CN103626871A - Monoclonal antibodies capable of specifically recognizing EGFR mutant protein, preparation method and application thereof - Google Patents

Abstract

The invention discloses monoclonal antibodies capable of specifically recognizing EGFR mutant protein, a preparation method and application thereof., and belongs to the field of biological science and drug carriers. The three monoclonal antibodies prepared by employing the preparation method provided by the invention can respectively specifically recognize ten kinds of mutant protein, and the mutant protein comprises EGFR G719A, EGFR G719 C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L. The ten monoclonal antibodies can used to detect whether the EGFR protein of animal or human tissues comprises G719A, G 719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769R mutation, and helps to provide basis on clinical diagnosis and treatment for multiple cancers.

Description

The monoclonal antibody of specific recognition EGFR mutain, preparation method and application thereof

Technical field

The invention belongs to bio-science and pharmaceutical carrier field, particularly the application of the monoclonal antibody of specific recognition EGFR mutain, preparation method and conduct diagnosis thereof and treatment reagent.

Background technology

EGFR albumen (Epidermal Growth Factor Receptor, EGF-R ELISA, also referred to as ErbB-1 or HER1) is the transmembrane receptor member of the Epidermal Growth Factor Receptor Family of cell surface.EGFR and extracellular Urogastron (EGF) combination, thus extracellular signal is transmitted in cell.At multiple human cancer, for example, in lung cancer, anus cancer, the rectum cancer, mammary cancer, all there are (Paez JG et al., Science, 304 (5676): 1497-500 (2004)) in the sudden change of EGFR albumen.These sudden changes cause the sustained activation of EGFR albumen, and then promote the propagation of cell to transform and growth of cancer cells.Common EGFR mutain has G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L.

Inhibitor medicaments for EGFR mutain goes on the market, and comprises Iressa ^tM(Iressa ^tM, chemical name Genfitinib), Erlotinib ^tM(Tarceva ^tM, chemical name Erlotinib) etc. (Mayumi Ono1and Michihiko Kuwano, Clin.Cancer Res.12:7242 (2006)).These inhibitor medicaments specificitys are for the EGFR albumen of sudden change, therefore before using, need to a kind of can specific binding sudden change EGFR albumen but in conjunction with the antibody of Wild type EGFR albumen, whether do not detect this patient with EGFR sudden change (Pallis AG et al., Br.J.Cancer.105 (1): 1-8. (2011)).This antibody can be used to a small amount of sample of taking from patient of rapid detection, for diagnosing cancer.Antibody that can specific binding EGFR mutain be also treatment cancer in the urgent need to.

Summary of the invention

The object of the invention is provides for addressing the above problem six kinds of monoclonal antibodies, preparation method and application thereof identifying specifically respectively EGFRG719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L mutant protein.Utilize the monoclonal antibody that method of the present invention is prepared can identify specifically respectively G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L mutant protein, and nonrecognition Wild type EGFR albumen.

In certain operations example, monoclonal antibody or the antigen-binding site of described specific recognition EGFR mutain, is characterized in that, a heavy chain and a light chain, consists of, the CDR1 of heavy chain and light chain, the aminoacid sequence of CDR2 and CDR3 is to be respectively CCTCC C201301 by Chinese Typical Representative culture collection center (CCTCC) preserving number, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCCC201320, the CDR1 of the monoclonal antibody that the hybridoma of CCTCC C201304 and CCTCC C201316 produces, the aminoacid sequence of CDR2 and CDR3 determines.In further operational instances, monoclonal antibody or the antigen-binding site of described specific recognition EGFR mutain, it is characterized in that, by the heavy chain and the light chain that comprise respectively the aminoacid sequence of the heavy chain of monoclonal antibody that described hybridoma produces and the Variable Area of light chain, formed.In operational instances further, antibody or antigen-binding site comprise the heavy chain of monoclonal antibody and the aminoacid sequence of variable region of light chain that above-mentioned hybridoma produces.

Monoclonal antibody of the present invention can be a kind of antibody of humanized or mosaic type.In some operational instances, this monoclonal antibody is IgG.

On the one hand, the present invention includes that to be kept at Chinese Typical Representative culture collection center (CCTCC) preserving number be the hybridoma of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.

The present invention includes a kind of medicine that contains described monoclonal antibody or antigen-binding site or can be used for the carrier of medicine.The present invention also comprises monoclonal antibody or its antigen-binding site diagnostic kit of mentioning in a kind of the present invention of containing.

In some application examples, the present invention can provide a kind of method (for example, lung cancer of cancer diagnosis, nonsmall-cell lung cancer, the rectum cancer, colorectal carcinoma, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, glioma, the cancer of the brain), its key step comprises: with monoclonal antibody contact of the present invention, come from the biological sample that detects target; Whether detect antibody or its part is combined with sample; If antibody and sample exist combination, show that detected object is cancer, or have the risk that develops into cancer.

In other application example, the invention provides a kind of method for the treatment of tumour patient, comprising: whether the EGFR albumen that detects G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant form is present in the sample that comes from patient; The existence if one of them suddenlys change, is used a kind of EGFR inhibitor (for example Genfitinib and Erlotinib) to treat.The present invention also provides a kind of method for the treatment of cancer patient (for example, the rectum cancer, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, lung cancer).This methods for the treatment of comprises and gives the medicine that contains monoclonal antibody of the present invention or its antigen-binding portion thereof in a kind of moiety of patient.The present invention further provides a kind of use monoclonal antibody of the present invention or its antigen-binding site as pharmaceutical treatment malignant melanoma, the rectum cancer, thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, mammary cancer, lung cancer, or hemopoietic tissue cancer.

The present invention includes a kind of nucleic acid molecule of purifying, its consist of can code book specific binding sudden change EGFR albumen in invention the heavy chain of monoclonal antibody or the nucleotide sequence of light chain or antigen-binding portion thereof.Further the present invention includes the carrier that contains this nucleotide sequence; This carrier is optimized for to contain and comprises the expression regulation sequence that can relate to this nucleic acid molecule.In a kind of application example, the invention provides a kind of host cell that contains this nucleic acid molecule.In Another application example, the invention provides and a kind ofly can produce described monoclonal antibody or the cell strain of antigen-binding site.The present invention also comprises and a kind ofly produces specific binding saltant type and not in conjunction with the monoclonal antibody of Wild type EGFR albumen or the method for antigen-binding site.Its step comprises: cultivate under suitable condition the cell strain of mentioning in host cell or the present invention; Antibody described in purifying.

The present invention is based on monoclonal antibody specificity in conjunction with the discovery of the EGFR albumen of saltant type.We find that these antibody can the very well preferential EGFR albumen in conjunction with sudden change (the EGFR albumen for example suddenling change with G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L).The diseases such as cancer can be diagnosed or treat to these antibody effectively.These antibody are the EGFR albumen of trace in detection of biological sample especially effectively.

EGFR

The described EGFR of this patent refers to EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) gene or albumen (Hunter T and Cooper JA, Annu Rev Biochem.54:897-930 (1985)).This gene or albumen are also referred to as ErbB-1 or HER1.The EGFR protein sequence of wild-type comprises SEQ ID NO:7, and the coding mRNA sequence of Wild type EGFR albumen comprises SEQ ID NO:8.

