CN108250297A - Anti-egfr antibodies, its preparation method and its application - Google Patents

Abstract

The present invention relates to anti-egfr antibodies, its preparation method and its applications.The present invention provides the albumen containing unique complementary determining region, and the function of EGFR can be specifically bound and effectively inhibited with EGFR, alleviate or treatment EGFR is overexpressed relevant disease, such as tumour.

Description

Anti-egfr antibodies, its preparation method and its application

Technical field

The invention belongs to biomedicine field, specifically, the present invention relates to anti-egfr antibodies, its preparation method and its applications.

Background technology

EGF-R ELISA (The epidermal growth factor receptor, EGFR, erbB1, HER1 it is) one of four members of HER/ErbB receptor families, belongs to tyrosine kinase growth factor receptor.EGFR is one Molecular weight is the transmembrane glycoprotein of 170KD, mainly by the extracellular region of participation ligand binding, transmembrane region and with tyrosine kinase The intracellular region three parts of activity are formed.The main ligand of EGFR is epidermal growth factor (epidermal growth Factor, EGF) and transforming growth factor α (transforming growth factor- α, TGF- α), EGFR and ligand knot After conjunction, homologous or Heterodimerization occurs, causes EGFR intracellular tyrosine kinase activities area that phosphorylation occurs, and cell is believed It number hands on, and then proliferation and differentiation, the decline of Apoptosis, revascularization, tumor invasion and metabasis for leading to cell etc..

Research shows that in many entity tumors, such as non-alveolar cells lung cancer, colon cancer, kidney, oophoroma, head and neck cancer, food (Woodburn JR, the Pharmacol Ther 1999 such as road cancer, prostate cancer, cancer of pancreas, breast cancer, glioma；82: 241-50；Yarden Y, Eur J Cancer 2001；37:S3-S8；Mendelsohn J etc., Oncogene 2000；19: High expression or unconventionality expression, the mechanism that EGFR signal paths are abnormal in 6550-65) there are EGFR have：EGFR's It is overexpressed；EGFR is mutated；The effect enhancing of autocrine loop；The increase of ligand expression；The destruction of receptor down-regulated mechanism；Abnormal letter Activation of number Signal Transduction Pathways etc. (Roza Zandi etc., Cellular Signalling, 2007,19:2013-2023).

At present, the drug of targeting EGFR treatment mainly has two classes：1. for the monoclonal antibody of EGFR extracellular domains part, Such as Cetuximab (IMC-225, Erbitux), Matuzumab (EMD72000), Panitumumab (ABX-EGF) and IMC- 11F8 can block ligand molecular to the activation of EGFR or interfere the formation of EGFR dimerizations；2. for small point of EGFR kinases area Sub- kinase activity inhibitor, such as Gefitinib (Iressa), Erlotinib (Tarceva) and Sorafenib (Nexavar), Analog competitive binding as ATP inhibits the transduction of EGFR signal paths in the TK areas of EGFR.

There are many antibody of targeting EGFR at present to have come into clinical trial or have been applied to clinic In treatment, but the indication of different antibodies has differences at present, as Cetuximab and Panitumumab monoclonal antibodies only should at present In several limited cancers such as colon cancer, head and neck cancer, lung cancer.

The Treatment need of the entity tumor of high expression or unconventionality expression in view of other more EGFR, this field has Necessity develops more EGFR antibody to meet this needs.

Invention content

The purpose of the present invention is to provide anti-egfr antibodies, its preparation method and its applications.

In the first aspect of the present invention, the binding protein of specific binding EGFR is provided, which has light chain can Become area and heavy chain variable region, and, the amino acid sequence such as SEQ ID NO of the CDR1 of heavy chain variable region:Shown in 7, the ammonia of CDR2 Base acid sequence such as SEQ ID NO:Shown in 8, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 9；Its light chain variable region The amino acid sequence of CDR1 such as SEQ ID NO:Shown in 10, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 11；CDR3's Amino acid sequence such as SEQ ID NO:Shown in 12；Or

The amino acid sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 19, the amino acid sequence of CDR2 is such as SEQ ID NO:Shown in 20, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 21；The amino of the CDR1 of its light chain variable region Acid sequence such as SEQ ID NO:Shown in 22, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 23；The amino acid sequence of CDR3 Such as SEQ ID NO:Shown in 24.

