CN105884897A

CN105884897A - Quick expression of slow viruses by aid of c-Met-resistant univalent antibody and application thereof

Info

Publication number: CN105884897A
Application number: CN201610253999.XA
Authority: CN
Inventors: 郭佳; 蒋明; 尹衍新; 于丽华; 蒋韵
Original assignee: SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
Current assignee: SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
Priority date: 2016-04-23
Filing date: 2016-04-23
Publication date: 2016-08-24

Abstract

The invention discloses a recombinant human c-Met-resistant univalent antibody. Nucleic acid sequences of variable regions of heavy chains of the recombinant human c-Met-resistant univalent antibody are shown as SEQ ID:1, and nucleic acid sequences of variable regions of light chains are shown as SEQ ID NO:2, amino acid sequences of the variable regions of the heavy chains of the recombinant human c-Met-resistant univalent antibody are shown as SEQ ID:3, and amino acid sequences of the variable regions of the light chains are shown as SEQ ID NO:4; nucleic acid sequences of complete heavy chains of the recombinant human c-Met-resistant univalent antibody are shown as SEQ ID:5, nucleic acid sequences of complete light chains are shown as SEQ ID:6, and nucleic acid sequences of complete Knob chains are shown as SEQ ID:7; amino acid sequences of the complete heavy chains of the recombinant human c-Met-resistant univalent antibody are shown as SEQ ID;8, amino acid sequences of the complete light chains are shown as SEQ ID:9, and amino acid sequences of the complete Knob chains are shown as SEQ ID:10. The recombinant human c-Met-resistant univalent antibody has the advantages that the recombinant human c-Met-resistant univalent antibody can be specifically combined with human c-Met, combination of HGF (hepatocyte growth factors) and the human c-Met can be blocked, HGF/c-Met signal transduction pathways can be screened and blocked, and accordingly important effects can be realized by the recombinant human c-Met-resistant univalent antibody in the aspect of research and development of neutralization antibody medicines for suppressing tumor growth.

Description

Anti- c-Met Univalent antibody slow virus is quickly expressed and applies

Technical field

The invention belongs to antibody drug technical field, be specifically related to a kind of restructuring univalent antibody for people's interstitial epidermis transforming factor c-Met molecule and application thereof.

Background technology

People interstitial epidermis transforming factor (c-Mesenchymal Epithelial transition factor, c-Met) be the class transmembrane receptor with autonomous phosphorylation activity, after be proved hepatocyte growth factor (hepatocyte Growth factor, HGF) it is its ligands specific, there is in Several Kinds of Malignancy develops important function.The heterodimer that the basic structure of c-Met is made up of the α chain of 50 kD and the β chain of 145 kD, including the domain that 3 functions are different, i.e. extracellular region, cross-film district and intracellular region.After c-Met is combined with its part, intracellular region has tyrosine kinase activity, can activate many A signal pathways, such as PI3K-Akt, Ras-MAPK and STAT3 etc..Cause extensive cell response, including the interaction between tumor cell, matrix attachment, cell migration, invasion and attack and angiogenesis.At present, block HGF/c-Met signal transduction and become a Critical policies of antitumor drug research.The clinical experiment report of c-Met inhibitor shows that it can make the existence of nonsmall-cell lung cancer some patients benefit.Gram azoles researched and developed by Pfizer obtains FDA approval for Buddhist nun's capsule (crizotinib) in August, 2011 and there is the advanced Non-small cell lung of EML4-ALK fusion gene for treatment, and this is first granted small molecule tyrosine kinase inhibitors for c-Met exploitation.The c-Met tyrosinase inhibitor card researched and developed by Exelixis company is rich obtains FDA approval listing for Buddhist nun (cabozantinib) in November, 2012, for treating the treatment of pernicious Locally Advanced or metastatic medullary thyroid carcinoma.At present, also there is no the neutralizing antibody medicine of the anti-C-Met that FDA ratifies.The present invention is directed to tradition neutralizing monoclonal antibody at present can be by c-Met dimerization and then activation this problem of tumor cell signal transduction, with the neutralizing antibody ch3E1D7 of anti-c-Met as parental antibody, building, express and the active univalent antibody that can block again c-Met dimerization of the existing neutralization of Preliminary Identification, the therapeutic antibodies blocking HGF/c-Met signal path and then suppression tumor growth for follow-up research and development lays the foundation.