EGFR is the transmembrane receptor member of the Epidermal Growth Factor Receptor Family of cell surface, is a kind of receptor tyrosine kinase., there is dimerization and activate self kinase activity in EGFR and extracellular Urogastron (EGF) combination, the intracellular a series of downstreams of phosphorylation albumen, thus extracellular signal is transmitted in cell.

The sudden change pattern of EGFR albumen comprises one or more amino acid whose insertion, deletion, replacement etc.The described EGFR G719A sudden change of this patent refers to the glycine (G) of the 719th by the sudden change of L-Ala (A) replacement; EGFR G719C refers to the glycine (G) of the 719th by the sudden change of halfcystine (C) replacement; EGFR G719S refers to the glycine (G) of the 719th by the sudden change of Serine (S) replacement; EGFR D761Y refers to the aspartic acid (D) of the 761st by the sudden change of tyrosine (Y) replacement; EGFR L858R refers to the leucine (L) of the 858th by the sudden change of arginine (R) replacement; EGFR L861Q refers to the leucine (L) of the 861st by the sudden change of glutamine (Q) replacement; EGFR A839T refers to the L-Ala (A) of the 839th by the sudden change of Threonine (T) replacement; EGFR N826S refers to the l-asparagine (N) of the 826th by the sudden change of Serine (S) replacement; EGFR G863D refers to the glycine (G) of the 863rd by the sudden change of aspartic acid (D) replacement; EGFR V769L refers to the α-amino-isovaleric acid (V) of the 769th by the sudden change of paddy leucine (L) replacement.

Antibody and antigen-binding site

The present invention includes specific binding mutant egf R albumen but not in conjunction with the monoclonal antibody of Wild type EGFR albumen." antibody " or " Mitochondrion IgG " refers to the tetramer being linked into by disulfide linkage by heavy chain (H, approximately 50-70kDa) and light chain (L, approximately 25kDa).Light chain can be lambda light chain or κ light chain.Heavy chain can be divided into μ, δ, γ, α, ε, the combination of different heavy chains and light chain determines that the type of antibody is respectively IgM, IgD, IgG, IgA or IgE(Fundamental Immunology Ch.7 jointly, Paul, W., ed., 2nd ed.Raven Press, N.Y. (1989)).

Every heavy chain (be sometimes referred to as H chain, or HC) all contains a variable region of heavy chain and (is also V _h) and a CH (be also C _h).CH by three structures with form, be respectively C _h1, C _h2 and C _h3.Every light chain (sometimes also become L chain, or LC) also have a variable region of light chain (to be also V _l) and constant region of light chain (C _l).Constant region of light chain only contains a structure and C _l, inner at light chain and heavy chain, variable region and constant region link by the structure of " J " shape of about 12 amino acid left and right.In inner " D " shape region that also has a 3 amino acid left and right of heavy chain.Variable region of heavy chain and variable region of light chain can further be divided into ，Ye Jiao complementary determining region, super variable region (CDR) and intert middle slightly conservative frame area (FR).These regions are arranged as FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from N end toward C end with this.The aminoacid sequence sequence of each structural region is according to Kabat(Proteins of Immunological Interest; National Institutes of Health; Bethesda; MD (1987and1991)) and Chothia & Lesk(Chothia & Lesk, J.Mol.Biol.196:901-917 (1987); Chothia et al., Nature342:878-883 (1989)) result of study classification.

One of content of the present invention be specific binding mutant egf R albumen the antigen-binding site of monoclonal antibody." antigen-binding site " refers to the one section of antibody fragment that makes it have the function of specific recognition conjugated antigen (polypeptide fragment that for example EGFR albumen the preceding paragraph contains specific sudden change) on antibody.Think at present, the antigen combined function of antibody can realize by the Partial Fragment of full length antibody." antigen-binding site " of antibody is characterised in that and comprises: (1) Fab fragment, one comprises V _l, V _h, C _l, C _hthe unit price fragment of 1 structural domain; (2) F (ab') ₂fragment, one contain by hinge area the divalence fragment of the Fab fragment that couples together of disulfide linkage; (3) one comprise V _hand C _hthe Fd fragment of 1 structural domain; (4) antibody single armed comprises a V _l, V _hthe Fv fragment of structural domain; (5) one by V _hthe dAb fragment (Ward et al., (1989) Nature341:544-546) that structural domain forms; (6) one independently complementary determining region (CDR).Further, although F _vtwo structural domain V of fragment _hand V _lbe to be encoded by different separate gene, but can by a synthetic link area, they be coupled together by the mode of gene recombination, form the V by single chain protein inside _hand V _lbe paired into monovalent molecule, also referred to as strand F _v(scF _v) (Bird et al.Science242:423-426 (1988) and Huston et al.Proc.Natl.Acad.Sci.USA85:5879-5883 (1988)).This single-chain antibody is also included within the scope of " antigen-binding site ".A part for antibody, such as Fab and F (ab') ₂fragment, can be from the antibody of total length by conventional technology, such as the method acquisition of papain (papain) or stomach en-(Pepsin) enzymic digestion.They also can obtain by the DNA recombinant technology of the standard that the following describes.

Anti-egfr antibodies and antigen-binding site thereof

Antibody of the present invention can for example, be prepared with the inhuman animal of the antigen immune that comes from human EGFR mutain (, mouse, rabbit, rat or hamster).These antibody also can obtain by display technique of bacteriophage or other known antibody production techniques.Antigen can be multimer polypeptide or the polypeptide of purifying, can be also multimer polypeptide or the polypeptide at cell inner expression.

EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or the V769L of the sudden change that specified disease is relevant.

In the present invention, the antibody of specific binding EGFR G719A mutain can obtain by polypeptide ETEFKKIKVLASGAFGTV (SEQ ID NO:1) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201301, and name is called hybridoma cell strain M116.

In the present invention, the antibody of specific binding EGFR G719C mutain can obtain by polypeptide ETEFKKIKVLCSGAFGTV (SEQ ID NO:2) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C2012127, and name is called hybridoma cell strain M32-8.

In the present invention, the antibody of specific binding EGFR719S mutain can obtain by polypeptide ETEFKKIKVLSSGAFGTV (SEQ ID NO:3) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C2012118, and name is called hybridoma cell strain M20-18.

In the present invention, the antibody of specific binding EGFR D761Y mutain can obtain by polypeptide EATSPKANKEILYEAYVM (SEQ ID NO:4) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201303, and name is called hybridoma cell strain M121#1.

In the present invention, the antibody of specific binding EGFR L858R mutain can obtain by polypeptide KTPQHVKITDFGRAKLLG (SEQ ID NO:5) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C2012125, and name is called hybridoma cell strain M85-13.

In the present invention, the antibody of specific binding EGFR L861Q mutain can obtain by polypeptide ITDFGLAKQLGAEEKE (SEQ ID NO:6) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201315, and name is called hybridoma cell strain M80-14.

In the present invention, the antibody of specific binding EGFR A839T mutain can obtain by polypeptide DRRLVHRDLTARNVLVKTP (SEQ ID NO:7) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201302, and name is called hybridoma cell strain M118#7.

In the present invention, the antibody of specific binding EGFR N826S mutain can obtain by polypeptide IAKGMSYLEDRRLVHR (SEQ ID NO:8) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201320, and name is called hybridoma cell strain M117-10.