In a preference, the amino acid sequence such as SEQ ID NO of the protein-bonded heavy chain variable region: 26 Shown, the amino acid sequence of light chain variable region is as indicated at 28；Or the amino acid sequence of its heavy chain variable region such as SEQ ID NO: Shown in 30, the amino acid sequence such as SEQ ID NO of light chain variable region:Shown in 32.

In another preferred example, the binding protein is monoclonal antibody.

In another aspect of this invention, the protein-bonded polynucleotides of the coding specific binding EGFR are provided.

In a preference, the nucleotide sequence such as SEQ ID of the CDR1 of the heavy chain variable region of the polynucleotides NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of CDR2:Shown in 2, the nucleotide sequence such as SEQ ID NO of CDR3:3 institutes Show；The nucleotide sequence of the CDR1 of its light chain variable region such as SEQ ID NO:Shown in 4, the nucleotide sequence such as SEQ ID of CDR2 NO:Shown in 5；The nucleotide sequence of CDR3 such as SEQ ID NO:Shown in 6；Or

The nucleotide sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 13, the nucleotide sequence of CDR2 is such as SEQ ID NO:Shown in 14, the nucleotide sequence such as SEQ ID NO of CDR3:Shown in 15；The nucleosides of the CDR1 of its light chain variable region Acid sequence such as SEQ ID NO:Shown in 16, the nucleotide sequence such as SEQ ID NO of CDR2:Shown in 17；The nucleotide sequence of CDR3 Such as SEQ ID NO:Shown in 18.

In another preferred example, the nucleotide sequence of the heavy chain variable region of the polynucleotides such as SEQ ID NO: 25 It is shown, the nucleotide sequence such as SEQ ID NO of light chain variable region:Shown in 27；Or the nucleotide sequence of its heavy chain variable region is such as SEQ ID NO:Shown in 29, the nucleotide sequence such as SEQ ID NO of light chain variable region:Shown in 31.

In another aspect of this invention, a kind of expression vector is provided, it includes the polynucleotides.

In another aspect of this invention, a kind of host cell is provided, it includes whole in the expression vector or genome Conjunction has the polynucleotides.

In another aspect of this invention, a kind of binding protein composition is provided, the composition contains Binding Protein 1 With binding protein 2, which has light chain variable region and heavy chain variable region, and the amino of the CDR1 of its heavy chain variable region Acid sequence such as SEQ ID NO:Shown in 7, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 8, the amino acid sequence of CDR3 is such as SEQ ID NO:Shown in 9；The amino acid sequence of the CDR1 of its light chain variable region such as SEQ ID NO:Shown in 10, the amino of CDR2 Acid sequence such as SEQ ID NO:Shown in 11；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 12；The binding protein 2 has Light chain variable region and heavy chain variable region, and the amino acid sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 19, The amino acid sequence of CDR2 such as SEQ ID NO:Shown in 20, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 21；Its light chain The amino acid sequence of the CDR1 of variable region such as SEQ ID NO:Shown in 22, the amino acid sequence such as SEQ ID NO of CDR2:23 institutes Show；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 24.

In a preference, the Binding Protein 1 and binding protein 2 are with 1：10~10：1 ratio is present in group It closes in object；Preferably, with 1：5~5：1 ratio is present in composition；Such as it can also be 1：4~4：1 ratio, 1：3 ~3：1 ratio, 1：2~2：1 ratio.

In another aspect of this invention, the binding protein of the specific binding EGFR or the combination egg are provided White composition is preparing for the purposes in diagnosing, treat and/or preventing the drug of EGFR overexpression related diseases.

In a preference, the EGFR overexpression related diseases are：There are the entity tumors that EGFR is overexpressed.