Summary of the invention

One of solution problem of the present invention is the variable region sequences using degenerate primer method accurately to clone c-Met Mus monoclonal antibody 3E1D7.

The recombinant anti human c-Met univalent antibody that the present invention provides, the nucleotide sequence of its variable region of heavy chain is as shown in SEQ ID NO:1, and the nucleotide sequence of its variable region of light chain is as shown in SEQ ID NO:2.

The recombinant anti human c-Met univalent antibody that the present invention provides, the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID NO:3；The aminoacid sequence of its variable region of light chain is as shown in SEQ ID NO:4.

The two of the problem that the present invention solves are to use overlap extension pcr, with the neutralizing antibody ch3E1D7 of anti-c-Met as parental antibody, build, express and the existing activity that neutralizes of Preliminary Identification can block again the anti-human c-Met univalent antibody with independent intellectual property right of c-Met dimerization.

The present invention provides recombinant anti human c-Met univalent antibody, and its entire heavy chain nucleotide sequence is as shown in SEQ ID:5, and Whole light chains nucleotide sequence is as shown in SEQ ID:6, and complete Knob chain nucleic acid sequence is as shown in SEQ ID:7.

The present invention provides recombinant anti human c-Met univalent antibody, and its entire heavy chain aminoacid sequence is as shown in SEQ ID:8, and Whole light chains aminoacid sequence is as shown in SEQ ID:9, and complete Knob chain amino acid sequence is as shown in SEQ ID:10.

The recombinant anti human c-Met univalent antibody gene order that the present invention provides is expressed in mammal, and obtains purpose antibody.

Human antibody or humanized antibody and the various product with application prospect can be evolved into, such as scFv, Fab etc. on the premise of keeping specificity and affinity for template with this sequence.

Recombinant anti human c-Met univalent antibody provided by the present invention, can specific be combined with Hepatic growth factor receptor c-Met.And HGF with c-Met can be blocked be combined, the therapeutic antibodies blocking HGF/c-Met signal path and then suppression tumor growth for follow-up research and development lays the foundation.

Accompanying drawing explanation

Fig. 1 designs univalent antibody schematic diagram, wherein VH: variable region of heavy chain for genetic engineering；CH1: CH1；VL: variable region of light chain；CL: constant region of light chain；Knobs into holes: " pestle mortar " structure.

Fig. 2 is anti-human C-Met chimeric antibody ch3E1D7 variable region of heavy chain, the CH-hole of IgG1-hole and the PCR primer electrophoretogram of univalent antibody entire heavy chain gene, and wherein (a) is that (swimming lane 1 is DL2000 Marker for the CH-hole of ch3E1D7 variable region of heavy chain and IgG1-hole;Swimming lane 2 is the CH-hole gene about 1000bp of IgG1-hole;Swimming lane 3 is variable region of heavy chain band 339bp), (b) is that over-lap PCR is by CH-hole and V_HTwo fragments connect, and (swimming lane 1 is DL2000 Marker to form the entire heavy chain of c-Met univalent antibody;Gene for the purpose of swimming lane 2).

The SDS-PAGE spectrum of Fig. 3 univalent antibody, wherein (a) is that (wherein swimming lane 1 is albumen marker to reproducibility SDS-PAGE;Swimming lane 2 is univalent antibody), (b) is that (wherein swimming lane 1 is albumen marker to irreducibility SDS-PAGE;Swimming lane 2 is univalent antibody).

Fig. 4 is the combination activity of ELISA detection univalent antibody and people c-Met, and wherein abscissa is univalent antibody concentration (ng/ml), and vertical coordinate is the photometric absorbance value (OD450) for measuring under the conditions of 450nm Single wavelength.

Fig. 5 is in competitive ELISA detection univalent antibody and activity, and wherein abscissa is univalent antibody concentration (ng/ml), and vertical coordinate is the photometric absorbance value (OD450) for measuring under the conditions of 450nm Single wavelength.

Detailed description of the invention

Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention rather than limit the scope of the present invention.In addition, it is to be understood that after having read the content that the present invention lectures, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within the application appended claims limited range equally.