In the present invention, the antibody of specific binding EGFR G863D mutain can obtain by polypeptide ITDFGLAKLLDAEEKE (SEQ ID NO:9) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201304, and name is called hybridoma cell strain M120.

In the present invention, the antibody of specific binding EGFR V769L mutain can obtain by polypeptide EAYVMASLDNPHV (SEQ ID NO:10) immune mouse.The hybridoma that this polypeptide immune obtains that passes through in the present invention has been deposited in the Chinese Typical Representative culture collection center that meets Budapest agreement, and preserving number is CCTCC C201316, and name is called hybridoma cell strain M122#1.

The present invention further comprises a kind of monoclonal antibody or its antigen-binding site, heavy chain amino acid sequence and the light-chain amino acid sequence of the monoclonal antibody that the hybridoma that they comprise by preserving number is CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 produces.In certain operations example, heavy chain amino acid sequence or the light-chain amino acid sequence of the monoclonal antibody that the hybridoma that it is CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 that the monoclonal antibody in the present invention perhaps only comprises by preserving number produces.

In some operational instances, anti-egfr antibodies of the present invention or antigen-binding site comprise the heavy chain of monoclonal antibody or the aminoacid sequence of light chain or CDR that the one or more of hybridomas that are CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 by preserving number produce.In some operational instances, the heavy chain of monoclonal antibody that the hybridoma that it is CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 that antibody or antigen-binding site comprise by preserving number produces and all CDR1, the CDR2 of light chain and the aminoacid sequence of CDR3.

In some operational instances, the heavy chain of monoclonal antibody or the aminoacid sequence of variable region of light chain that the hybridoma that it is CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 that anti-egfr antibodies of the present invention or antigen-binding site comprise by preserving number produces.In specific operational instances, the heavy chain of monoclonal antibody or the aminoacid sequence of variable region of light chain that the hybridoma that it is C CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316 that antibody or antigen-binding site comprise by preserving number simultaneously produces.

The binding specificity of EGFR

Antibody of the present invention or antigen-binding site can be distinguished specifically EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and the EGFR V769L albumen in conjunction with saltant type, but not in conjunction with the EGFR albumen of wild-type (completely not in conjunction with or to a certain extent not in conjunction with).In other words, this antibody or antigen-binding site can be distinguished the human EGFR albumen of saltant type and wild-type.The specificity of this combination can be confirmed by known conventional detection means, immunoblot experiment (Western blot) for example, enzyme-linked immunosorbent assay (ELISA), fluidic cell sorting, co-immunoprecipitation, immunofluorescence, immunohistochemical staining, surface ion resonance (BIACORE for example ^tM) or the method such as radioimmunoassay experiment.

In certain operations example, this antibody is the preferential EGFR albumen in conjunction with saltant type specifically, for at least 10 times of the binding abilities of mutant egf R albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its binding ability to Wild type EGFR albumen.In some operational instances, the dissociation equilibrium constant K of the antibody in the present invention or antigen-binding site specific binding mutant egf R albumen _dat least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 250 times, at least 500 times, or at least 1000 times of dissociation equilibrium constant (K that are better than the combination of Wild type EGFR albumen _d).Dissociation equilibrium constant (K _d) can resonate or flow cytometry records by surface ion.

Antibody of the present invention or antigen-binding site perhaps can only can not for example, in conjunction with other EGFR mutains (EGFR G719A, EGFR G719C, EGFR719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L's is a kind of) in conjunction with a kind of EGFR mutain.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR G719A albumen, and at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its binding ability to other any sudden change EGFR albumen.

In certain operations example, this antibody perhaps can only be in conjunction with EGFR G719A albumen not for example, in conjunction with the EGFR albumen (EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR G719A albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR G719C albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR G719C albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR G719S albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR G719S albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFRN826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR D761Y albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR D761Y albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR L861Q albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR L861Q albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR A839T, EGFR N826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR A839T albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR N826S, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR A839T albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR N826S, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR N826S albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR G863D and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR N826S albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR G863D and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR G863D albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S and EGFR V769L) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR G863D albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S and EGFR V769L).

In certain operations example, this antibody perhaps can only be in conjunction with EGFR V769L albumen not for example, in conjunction with the EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S and EGFR G863D) of other saltant types.For instance, this antibodies specific ground is in conjunction with perhaps at least 10 times of the abilities of EGFR V769L albumen, at least 20 times, at least 50 times, at least 100 times, at least 250 times, or at least 500 times be better than its for example, binding ability to other any sudden change EGFR albumen (EGFR G719A, EGFR G719C, EGFR G719S, EGFR D761Y, EGFR L858R, EGFR L861Q, EGFR A839T, EGFR N826S and EGFR G863D).

In certain operations example, the antibody in the present invention or antigen-binding site can contain SEQ ID NO:1 by specific binding, 2,3,4,5,6,7,8,9 or 10 EGFR polypeptide.In certain operations example, antibody of the present invention or antigen-binding site specific binding preserving number are the epitope of the monoclonal antibody that produces of the hybridoma of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 or CCTCC C201316 or the epitope overlapping with it.

Produce the recombination method of antibody

The present invention includes EGFR albumen that coding can specific binding human mutant type but can not be in conjunction with the monoclonal antibody of Wild type EGFR albumen and the nucleotide sequence of antigen-binding site thereof.The EGFR albumen of human mutant comprises: G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D and V769L.In the middle of certain operations example, this nucleic acid molecule only encode heavy chain or the light chain of monoclonal antibody or antigen-binding site.In the middle of other operational instances, this nucleic acid molecule encode heavy chain and the light chain of monoclonal antibody and antigen-binding site thereof simultaneously.In certain operations example, this nucleic acid molecule encoding preserving number is heavy chain, light chain, variable region of heavy chain or the variable region of light chain of the monoclonal antibody that produces of the hybridoma of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.Accounting can be the nucleic acid molecule of purifying.

The nucleic acid molecule of code book invention antibody can obtain from any source that can produce this antibody.For example, nucleic acid can obtain from preserving number is the hybridoma of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.The method of the nucleic acid of separation and purification encoding antibody is a kind of known common technology.Concrete grammar can be referring to books: [U.S.] J. Sha nurse Brooker D.W. Russell work, Huang Peitang etc. translate, < < molecular cloning experiment guide > >, Science Press, the third edition, 2002.

In certain operations example, the nucleotide sequence of the heavy chain of the monoclonal antibody in code book invention can be the V by code book invention antibody _hthe same frame encoding sequence of the composition of the CH of the nucleotide sequence of structural domain and any other source antibody of coding.Similarly, the nucleic acid molecule of the light chain of a coding anti-EGFR albumen of the present invention can be also the V by code book invention antibody _lthe same frame encoding sequence of the composition of the constant region of light chain of the antibody in the nucleotide sequence of structural domain and any other source of coding.