In another preferred example, the entity tumor includes：Non- alveolar cells lung cancer, colon cancer, kidney, oophoroma, Head and neck cancer, cancer of the esophagus, prostate cancer, cancer of pancreas, breast cancer, glioma.

In another aspect of this invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition contains：A effective amount of institute The binding protein of specific binding EGFR stated；And pharmaceutically acceptable carrier.

In another aspect of this invention, a kind of pharmaceutical composition is provided, described pharmaceutical composition contains：A effective amount of institute The binding protein composition and pharmaceutically acceptable carrier stated.

In another aspect of this invention, a kind of medicine for being used to treating and/or preventing EGFR overexpression related diseases is provided Box, the medicine box include：The binding protein of the specific binding EGFR；The binding protein composition；It is or described Pharmaceutical composition.

In another aspect of this invention, a kind of immune stop is provided and closes object, the immune conjunction object that stops includes：The spy The opposite sex combines the binding protein of EGFR；And detectable marker；Preferably, the detectable marker includes：Fluorescent marker Object, chromogenic label.

In another aspect of this invention, a kind of detection kit for being used to detect EGFR levels, the detection kit are provided Include：The binding protein of the specific binding EGFR；Or immune stop closes object.

The other aspects of the present invention are apparent to those skilled in the art due to this disclosure 's.

Description of the drawings

Fig. 1, EGFR monoclonal antibody FACS results.Shown in figure：Mouse source antibody Mab79, Mab87 flow cytometry results.Black Represent control group, green line represents antibody group, and M1 represents control group fluorescence shift amount, and M2 represents antibody assay group fluorescence shift Amount.

Fig. 2, EGFR antibody Mab79, Mab87 and western appropriate former times in vitro act on A431 cell inhibitory effects.

Fig. 3, A431 neoplasm transplantation are tested.

Specific embodiment

The present inventor after extensive and in-depth study, obtains a kind of spy for containing unique complementary determining region (CDR region) The opposite sex combines the binding protein of EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR), Can with EGFR specifically bind and effectively inhibit EGFR, can be applied to treatment it is related to EGFR overexpressions or by The disease of EGFR function effects.

The different characteristics that different antibody has such as combine epitope, affinity, Antibody types, frequently can lead to The different direct or indirect effects for inhibiting EGFR downstream signal transductions, this is exactly the starting point of the present invention.The present inventor endeavours In the monoclonal antibody for finding the new epitope of targeting EGFR, monoclonal antibody epitope of the invention is in EGFR the IIIth relevant with ligand binding Structural domain, and it is different from other existing monoclonal antibodies.The anti-Mab87 epitope sites of mouse：N 389, N 420, T 422, mouse resists Mab79 epitope sites:F 352、R 353、G 354、F 357、H 359、 L 363、W 386.

The present inventor has been unexpectedly discovered that, Mab79 of the invention and Mab87 use in conjunction, compared with being applied alone, Neng Goucheng Existing synergistic effect, becomes apparent the effect for inhibiting tumour.

Binding protein

The present invention provides the binding proteins that can specifically bind EGFR.The binding protein of the present invention can be complete Immunoglobulin molecules or antigen-binding fragment, including but not limited to Fab segments, Fd segments, Fv segments, F (ab’)₂Segment, complementary determining region (CDR) segment, single-chain antibody (scFv), domain antibodies, bivalent single-chain antibodies single-stranded are bitten Somatic antibody, double specific duplex antibody, three chain antibodies, four chain antibodies etc..

CDR region is the sequence of the interested protein of immunology.Binding protein of the present invention may include disclosed herein Two, three, four, five or all six CDR regions.Preferably, binding protein of the present invention include it is disclosed herein at least Two CDR.