Embodiment 1

293T cell is purchased from Shanghai Inst. of Cytobiology, Chinese Academy of Sciences；The plasmids such as CMV/IgG1-hole-flag, CMV/IgG1-knob-his, CMV/ch3E1D7-L, CMV/ ch3E1D7-H, pRRL-CMV and c-Met extracellular region fusion protein (see and publish an article) are that this laboratory preserves；E. coli competent DH5 α is purchased from Beijing Quanshijin Biotechnology Co., Ltd；Cell culture medium DMEM and hyclone are purchased from Hyclone company；Various restriction endonuclease, phusion surpass fidelity PCR kit and are U.S.'s NEB Products；DNA gel recovery test kit, mini-scale plasmid extraction agent box, endotoxin purification take out test kit and TMB nitrite ion is purchased from Beijing Tian Gen biochemical technology company limited；NovoRec PCR mono-step directed cloning test kit is purchased from Shanghai Xinbainuo Biology Science Co., Ltd；Rabbit anti-Flag-HRP polyclonal antibody is Huaan biological product；Goat anti human's HGF biotinylated polyclonal antibody and recombined human HGF albumen are U.S.'s R&D Products；Streptavidin-HRP antibody is purchased from U.S. Cell Signaling Technology company；Protein A is purchased from Amersham company.

The design of c-Met univalent antibody structure

With the neutralizing antibody ch3E1D7 of anti-c-Met as parental antibody, design univalent antibody mono1H9F6.Following three chains of this research design: wherein antibody arm in side derives from parent chimeric antibody ch3E1D7, i.e. an original light chain of ch3E1D7 is constant；And the CH3 domain of ch3E1D7 original heavy chain replaces three macromole aminoacid with three little molecule aminoacid, and i.e. T366S: L368A: Y407V, make to be formed on CH3 domain a mortar shape structure, i.e. " hole "；.Opposite side Knob chain is the CH making human normal immunoglobulin's IgG1 antibody₃Domain highlights one " knob ", i.e. replaces a little molecule aminoacid Thr with a macromole aminoacid Try, such as T366Y, forms a pestle shape structure.Improved two CH₃Space conformation forms corresponding relation, only contains the CH of " hole "₃Domain and can form stable heterodimer containing between the CH3 domain of " knob ", it is to avoid the by-product occurred during antibody expression assembles, and antibody has the cytotoxicity that the antibody dependent cellular similar to wild type mediates after suddenling change.Univalent antibody structure chart is as shown in Figure 1.

Embodiment 2

Three chain Lentivirals of c-Met univalent antibody build

The original light chain of the light chain of Mono3E1D7, i.e. ch3E1D7, its Lentiviral CMV/ch3E1D7-L is built by this laboratory and preserves.Opposite side Knob chain, is the CH making IgG1₃Domain highlights the pestle shape structure of " knob ", and adds His label at its C end, and its Lentiviral CMV/IgG1-knob-his is built by this laboratory and preserves.This laboratory preserves CMV/IgG1-hole-flag plasmid, forming " hole " mortar shape structure on its CH3 domain, therefore the heavy chain of unit price needs the constant region by ch3E1D7 variable region of heavy chain with IgG1-hole-flag to be stitched together by overlap extension pcr (SOE-PCR).Primer is synthesized by Jin Weizhi bio tech ltd, Suzhou, and its sequence is as follows:

P1 primer sequence (5 '-3 '): CACCGACTCTAGAGGATCCACCGGTIt is AgeI restriction enzyme site shown in CGCCACCATGGTCAGCTACTGGGACACC(underscore)；

P2 primer sequence (5 '-3 '): GTGAGTTTTGTCACAAGATTTGGGCTCAACTTTCT；

P3 primer sequence (5 '-3 '): CCCAAATCTTGTGACAAAACTCACACATGCCCACC；

P4 primer sequence (5 '-3 '): TCCAGAGGTTGATTGTCGACCTACTTATCGTCGTCATCCTTGTAATC(being SalI restriction enzyme site shown in underscore, italic is Flag label).

