detection A549 cell surfaces AXL and 9A.Exponential phase A549 cells are collected, Cell density is adjusted to 5 × 10⁶Cell/mL, on ice precooling.The normal saline dilution 9A antibody of the precooling containing 2%FBS is to 20 μ g/ ml.100 μ l cells are taken, add isometric foregoing dilution 9A antibody, 4 DEG C of lucifuges react 30min.After, with pre- containing 2%FBS Cold physiology salt washing is twice (6000rpm, 45s).1 is pressed with the physiological saline of the precooling containing 2%FBS：The mouse of 5 dilution PE marks Anti-human igg secondary antibody (PE Mouse Anti-Human IgG, BD Pharmingen), takes 100 μ L that cell is resuspended, and 4 DEG C of lucifuges are anti- Answer 30min.After reaction, washed twice (6000rpm, 45s) with the physiology salt of the precooling containing 2%FBS.With 400 μ l more than 1% Cell is resuspended in polyformaldehyde.Flow cytometer (BD Calibur) analyzes the binding ability of antibody and cell surface antigen.

The results show that 9A antibody energy specific recognition A549 cell surface AXL, the results are shown in Figure 4.

Although above only describes the embodiment example of the present invention, those skilled in the art should manage Solution, these are merely illustrative of, and protection scope of the present invention is defined by the appended claims.Those skilled in the art On the premise of without departing substantially from the principle of the present invention and essence, various changes or modifications, but this can be made to these embodiments A little changes or modification each fall within protection scope of the present invention.

Sequence table

<110>Hangzhou Shang Jian Bioisystech Co., Ltd

<120>Monoclonal antibody 9A and its application

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Ser Tyr Ala Ile Ser

1 5

<210> 2

<211> 17

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 2

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

1 5 10 15

Gly

<210> 3

<211> 11

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 3

Ala Tyr Tyr Asp Phe Trp Ser Gly Tyr Glu Pro

1 5 10

<210> 4

<211> 16

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 4

Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp

1 5 10 15

<210> 5

<211> 7

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 5

Leu Gly Ser Asn Arg Ala Ser

1 5

<210> 6

<211> 9

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 6

Met Gln Ala Leu Gln Thr Met Tyr Thr

1 5

<210> 7

<211> 120

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 7

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ala Tyr Tyr Asp Phe Trp Ser Gly Tyr Glu Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 8

<211> 111

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

20 25 30

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

85 90 95

Gln Thr Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu

100 105 110

<210> 9

<211> 360

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 9

caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ctggcagcag cgtgaaggtg 60

agctgcaagg ccagcggcgg caccttcagc agctacgcca tcagctgggt gaggcaggcc 120

cctggccagg gcctggagtg gatgggcggc atcatcccta tcttcggcac cgccaactac 180

gcccagaagt tccagggcag ggccaccatc accgccgacg agagcaccag caccgcctac 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcgc cagggcctac 300

tacgacttct ggagcggcta cgagccttgg ggccagggca ccctggtgac cgtgagcagc 360

<210> 10

<211> 333

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 10

cagagcctgg cccagacccc tctgagcctg cctgtgaccc ctggcgagcc tgccagcatc 60

agctgcagga gcagccagag cctgctgcac agcaacggct acaactacct ggactggtac 120

ctgcagaagc ctggccagag ccctcagctg ctgatctacc tgggcagcaa cagggccagc 180

ggcgtgcctg acaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 240

agggtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gaccatgtac 300

accttcggcc agggcaccaa gctgaccgtg ctg 333

<210> 11

<211> 218

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 11

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

1 5 10 15

Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser Asn

20 25 30

Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro

35 40 45

Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro Asp

50 55 60

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

65 70 75 80

Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala Leu

85 90 95

Gln Thr Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Thr Val Leu Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 12

<211> 657

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 12

cagagcctgg cccagacccc tctgagcctg cctgtgaccc ctggcgagcc tgccagcatc 60

agctgcagga gcagccagag cctgctgcac agcaacggct acaactacct ggactggtac 120

ctgcagaagc ctggccagag ccctcagctg ctgatctacc tgggcagcaa cagggccagc 180

ggcgtgcctg acaggttcag cggcagcggc agcggcaccg acttcaccct gaagatcagc 240

agggtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gaccatgtac 300

accttcggcc agggcaccaa gctgaccgtg ctgcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgttag 657

<210> 13

<211> 450

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 13

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

1 5 10 15

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

20 25 30

Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Gln Gly Arg Ala Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Arg Ala Tyr Tyr Asp Phe Trp Ser Gly Tyr Glu Pro Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 14

<211> 993

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 14

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420

gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ccgaggtcaa gttcaactgg 480

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960

cagaagagcc tctccctgtc tccgggtaaa tga 993

<210> 15

<211> 21

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 15

Ser Gly Gly Ser Thr Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu

1 5 10 15

Ser Ser Ser Gly Thr

20

Claims

1. a kind of separated binding molecule, it is characterised in that the binding molecule includes：

(1)SEQ ID NO:Heavy chain CDR1, SEQ ID NO shown in 1:Heavy chain CDR2, SEQ ID NO shown in 2:Shown in 3 Heavy chain CDR3；And/or

(2)SEQ ID NO:Light chain CDR1, SEQ ID NO shown in 4:Light chain CDR2, SEQ ID NO shown in 5:Shown in 6 Light chain CDR3.

2. binding molecule according to claim 1, it is characterised in that the binding molecule includes：

3. binding molecule according to claim 1, it is characterised in that the binding molecule includes：

4. encode the nucleic acid molecules of the binding molecule any one of claim 1-3.

5. nucleic acid molecules according to claim 4, it is characterised in that the nucleic acid molecules include SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:At least one nucleotide sequence in nucleotide sequence shown in 12.

A kind of 6. expression vector of the nucleic acid molecules including described in claim 4 or 5.

7. the host cell of the expression vector described in a kind of nucleic acid molecules or claim 6 including described in claim 4 or 5.

8. a kind of pharmaceutical composition of the binding molecule including any one of claim 1-3, detection product, diagnosis production Product.

9. application of the binding molecule in detection product or diagnostic products are prepared any one of claim 1-3；It is described Detection product is the product for detecting AXL expressing quantities, and the diagnostic products are disease of the diagnosis using AXL protein overexpressions as characterization The product of disease；Preferably, the disease using AXL protein overexpressions as characterization includes tumour, fibrosis or hepatic sclerosis.

10. the binding molecule any one of claim 1-3 is preparing prevention or is treating what is induced by AXL protein overexpressions Application in the pharmaceutical preparation of disease；Preferably, the disease induced by AXL protein overexpressions includes tumour, or tumour is resistance to Pharmacological property.