Another aspect of the present invention includes protein-bonded functional variety described herein.If become physical efficiency to be combined with parental generation Protein competition specifically binds EGFR, then it is assumed that the Variant molecules are the protein-bonded functional varieties of the present invention.In other words, The functional variety remains to combine or its segment.It is substantially similar but contain that functional variety includes but not limited to primary structural sequence There is the not found derivative of chemistry and/or biochemical modification in vitro or in vivo for example in parental generation binding protein.It is this to repair Decorations include the covalent of second phthalein, phthalein, the nucleotide either covalent attachment of nucleotide derivative, lipid or lipid derivate Attachment, crosslinking, disulfide bond formation, glycosylation, hydroxylating, methylate, aoxidize, Pegylation, proteolysis processing, phosphoric acid Change etc..In other words, the modification in the protein-bonded amino acid of parental generation and/or nucleotide sequence do not significantly affect or change by The protein-bonded binding characteristic described nucleotide sequence coded or containing the amino acid sequence, i.e., described knot Hop protein remains to identify and with reference to its target position.

The functional variety can have conserved sequence modification, including nucleotide and amino acid substitution, addition and missing. These modifications can be imported by the known standard technique in this field, such as the mutagenesis of directed mutagenesis and random PCR mediation, and And it may include natural and non-natural nucleotides and amino acid.

Conserved amino acid substitution includes wherein amino acid residue by another amino with similar structure or chemical property The substitution of sour residue displacement.The family of amino acid residue with similar side chain oneself through limiting in the art.These families wrap Include the amino acid (such as lysine, arginine, histidine) with basic side chain, acidic side chains (such as asparagus fern ammonia Acid, glutamic acid), without charge polarity side chain amino acid (such as asparagus fern phthalein amine, paddy ammonia phthalein amine, serine, threonine, tyrosine, Cysteine, tryptophan), nonpolar side chains (such as glycine, alanine, valine, leucine, isoleucine, Proline, phenylalanine, methionine), branched side chains (such as threonine, valine, isoleucine) and fragrance Side chain amino acid (such as tyrosine, phenylalanine, tryptophan).It will be appreciated that it can also use in addition to above-mentioned Other amino acid residue families mode classifications except family.In addition, variant can have non-conservative amino acid substitution, such as Amino acid is by another radical amino acid replacement with different structure or chemical property.Similar small variation may also comprise ammonia Base acid missing is either inserted into or both.It can be found which amino acid determined using computer program well known in the art The guidance that residue can be substituted, be inserted into or be lacked without eliminating immunologic competence.

Functional variety may include amino acid sequence in amino terminal either carboxyl terminal or the truncate at this both ends.This The functional variety of invention can have identical or different, higher or lower binding affinity compared with parental generation binding protein, but It is to remain to combine EGFR or its segment.For example, the present invention functional variety compared with parental generation binding protein for EGFR or its piece Section can have the binding affinity for increasing or decreasing and (preferably increasing).Preferably, variable region includes but not limited to framework region, height The amino acid sequence for becoming area or CDR region is modified.In general, light chain and heavy chain variable region include three hypervariable regions, including three CDR and more conservative region, i.e., so-called framework region (FR).Hypervariable region includes the amino acid residue from CDR and comes from The amino acid residue of hypervariable loop.Functional variety within the scope of the present invention has at least big with parental generation binding protein described herein About 50% to about 99%, be preferably at least about 60% to about 99%, more preferably at least about 70% to about 99%, very To more preferably at least about 80% to about 99%, most preferably at least about 90% to about 99%, particularly at least about 95% to about 99% and the especially at least amino acid sequence homology of about 97% to about 99%.Art technology Computerized algorithm known to personnel such as Gap or Bestfit can be used for most preferably arranged amido acid sequence to be compared and Specify similar or identical amino acid residue.Functional variety can be by using the known common molecular biology side in this field Method changes parental generation binding protein or part of it and obtains, the method includes but be not limited to fallibility PCR, oligonucleotides guidance Mutagenesis, direct mutagenesis and heavy chain and/or light chain reorganization method.