In further operational instances, encoding heavy chain variable region (V _h) and/or variable region of light chain (V _l) nucleic acid molecule be converted into the antibody coding gene of total length.In certain operations example, coding V _hor V _lthe nucleotide sequence of structural domain is contained CH (C by being inserted into respectively _h) or constant region of light chain (C _l) expression vector in the method for going be configured to a full length antibody gene.The C of the full length antibody gene building like this _hfragment and C _lfragment in carrier inside, be connected, and/or coding V _hfragment with coding V _lfragment in carrier inside, be connected.In other operational instances, coding V _hand/or V _lthe nucleotide sequence of structural domain is connected to a coding C by nucleic acid recombinant technology respectively _hfragment and/or coding C _lfragment above.The gene of the constant domain of encoding human immunoglobulin like protein heavy chain and light chain is known.Specifically referring to works Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., NIH Publ.No.91-3242,1991.The nucleic acid molecule of coding total length heavy chain and/or light chain can be expressed in the cell strain being introduced into purifying EGFR antibody.

Can use nucleic acid molecule to realize recombinant expressed a large amount of EGFR antibody.Also can produce chimeric antibody with nucleic acid molecule, dual specific binding antibody, single-chain antibody, immune adherence agent, double end antibody, sudden change antibody and antibody derivatives.If this nucleic acid molecule comes from non-human, non-transgenic animal, this nucleic acid molecule also may be used to realize the humanization of antibody.

The present invention also comprises the nucleic acid molecule that is inserted with encoding antibody heavy chain, light chain or simultaneously encode EGFR antibody of the present invention or its antigen-binding site.This carrier may be suitable at prokaryotic host cell, for example, in yeast cell, insect cell or mammalian cell, express antibody or its antigen-binding site in the present invention.

Common mammalian cell comprises that a lot of immortal cell lines can be used as expressive host.These clones include but not limited to: Chinese hamster ovary cell (CHO), NS0 cell, SP2 cell, HEK293T cell, 293Freestyle(Invitrogen company), NIH3T3 cell, HeLa cell, baby hamster kidney cell (BHK), African green monkey kidney cell, human liver cancer cell (as HepG2), and A549 cell.Other can have insect cell for the cell of expressing, and comprises sf9 and sf21 cell.Plant host cell comprises: tobacco, Arabidopis thaliana, duckweed, corn, wheat, tomato, the cell in paddy rice source.Bacterial host cell comprises intestinal bacteria and actinomycetes.Yeast host comprises fission yeast, yeast saccharomyces cerevisiae, pichia spp.

Utilize different clone under different culture condition, or the antibody that utilizes transgenic animal to express can have different glycosylation patterns.But no matter what kind of its glycosylation pattern is, all antibody by described nucleic acid or the sequence encoding that contains described nucleic acid is all content of the present invention.

Chimeric and humanized antibody

Here said chimeric antibody, is characterized in that the antibody being comprised of the structural domain that comes from two kinds or more kinds of different antibody.For example, the variable region structural domain of chimeric antibody may be to come from the monoclonal antibody that preserving number is the hybridoma generation of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316, but its constant region may be to come from human antibodies.This chimeric antibody has less immunogenicity when the medicine as the mankind.

In certain operations example, the humanized antibody of antibody of the present invention is that a kind of CDR structural domain by the antibody that derives from mouse in the present invention is inserted in human receptor's antibody and goes.Therefore, the FR of chimeric antibody and constant region come from the mankind.The chimeric antibody that the humanization EGFR antibody in this mankind of containing FR region comes from the mankind than constant region just on mankind has less immunogenicity.In certain operations example, the CDR sequence of humanized antibody can be to come from the monoclonal antibody that preserving number is the hybridoma generation of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.Antibody humanization's method is a kind of known mature technology.

Modify and tag antibody

The present invention also comprises that monoclonal antibody against EGFR described above or its antigen-binding site modified by least one additional molecules or group the antibody forming.This modification can or detect this antibody for purifying, and/or strengthens its result for the treatment of.For example, antibody or antigen-binding site can be connected to a kind of detection reagent, label, cytotoxic reagent, drug molecule and/or albumen or polypeptide, for example, to receive the combination of this antibody or its antigen-binding site and other a kind of molecule (avidin nucleus, or poly is histidine-tagged).Common detection label includes but not limited to: radio isotope (for example ¹²⁵i, ¹³¹i, ³⁵s or ³h), fluorescent composition (fluorescein for example, dichlorotriazine ammonia fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalene sulfonyl chlorobenzene, fluorescein PE, rare earth luminous thing, Umbelliferone, and enzyme (horseradish peroxidase for example, O-tilactase dansyl chloride),, beta-galactosidase enzymes, luciferase, alkaline phosphatase, glucose oxidase, acetylcholinesterase).In further operational instances, antibody of the present invention or its part can be labeled vitamin H, or verified can for example, by another reporter molecules (leucine zipper pairing region, two anti-binding sites, metal collecting structure territory, epitope tag) polypeptide epitope of identification.In operational instances further, any one antibody or its part can be utilized polyvinyl alcohol (PEG), and methyl or ethyl or glycosyl are modified through row.

The diagnostic uses of anti-egfr antibodies or its antigen-binding site

One aspect of the present invention is to use described anti-egfr antibodies or its antigen-binding site as a kind of cancer diagnosis reagent.Cancer refers to any one malignant tumour, comprises any stage and other cancer of level.May include, but are not limited to the cancer in particular example of monoclonal antibody diagnosis of the present invention: lung cancer, nonsmall-cell lung cancer, the rectum cancer, colorectal carcinoma, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, glioma, the cancer of the brain.

Diagnosis comprises the development degree that detects cancer, confirms the existence of cancer, or the somatotype of cancer or classification.In certain operations example, the present invention can provide a kind of method of diagnosing cancer in target, and its step comprises: with monoclonal antibody against EGFR contact of the present invention, come from the biological sample that detects target; Whether detect antibody or its part is combined with sample; If antibody and sample exist combination, show that detected main body is cancer, or have the risk that develops into cancer.The sample detecting can be blood, serum, lymph, tissue (for example fixing coated section of puncture or formaldehyde and paraffin).

Antibodies, to whether detecting with common known technology on sample, includes, but are not limited to: ELISA, RIA, fluidic cell, immunocytochemistry, immunohistochemistry, immunofluorescence, immunoblotting, or co-immunoprecipitation.Anti-egfr antibodies or its part also can be with the direct marks of a kind of marker that can detect.If antibody is not labeled, can use a kind of two anti-or other molecules that can also can be detected in conjunction with EGFR antibody.According to existing general knowledge, the primary antibodie of the specific two specific kinds of anti-combination specifically and hypotype.For example, if anti-egfr antibodies is a kind of mouse IgG, two anti-must be a kind of antibody of anti-mouse IgG of mark.Other can binding antibody molecule include, but are not limited to Protein A and Protein G.They have multiple business-like form.For example, from Protein A and the Protein G of Pierce company.Be applicable to including, but are not limited to for traget antibody or two anti-molecules: enzyme, prothetic group, fluorescent material, luminescent material, magneticsubstance and radio active material (description sees above).In certain operations example, antibody of the present invention or its part can be provided in a kind of test kit of inclusion test antibodies reagent.