First for primer, the constant region of ch3E1D7 variable region of heavy chain and IgG1-hole-flag being carried out PCR amplification with P1/P2 and P3/P4 respectively, product purification after 1.5% agarose gel electrophoresis reclaims.Again by ch3E1D7 variable region of heavy chain V_HBeing connected by SOEPCR with two fragments of constant region CH-hole of IgG1-hole, electrophoresis and PCR primer reclaim again, and PCR primer electrophoretogram is as shown in Figure 1.Slow virus expression plasmid pRRL-CMV is reclaimed by AgeI and SalI-HF double digestion, rubber tapping.With carrier NovoRec PCR mono-step directed cloning test kit, purpose fragment being carried out homologous recombination be connected, connect product transformed competence colibacillus bacillus coli DH 5 alpha, screening positive clone carries out order-checking to be identified.It is standby that new univalent antibody heavy chain pRRLCMV-mono3E1D7-H removes endotoxic plasmid extraction kit purification.

Embodiment 3

Expression, purification and the qualification of c-Met univalent antibody mono3E1D7

Inoculation is in the 293T cell of exponential phase; transfect when cell grows to 50% ~ 60% fusion; after 1.8 μ g slow virus packaging plasmid mixture and 1.9 μ g univalent antibody expression plasmids (CMV/ch3E1D7 L, pRRLCMV-mono3E1D7-H and CMV/IgG1-knob-his mix according to the ratio of 1:1:1) are used basal medium resuspended by univalent antibody group, utilize calcium phosphate method that the Lentiviral plasmid of tri-chains of mono3E1D7 is transfected 293T cell.293T cell is transfected as positive control, using the 293T cell of untransfected as blank using the carrier p RRL-CMV-GFP containing green fluorescent protein.Slow virus expression plasmid and packaging plasmid mixture cotransfection 293T cell; formation slow virus can be packed after 24h; virion infects 293T cell further, is effectively incorporated on host chromosome by univalent antibody gene, thus reaches the effect of expression destination protein quick, lasting.Virus collects 293T cell culture fluid after infecting 48h, and 10000 rpm are centrifuged 20 min, and centrifugal rear supernatant is through the membrane filtration of 0.45 μm.The culture fluid filtered is mixed with PBS equal-volume, by protein A affinitive layer purification antibody.BCA test kit carries out concentration mensuration, and univalent antibody after purification uses polyacrylamide gel electrophoresis respectively under non-reduced (7.5%) and reducing condition (10%), and coomassie brilliant blue staining is identified antibody, detected its purity and molecular size range.SDS-PAGE under reducing condition, the disulfide bonds of univalent antibody, its three chains occur in that three bands, the most corresponding heavy chain of molecular size range about 55KDa, the light chain of 25KDa and the Knob chain of 30KDa.Under non reducing conditions, disulfide bond will not rupture, and three chains are by disulfide formation heterodimer, and therefore univalent antibody presents a band, size about 110KDa.And due to by-product assembling when Knobs into holes " pestle mortar " structure avoids antibody expression, present the most single band, as shown in Figure 3.In sum, the success of univalent antibody purification, and ensure that correctness and the integrity of structure.

Embodiment 4

The Preliminary detection of c-Met univalent antibody biological function

With CBS, c-Met extracellular region fusion protein being diluted to 5 μ g/ml, 4 DEG C are overnight coated.PBST washs 3 times, closes 2 h with 1%BSA 37 DEG C.C-Met univalent antibody is added in ELISA Plate, 37 DEG C of effect 2 h, wash 3 times as negative control, PBST using PBS simultaneously.Using rabbit anti-Flag-HRP polyclonal antibody as surveying antibody, after 37 DEG C of effect 1h, PBST washings, every hole adds 100 μ l TMB nitrite ions, and 37 DEG C of lucifuges develop the color 10 min, and after terminating reaction, microplate reader measures A450 numerical value.As shown in Figure 4, univalent antibody can occur specific combination with antigen c-Met albumen to ELISA testing result, and along with the increase of concentration, signal gradually strengthens, and is significantly higher than the PBS group as negative control.Thus prove, successfully build and have expressed and can be combined univalent antibody with people's c-Met antigenic specificity.