Anti-egfr antibodies or its part can also be used to check the development process of cancer patient, then determine to take any treatment treatment plan.Following in detection at treatment plan for example.Medical worker may decide the treatment plan to given patient optimum according to the detected result of EGFR sudden change.Treatment plan can be operation, radiation treatment, pharmacological agent (comprising chemotherapy and targeted therapy), or the combination of above multiple therapeutic modality.For instance, the present invention includes a kind of method for the treatment of cancer patient, it is characterized in that comprising: check whether the biological specimen from patient suddenlys change with EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L; If there is certain sudden change, give EGFR inhibitor for treating to patient.EGFR inhibitor may be a kind of sense-rna (antisense RNA), little intervening rna (siRNA), Microrna (miRNA) medicine, monoclonal antibody against EGFR medicine.Before the targeted therapy that gives the anti-EGFR thing of patient, detect and whether exist EGFR sudden change very meaningful for treatment.

The treatment application of anti-egfr antibodies and antigen-binding site thereof

The present invention provides a kind of antibody and antigen-binding site thereof to comprise the application as the treatment mankind by antibody of the present invention or its part as the application of curative drug equally, and uses antibody or its part as manufacture, to be used for the treatment of the application of human cancer medicine.These human cancers comprise the rectum cancer, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, lung cancer.Detailed cancer species also can be consulted description above.

In certain operations example, medical worker gives to reach to specified disease antibody of the present invention or the part of effective dose, be generally a kind of chimeric or humanized antibody or part, thereby realize, slow down or treat this disease, prevent cancer metastasis or further develop.The administering mode of antibody for example can be: inject or impregnate.Antibody or part dosage can be determined by medical worker, approximate range is 0.1 to 100 mg/kg of body weight, is more preferably 0.5 to 50 mg/kg of body weight, is more preferably 1 to 20 mg/kg of body weight, is more preferably 1 to 10 mg/kg of body weight.The effect for the treatment of can illustrate by monitoring the degree that for example gross tumor volume dwindles.

The composition of curative drug of the present invention, except described monoclonal antibody or its part, may comprise a kind of acceptable medicament carrier.Acceptable medicament carrier is characterized in that, can be a kind of solvent, dispersion agent, and coating agent, antiseptic-germicide and anti-mycotic agent, osmotic equilibrium agent and absorption delayer, is characterized in that having physiology compatibility.Under many circumstances, preferential carrier comprises osmotic equilibrium agent, carbohydrate for example, and poly alcohol is as N.F,USP MANNITOL, sorbyl alcohol, sodium-chlor component.Other acceptable medicament material comprises wetting agent or a small amount of assistant agent, wetting agent for example, and emulsifying agent, protective material or damping fluid, they can increase quality guaranteed period or the effect of antibody.

In certain operations example, anti-egfr antibodies of the present invention or its part by with other a kind of and/or multiple medicine, diagnostic medicine, or preventive mixes.Medicine includes but not limited to have the anti-egfr antibodies of the different targets of combination of different trickle specificity differences, and EGFR inhibitor, for example inhibitor can be Cetuximab (cetuxmab), Victibix (panitumumab), Necitumumab, Nimotuzumab, Gefitinib (gefitinib), Tarceva (erlotinib), lapatinibditosylate (lapatinib), ZD6474 (Vandetanib), PF299804, RO5083945, ABT-806, AP-26113, Afatinib (BIBW2992), Canertinib (CI-1033), Neratinib (HKI-272), CP-724714, TAK-285, AST-1306, ARRY334543, Lapatinib, Arry-380, Dacomitinib (PF-299804), WHI-P154, Desmethyl Erlotinib (CP-473420), Desmethyl Erlotinib(OSI-420), AZD8931, AEE788 (NVP-AEE788), Pelitinib (EKB-569), CUDC-101, WZ804, WZ4002, WZ3146, XL647, PD-153035, PD-174265, PD158780, BMS-599626 (AC480), Lavendustin A, Lavendustin C, BIBU-1361, Hypericin, BIBX-1382, DAPH, Erbstatin Analog, JNJ28871063, RO106-9920, GW2974, GW583340, PD-153035, PD-158780, PD-168393, Tyrphostin AG-183, Tyrphostin AG-99, Tyrphostin AG-825, Tyrphostin B42(AG-490), Tyrphostin AG-494, Tyrphostin AG-555, Tyrphostin AG-527, Tyrphostin AG-528, Tyrphostin AG-835, TyrphostinAG-1478, Tyrphostin RG-14620, Daphnetin, BPIQ, BPIQ-I, BPIQ-II, CL-387, 785, EGFR Inhibitor(K00598a), LFM-A12, Pelitinib, PKC-412, PP3(5334-30-5), SU5402, HDS029, WHI-P154, TAK165, Vatalanib.

Medicament of the present invention forms antibody of the present invention or the antigen-binding site that may contain a kind of " treatment effective dose " or " prevention significant quantity "." treatment effective dose " refers under certain course for the treatment of and dosage, reach and have the active drug of result for the treatment of amount.The treatment effective dose of antibody and antigen-binding portion thereof thereof may be according to many factors, disease process for example, the age, sex, body weight, and antibody or antibody moiety can be in individual caused response capacity and different.The advantageous effect that treatment effective dose has also comprised this antibody or antigen-binding site is better than the meaning of any toxicity or detrimental action." prevention significant quantity " refers under certain course for the treatment of and dosage, reach and have the active drug of preventive effect amount.Conventionally because prophylactic agent is all before disease occurs or disease early application, therefore prevent significant quantity may be less than treatment significant quantity.

Unless specifically stated otherwise, all technology used or science belong to all consistent with personnel's the general knowledge of generally knowing field of the present invention here.The method that can imitate and material will be described in detail below, although also can be used for checking the present invention to similar or same method described herein and material.All reference are all listed in detail.Although quoted a large amount of documents, these are quoted and are not shown to admit that any document is the general knowledge in this field.This patent is described " the comprising " of in process, using and " is comprised " and be appreciated that and show to comprise described entire object or colony's object, rather than gets rid of any other object or colony's object.Material, method and example are only for technical scheme of the present invention is described, and unrestricted.

Following instance is in order to describe method of the present invention and material.The suitable modification of the conditioned disjunction parameter of describing or equal replacement are met to those skilled in the art's obvious general knowledge conventionally, so still belong to the spirit and scope of the present invention.

Accompanying drawing explanation

Fig. 1 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201301 in conjunction with EGFR G719A albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R G719A albumen that swimming lane 2 contains escherichia coli expression;

Fig. 2 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012127 in conjunction with EGFR G719C albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R G719C albumen that swimming lane 2 contains escherichia coli expression;

Fig. 3 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012118 in conjunction with EGFR G719S albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R G719S albumen that swimming lane 2 contains escherichia coli expression;

Fig. 4 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201303 in conjunction with EGFR D761Y albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R D761Y albumen that swimming lane 2 contains escherichia coli expression;

Fig. 5 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012125 in conjunction with EGFR L858R albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R L858R albumen that swimming lane 2 contains escherichia coli expression;

Fig. 6 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201315 in conjunction with EGFR L861Q albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R L861Q albumen that swimming lane 2 contains escherichia coli expression;

Fig. 7 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201302 in conjunction with EGFR A839T albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R A839T albumen that swimming lane 2 contains escherichia coli expression;

Fig. 8 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201320 in conjunction with EGFR N826S albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R N826S albumen that swimming lane 2 contains escherichia coli expression;

Fig. 9 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201304 in conjunction with EGFR G863D albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R G863D albumen that swimming lane 2 contains escherichia coli expression;

Figure 10 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201316 in conjunction with EGFR V769L albumen, but can not be in conjunction with the immunity printing and dyeing experimental patterns of the EGFR albumen of wild-type.The Wild type EGFR albumen that swimming lane 1 contains escherichia coli expression.The mutant egf R V769L albumen that swimming lane 2 contains escherichia coli expression;

Figure 11 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201301 in conjunction with EGFR G719A albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R G719A albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR G719A albumen (below) in occur;

Figure 12 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012127 in conjunction with EGFR G719C albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R G719C albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR G719C albumen (below) in occur;

Figure 13 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012118 in conjunction with EGFR G719S albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R G719S albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR G719S albumen (below) in occur;

Figure 14 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201303 in conjunction with EGFR D761Y albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R D761Y albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR D761Y albumen (below) in occur;

Figure 15 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C2012125 in conjunction with EGFR L858R albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R L858R albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR L858R albumen (below) in occur;

Figure 16 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201315 in conjunction with EGFR L861Q albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R L861Q albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR L861Q albumen (below) in occur;

Figure 17 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201302 in conjunction with EGFR A839T albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R A839T albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR A839T albumen (below) in occur;

Figure 18 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201320 in conjunction with EGFR N826S albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R N826S albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR N826S albumen (below) in occur;

Figure 19 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201304 in conjunction with EGFR G863D albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R G863D albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR G863D albumen (below) in occur;

Figure 20 show preserving number be the antibodies specific that produces of the cell strain of CCTCC C201316 in conjunction with EGFR V769L albumen, but can not be in conjunction with the immunofluorescence experiment collection of illustrative plates of the EGFR albumen of wild-type.Green fluorescent protein (GFP) signal is indicated respectively the expression of Wild type EGFR (above) and mutant egf R V769L albumen (below).Red fluorescence signal can be indicated the combination of this antibody and EGFR albumen, and only express the cell of EGFR V769L albumen (below) in occur.

Embodiment

Embodiment 1

The production of the monoclonal antibody of specific binding EGFR mutain

The polypeptide that contains EGFR mutain aminoacid sequence obtains by synthetic.Detailed peptide sequence is listed in following table, and the mutating acid of wild-type protein underlines sign relatively:

The EGFR polypeptide of table 1 sudden change

EGFR sudden change	Peptide sequence (mutating acid underlines)
		G719A	ETEFKKIKVL ASGAFGTV(SEQ ID NO:1)
G719C	ETEFKKIKVL CSGAFGTV(SEQ ID NO:2)
		G719S	ETEFKKIKVL SSGAFGTV(SEQ ID NO:3)
D761Y	EATSPKANKEIL YEAYVM(SEQ ID NO:4)
		L858R	KTPQHVKITDFG RAKLLG(SEQ ID NO:5)
L861Q	ITDFGLAK QLGAEEKE(SEQ ID NO:6)
		A839T	DRRLVHRDLTARNVLVKTP(SEQ ID NO:7)
N826S	IAKGMSYLEDRRLVHR(SEQ ID NO:8)
		G863D	ITDFGLAKLLDAEEKE(SEQ ID NO:9)
V769L	EAYVMASLDNPHV(SEQ ID NO:10)

It is upper that every synthetic polypeptide is all coupled to succinylation key hole Qi hemocyanin (keyhole limpet hemocyanin, KLH), is used for immune mouse, and utilize freund's adjuvant to mix to strengthen immune effect.The spleen cell of anti-EGFR G719A, G719C, G719S, D761Y, L858R and L861Q mouse with above polypeptide immune is passed through to polyvinyl alcohol (PEG) mediates fusion with mouse myeloma SP2/O cell respectively.With ELISA experiment screening positive colony.Positive colony cell is expelled to the mouse peritoneal of identical strain, produces ascites.Antibody purification from ascites.

The hybridoma that the polypeptide immune mouse of the EGFR albumen that contains sudden change by use produces is deposited in Chinese Typical Representative culture collection center (CCTCC, Wuhan City, Hubei Province Wuhan University, 430072). title and the deposit number of hybridoma are listed in following table.

The anti-EGFR hybridoma cell strain of table 2 and deposit number

Antibody and specificity thereof	Deposit number
		Anti-EGFR G719A	CCTCC C201301
Anti-EGFR G719C	CCTCC C2012127
		Anti-EGFR G719S	CCTCC C2012118
Anti-EGFR D761Y	CCTCC C201303
		Anti-EGFR L858R	CCTCC C2012125
Anti-EGFR L861Q	CCTCC C201315
		Anti-EGFR A839T	CCTCC C201302
Anti-EGFR N826S	CCTCC C201320
		Anti-EGFR G863D	CCTCC C201304
Anti-EGFR V769L	CCTCC C201316

Embodiment 2

The mutain of immunoblot experiment analysis list clonal antibody specific binding intestinal bacteria and animal cell expression

While expressing EGFR albumen in intestinal bacteria, by the cDNA sequence clone of coding EGFR wild-type and EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutein to on 6 histidine-tagged bacterial expression vector pET28a.The plasmid building is transformed in e. coli bl21 (DE3) bacterial strain, and the bacterium of conversion is applied on the LB culture medium flat plate that contains kantlex, 37 ℃ of cultivations.From flat board, select mono-clonal bacterium colony, put into the Erlenmeyer flask that contains 500 milliliters of LB substratum that contain kantlex.Erlenmeyer flask is placed in the shaking table of 37 ℃ and cultivates and approach 1.0 to OD600.Then toward the IPTG that adds 0.2mM in Erlenmeyer flask 37 ℃ of inducing culture 3 hours.The bacterium that centrifugal collection is cultivated, then adds lysate (20mM Tris, 100mM NaCl, 1%Triton ^tMx-100, and proteinase inhibitor).The bacterium liquid of cracking under the speed of 12000g centrifugal 30 minutes, collects supernatant.Supernatant is joined on a nickel post, according to nickel post manufacturer's explanation purifying, obtain pure EGFR albumen.Then the EGFR albumen of every kind of purifying of 0.1 μ g is mixed with isopyknic 2 * SDS sample-loading buffer respectively, and 95 ℃ of heating 10 minutes.

When using mammalian cell expression, the cDNA sequence clone of coding EGR wild-type and EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutein, in mammalian expression vector pCNDA3.0, and is merged and forms an open reading frame with the sequence of an encoding green fluorescent protein (GFP).The plasmid building is transformed in DH5 α intestinal bacteria, to increase and extracting plasmid.In transfection the day before yesterday, HEK293T cell is taped against in 24 porocyte culture plates with 30% density, be placed on the 5%CO of 37 ℃ ₂in incubator, cultivate.Then the plasmid that contains every kind of EGFR albumen of 0.5 μ g expression is transfected in HEK293T cell by the method for the calcium phosphate transfection of standard.The cell of transfection continues to cultivate 24 hours.Then by the cell harvesting of transfection, every porocyte adds the cell pyrolysis liquid of 20 μ l, places 10 minutes on ice.Then by cell under 12000 speed that turn centrifugal 10 minutes, collect supernatant.Get respectively the supernatant of every kind of cell pyrolysis liquid of 10 μ l, mix with isopyknic 2 * SDS sample-loading buffer, and 95 ℃ of heating 10 minutes.