Embodiment 5

The biologic activity that univalent antibody extracorporeal blocking c-Met with HGF is combined

With CBS, c-Met extracellular region fusion protein being diluted to 5 μ g/ml, 4 DEG C are overnight coated.After PBST washs 3 times, close 2 h with 1%BSA 37 DEG C.The univalent antibody of variable concentrations is added in ELISA Plate, 37 DEG C of effect 1.5 h, wash 3 times as negative control, PBST using PBS simultaneously.Commercialization recombined human HGF albumen is diluted to 15ng/ml, every hole 100 μ l adds in ELISA Plate, after 37 DEG C of effects 2 h, PBST wash 3 times, using Goat anti human's HGF biotinylated polyclonal antibody as detection antibody, after 37 DEG C of effects 1h, PBST wash 3 times, add the Streptavidin-HRP of working concentration, 37 DEG C of effect 0.5h, after PBST washing, every hole adds 100 μ l TMB nitrite ions, and 37 DEG C of lucifuges develop the color 10 min, and after terminating reaction, microplate reader measures A450 numerical value.Whether detection univalent antibody has and the ability of people's HGF competitive binding c-Met albumen.ELISA result shows, reaction numerical value presents downward trend with the increase of univalent antibody concentration, i.e. with the increase of univalent antibody concentration, significantly suppress the combination activity of human hepatocyte growth factor and c-Met, as shown in Figure 5.Result explanation c-Met univalent antibody has neutralization activity.

SEQUENCE LISTING

<110>Tongji University Suzhou academy

<120>anti-c-Met univalent antibody slow virus is quickly expressed and applies

<130> 2016

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<170> PatentIn version 3.5

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

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

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

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

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

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

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Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu

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Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

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Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

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Leu Ser Pro Gly Lys Asp Tyr Lys Asp Asp Asp Asp Lys

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<213>synthetic

<400> 9

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser

1 5 10 15

Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Asp Ile Val Ile Thr Gln

20 25 30

Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser

35 40 45

Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Phe Gln Gln

50 55 60

Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu

65 70 75 80

His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

85 90 95

Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr

100 105 110

Phe Cys Gln Gln Gly Asn Thr Phe Pro Pro Thr Phe Gly Gly Gly Thr

115 120 125

Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe

130 135 140

Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys

145 150 155 160

Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val

165 170 175

Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln

180 185 190

Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser

195 200 205

Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His

210 215 220

Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235 240

<210> 10

<211> 259

<212> PRT

<213>synthetic

<400> 10

Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser

1 5 10 15

Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Asp Lys Thr His Thr Cys

20 25 30

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

35 40 45

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

50 55 60

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

65 70 75 80

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

85 90 95

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

100 105 110

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

115 120 125

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

130 135 140

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

145 150 155 160

Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys

165 170 175

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

180 185 190

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

195 200 205

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

210 215 220

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

225 230 235 240

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His

245 250 255

His His His

Claims

1. a recombinant anti human c-Met univalent antibody, the nucleotide sequence of its variable region of heavy chain is as shown in SEQ ID NO:1;The nucleotide sequence of its variable region of light chain is as shown in SEQ ID NO:2.

2. a recombinant anti human c-Met univalent antibody, the aminoacid sequence of its variable region of heavy chain is as shown in SEQ ID NO:3；The aminoacid sequence of its variable region of light chain is as shown in SEQ ID NO:4.

3. a DNA molecular, encodes the recombinant anti human c-Met univalent antibody described in claim 1 or 2.

4. the DNA molecular described in claim 3, the entire heavy chain nucleotide sequence of described recombinant anti human c-Met univalent antibody is as shown in SEQ ID NO:5；Whole light chains nucleotide sequence is as shown in SEQ ID NO:6；Complete Knob chain nucleic acid sequence is as shown in SEQ ID:7.

5. the DNA molecular described in claim 3, the entire heavy chain aminoacid sequence of described recombinant anti human c-Met univalent antibody is as shown in SEQ ID:8, and Whole light chains aminoacid sequence is as shown in SEQ ID:9, and complete Knob chain amino acid sequence is as shown in SEQ ID:10.

6. encode the plasmid of the arbitrary described recombinant anti human c-Met univalent antibody DNA of requirement 1-5 that has the right.

7. encode the expression of the arbitrary described recombinant anti human c-Met univalent antibody of requirement 1-5 of having the right, be included in the expression of protokaryon and eukaryotic cell.

8. according to the recombinant anti human c-Met univalent antibody one of claim 1-5 Suo Shu, it is characterised in that it is specific binding with Hepatic growth factor receptor c-Met, and it can block HGF with c-Met and be combined.

9. the application of the recombinant anti human c-Met univalent antibody as described in claim 4-5, it is characterised in that: for diagnosis and the treatment of tumor.