Every kind of ready EGFR protein sample of 10 μ l is joined on 10% SDS-PAGE albumin glue.Under 90 volts of voltages, electrophoresis is 90 minutes.Then by protein delivery separated on glue to pvdf membrane.Skimmed milk sealing pvdf membrane with 5%.Then transfer printing being had the pvdf membrane of EGFR albumen to be put in the TBS damping fluid of the antibody that contains purifying in embodiment 1 hatches 16 hours at 4 ℃.Then remove antibody, with containing 0.1% tween

three pvdf membranes of TBS damping fluid rinsing of-20, each rinsing 5 minutes.Then film is put into two anti-the hatching 30 minutes of the anti-mouse that contains HRP-mark.Remove two and resist, again with containing 0.1% tween

three pvdf membranes of TBS damping fluid rinsing of-20, each rinsing 5 minutes.Then film is put in the solution that contains ECL substrate and infiltrated, and use light reaching the film in darkroom.Develop a film, develop, photographic fixing.

Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9 and Figure 10 show, in immunoblot experiment, the anti-EGFR G719A producing in embodiment 1, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L monoclonal antibody can be distinguished specifically in conjunction with EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutain, but not in conjunction with the EGFR albumen of wild-type, no matter and this albumen is at expression in escherichia coli, or express in mammalian cell.

Embodiment 3

Immunofluorescence experiment is analyzed these monoclonal antibody specificities in conjunction with the EGFR albumen of expressing in mammalian cell

Can express with the wild-type of GFP label and the pCDNA3.0 plasmid of EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L mutant egf R albumen and build according to the method for embodiment 2.Before transfection, the slide glass of aseptically process is put in 24 porocyte culture plates, and HEK293T cell is taped against in these holes with 30% density, be placed on the 5%CO of 37 ℃ ₂in incubator, cultivate.Then the plasmid that contains every kind of EGFR albumen of 0.5 μ g expression is transfected in HEK293T cell by the method for the calcium phosphate transfection of standard.The cell of transfection continues to cultivate 36 hours.Then cell is fixed to 10 minutes with 3.7% paraformaldehyde.And contain 1%Triton ^tMthe PBS damping fluid of X-100 is processed cell 10 minutes, to increase its permeability.Then with contain 5% what is said or talked about bovine serum PBS sealing treatment 10 minutes.Then the cell of these processing is placed on respectively in the damping fluid that contains the monoclonal antibody against EGFR that embodiment 1 produces and hatches 16 hours at 4 ℃.Then remove antibody, by PBS damping fluid rinsing three times for cell, each rinsing 5 minutes.Then add two the hatching 30 minutes in anti-of anti-mouse that is marked with fluorescence radiation thing.In two anti-solution, add DAPI dyestuff with staining cell core simultaneously.Remove two and resist, again use PBS damping fluid rinsing three times, each rinsing 5 minutes.Then the slide glass that is attached with cell is taken out, be fixed on slide glass and with nail varnish and seal.Observation of cell taking pictures under fluorescent microscope.

Figure 11, Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17, Figure 18, Figure 19 and Figure 20 show, in immunofluorescence experiment, anti-EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or the V769L monoclonal antibody of in embodiment 1, producing can be combined in respectively anti-EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or the V769L mutain of expressing in mammalian cell specifically, but not in conjunction with the EGFR albumen of wild-type.

Above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can modify or be equal to replacement technical scheme of the present invention, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in the middle of claim scope of the present invention.

SWQMENCE LISTINE

<110> Wuhan Niu Site Bioisystech Co., Ltd

Monoclonal antibody, preparation method and the application thereof of <120> specific recognition EGFR mutain

<160> 10

<210> 1

<211> 18

<212>　PRT

<213> artificial sequence

<400> 1

ETEFKKIKVLASGAFGTV 18

<210> 2

<211> 18

<212> PRT

<213> artificial sequence

<400> 2

ETEFKKIKVLCSGAFGTV 18

<210> 3

<211> 18

<212> PRT

<213> artificial sequence

<400> 3

ETEFKKIKVLSSGAFGTV 18

<210> 4

<211> 18

<212> PRT

<213> artificial sequence

<400> 4

EATSPKANKEILYEAYVM 18

<210> 5

<211> 18

<212> PRT

<213> artificial sequence

<400> 5

KTPQHVKITDFGRAKLLG 18

<210> 6

<211> 16

<212> PRT

<213> artificial sequence

<400> 6

ITDFGLAKQLGAEEKE 16

<210> 7

<211> 19

<212> PRT

<213> artificial sequence

<400> 7

DRRLVHRDLTARNVLVKTP 19

<210> 8

<211> 16

<212> PRT

<213> artificial sequence

<400> 8

IAKGMSYLEDRRLVHR 16

<210> 9

<211> 16

<212> PRT

<213> artificial sequence

<400> 9

ITDFGLAKLLDAEEKE 16

<210> 10

<211> 13

<212> PRT

<213> artificial sequence

<400> 10

EAYVMASLDNPHV 13

Claims (31)

1. the monoclonal antibody of specific recognition EGFR mutain, it is characterized in that, can not identify the monoclonal antibody of EGFR wild-type protein, or antigen-binding proteins, the EGFR mutain of this monoclonal antibody specificity identification comprises: the glycine (Glycine of the 719th, G) sport the mutain (EGFR G719A) of L-Ala (Alanine, A); The glycine (G) of the 719th sports the mutain (EGFR G719C) of halfcystine (C); The glycine (Glycine, G) of the 719th sports the mutain (EGFR G719S) of Serine (Serine, S); The aspartic acid of the 761st (Asparatic Acid, D) sports the mutain (EGFR D761Y) of tyrosine (Tyrosine, Y); The leucine (Leucine, L) of the 858th sports the mutain (EGFR L858R) of arginine (Arginine, R); The leucine (Leucine, L) of the 861st sports the mutain (EGFR L861Q) of glutamine (Glutamine, Q); The L-Ala (Alanine, A) of the 839th sports the mutain (EGFR A839T) of Threonine (Threonine, T); The l-asparagine (Asparagine, N) of the 826th sports the mutain (EGFR N826S) of Serine (Serine, S); The glycine (Glycine, G) of the 863rd sports the mutain (EGFR G863D) of aspartic acid (Asparatic Acid, D); The α-amino-isovaleric acid (Valine, V) of the 769th sports the mutain (EGFR V769L) of paddy leucine (Leucine, L).

2. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:1.

3. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:2.

4. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:3.

5. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:4.

6. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:5.

7. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:6.

8. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:7.

9. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:8.

10. the preparation method of the monoclonal antibody of specific recognition EGFR mutain according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:9.

The preparation method of the monoclonal antibody of 11. specific recognition EGFR mutains according to claim 1, is characterized in that, specific binding is in the EGFR albumen that contains peptide sequence SEQ ID NO:10.

The preparation method of the monoclonal antibody of 12. specific recognition EGFR mutains according to claim 1, is characterized in that, comprises antigen-binding site, a heavy chain and a light chain, consists of, the CDR1 of heavy chain and light chain, the aminoacid sequence of CDR2 and CDR3 is to be respectively CCTCC C201301 by Chinese Typical Representative culture collection center (CCTCC) preserving number, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCC C2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, the CDR1 of the monoclonal antibody that the hybridoma of CCTCC C201304 and CCTCC C201316 produces, the aminoacid sequence of CDR2 and CDR3 determines.

The preparation method of the monoclonal antibody of 13. specific recognition EGFR mutains according to claim 12, it is characterized in that, comprise antigen-binding site, the monoclonal antibody heavy chain that described hybridoma produces and the composition of light chain comprise respectively the Variable Area of heavy chain and light chain.

The preparation method of the monoclonal antibody of 14. specific recognition EGFR mutains according to claim 12, it is characterized in that, comprise antigen-binding site, heavy chain and the light-chain amino acid sequence of the monoclonal antibody that the hybridoma described in including produces.

The monoclonal antibody of 15. specific recognition EGFR mutains according to claim 1, is characterized in that, comprises antigen-binding site, is a kind of humanization or mosaic type antibody.

16. according to the preparation method of the monoclonal antibody of the specific recognition EGFR mutain described in claim 2 to 13 and 15 any one, it is characterized in that, described monoclonal antibody is a kind of IgG.

The monoclonal antibody of 17. specific recognition EGFR mutains according to claim 1, it is characterized in that, the preserving number that is kept at Chinese Typical Representative culture collection center (CCTCC) is ten strain of hybridoma of CCTCC C201301, CCTCC C2012127, CCTCC C2012118, CCTCC C201303, CCTCCC2012125, CCTCC C201315, CCTCC C201302, CCTCC C201320, CCTCC C201304 and CCTCC C201316.

The application of the monoclonal antibody of 18. specific recognition EGFR mutains, is characterized in that, the medicine of this monoclonal antibody or antigen-binding site, and carrier that can be medicinal.

The application of the monoclonal antibody of 19. specific recognition EGFR mutains, is characterized in that, a kind of diagnostic kit of diagnosing EGFR sudden change, and its composition contains any one monoclonal antibody or the antigen-binding site described in claim 1 to 14.

The application of the monoclonal antibody of 20. specific recognition EGFR mutains, is characterized in that, a kind of method of diagnosing tumour, is characterized in that, comprises the following steps:

(1) biological specimen detecting with any one monoclonal antibody described in claim 1 to 14 or antigen-binding site contact need;

(2) detect monoclonal antibody or antigen-binding site and whether be combined with sample, and have cancer or develop into the risk of cancer in conjunction with indication sample.

21. the application of the monoclonal antibody of specific recognition EGFR mutain according to claim 20, is characterized in that, in described diagnostic method, common cancer types comprises: lung cancer, nonsmall-cell lung cancer, the rectum cancer, colorectal carcinoma, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, glioma, the cancer of the brain.

The application of the monoclonal antibody of 22. specific recognition EGFR mutains, is characterized in that, a kind of method for the treatment of cancer patient, is characterized in that:

(1) detect in the biological sample come from patient, whether to contain EGFR G719A, G719C, G719S, D761Y, L858R, L861Q, A839T, N826S, G863D or V769L sudden change;

(2) if there is said mutation, this patient is given to the treatment of EGFR protein inhibitor.

The application of the monoclonal antibody of 23. specific recognition EGFR mutains according to claim 22, it is characterized in that, in described methods for the treatment of, EGFR protein inhibitor comprises: sense-rna (antisense RNA), little intervening rna (siRNA), Microrna (miRNA) medicine, monoclonal antibody against EGFR medicine.EGFR inhibitor can be Cetuximab (cetuxmab), Victibix (panitumumab), Necitumumab, Nimotuzumab, Gefitinib (gefitinib), Tarceva (erlotinib), lapatinibditosylate (lapatinib), ZD6474 (Vandetanib), PF299804, RO5083945, ABT-806, AP-26113, Afatinib (BIBW2992), Canertinib (CI-1033), Neratinib (HKI-272), CP-724714, TAK-285, AST-1306, ARRY334543, Lapatinib, Arry-380, Dacomitinib (PF-299804), WHI-P154, Desmethyl Erlotinib (CP-473420), Desmethyl Erlotinib(OSI-420), AZD8931, AEE788 (NVP-AEE788), Pelitinib (EKB-569), CUDC-101, WZ804, WZ4002, WZ3146, XL647, PD-153035, PD-174265, PD158780, BMS-599626 (AC480), Lavendustin A, Lavendustin C, BIBU-1361, Hypericin, BIBX-1382, DAPH, Erbstatin Analog, JNJ28871063, RO 106-9920, GW2974, GW583340, PD-153035, PD-158780, PD-168393, Tyrphostin AG-183, Tyrphostin AG-99, Tyrphostin AG-825, Tyrphostin B42(AG-490), Tyrphostin AG-494, Tyrphostin AG-555, Tyrphostin AG-527, Tyrphostin AG-528, Tyrphostin AG-835, Tyrphostin AG-1478, Tyrphostin RG-14620, Daphnetin, BPIQ, BPIQ-I, BPIQ-II, CL-387, 785, EGFR Inhibitor(K00598a), LFM-A12, Pelitinib, PKC-412, PP3(5334-30-5), SU5402, HDS029, WHI-P154, TAK165, Vatalanib.

The application of the monoclonal antibody of 24. specific recognition EGFR mutains according to claim 22, is characterized in that, the method for described treatment cancer patient gives the medicine of the moiety that patient contains claim 11.

25. according to the application of the monoclonal antibody of the specific recognition EGFR mutain described in claim 22 or 24, it is characterized in that, in the method for described treatment cancer patient, common cancer types comprises: the rectum cancer, papillary thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, lung cancer.

The application of the monoclonal antibody of 26. specific recognition EGFR mutains, is characterized in that, the production process by the monoclonal antibody of claim 1 to 15 or antigen-binding site for cancer treatment drugs, common cancer species comprises: malignant melanoma, the rectum cancer, thyroid carcinoma, carcinoma of the pancreas, esophagus cancer, prostate cancer, ovarian cancer, mammary cancer, lung cancer, or hemopoietic tissue cancer.

The application of the monoclonal antibody of 27. specific recognition EGFR mutains, it is characterized in that, a kind of nucleic acid molecule of purifying, the heavy chain or the one antigen-binding site that comprise monoclonal antibody described in the claim 1 to 15 of can encoding, or light chain or the one antigen-binding site of can encoding, or the two the nucleotide sequence of can simultaneously encoding.

The application of the monoclonal antibody of 28. specific recognition EGFR mutains according to claim 27, is characterized in that, a kind of carrier molecule contains the expression regulation sequence that can relate to this nucleic acid molecule.

29. according to the application of the monoclonal antibody of the specific recognition EGFR mutain of claim 27, it is characterized in that a kind of host cell contains this nucleic acid molecule.

The application of the monoclonal antibody of 30. specific recognition EGFR mutains, is characterized in that, a kind of cell strain that comprises claim 1 to 14, and it contains described monoclonal antibody or its antigen-binding site.

The application of the monoclonal antibody of 31. specific recognition EGFR mutains, it is characterized in that, a kind of method of manufacture order clonal antibody or its antigen-binding site, the EGFR albumen of the antibody of producing or antigen-binding site specific binding human mutant, and not in conjunction with the EGFR albumen of human wild type, its step comprises:

(1), according to claim 29, cultivate under suitable condition this host cell, or according to claim 30, under the condition discharging, cultivate this cell strain;

(2) the described antibody moiety of purification.

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