As the preferred embodiment of the present invention, the binding protein is monoclonal antibody.The antigenic binding property of antibody can It is described by 3 that are located at heavy chain and light chain variable region specific regions, referred to as complementary determining region (CDR), the CDR region Variable region is partitioned into 4 frame areas (FR), the amino acid sequence of 4 FR is relatively conservative, does not participate in directly combining anti- It should.These CDR form cyclic structure, and the β-pleated sheet formed by FR therebetween is close to each other on space structure, on heavy chain CDR on CDR and corresponding light chain constitutes the antigen binding site of antibody.The CDR region of the monoclonal antibody against EGFR of the present invention It is completely new, different from existing anti-egfr antibodies.

The binding protein of the present invention can also be the albumen after being blended with IgG Fc.

Another aspect of the present invention provides at least one binding protein of the coding present invention, its functional variety or immune The nucleic acid molecules of conjugate.This nucleic acid molecules may be used as intermediary to be cloned, such as affinity as described above In maturation method.In a preferred embodiment, the nucleic acid molecules are isolated or purifieds.The sequence of DNA molecular can To be obtained with routine techniques or using hybridoma technology.

Once obtain related sequence, it is possible to obtain related sequence in large quantity with recombination method.This is typically will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method Row.

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

At present, it is already possible to completely by chemical synthesis come obtain coding the present invention binding protein (or its segment or Its derivative) DNA sequence dna.Then the DNA sequence dna can be introduced to various existing DNA moleculars as known in the art (or such as Carrier) and cell in.In addition, it can will be also mutated by chemical synthesis in protein-bonded sequence incorporated in the present invention.

The invention further relates to include above-mentioned appropriate DNA sequence dna and the carrier of appropriate promoter or control sequence.This A little carriers can be used for converting appropriate host cell, allow it to expression protein.It is inserted respectively in the expression vector Enter the mouse resource monoclonal antibody heavy chain variable region (VH) of anti-EGFR and the Human monoclonal antibody heavy chain constant region (perseverance from humanized IgG 1 Determine area) the mouse resource monoclonal antibody light chain variable region VL of fusion sequence and anti-EGFR with Human monoclonal antibody constant region of light chain (from people The constant region of source Iglambda) fusion sequence.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；It is or high Wait eukaryocytes, such as mammalian cell.Representative example has：Bacterial cell such as Escherichia coli, streptomyces；Mouse typhus is husky Door Salmonella；Fungal cell's such as yeast；Plant cell；Insect cell such as drosophila S2 or Sf9；Zooblast such as CHO, COS7, NSO Or Bowes melanoma cells etc..Host cell especially suitable for the present invention is eukaryotic host cell, especially mammal Cell, such as Chinese hamster ovary celI, 293 cells.

If desired, weight can be separated by various separation methods and purified using its physics, chemical and other characteristics The binding protein of group.These methods are well-known to those skilled in the art.The example of these methods includes but is not limited to： The renaturation process of routine handles (salting-out method), centrifugation, the broken bacterium of infiltration, supersound process, ultracentrifugation, molecule with protein precipitant Sieve chromatography (gel filtration), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and other various liquid chromatographies The combination of technology and these methods.

Pharmaceutical composition

The binding molecule of the present invention can be used for preparing diagnosis, treatment and/or prevention EGFR expression or the related disease of activity imbalance The pharmaceutical composition of disease.

" the EGFR overexpression related diseases " is including there are the entity tumors that EGFR is overexpressed.It is preferably, described " EGFR overexpression related diseases " includes but not limited to：Non- alveolar cells lung cancer, colon cancer, kidney, oophoroma, head and neck cancer, food Road cancer, prostate cancer, cancer of pancreas, breast cancer, glioma.

The EGFR monoclonal antibodies can also be used to detecting and quantifying EGFR, for various diagnostic purposes.

Based on the new discovery of the present invention, additionally provide a kind of diagnosable, treatment and/or prevention EGFR is overexpressed related disease The pharmaceutical composition of disease, it includes：A effective amount of binding molecule of the present invention；And pharmaceutically acceptable carrier.

The term as used herein " pharmaceutically acceptable " refer to when biomolecule ontology and composition suitably give animal or During people, unfavorable, allergy or other adverse reactions that they will not be generated." pharmaceutically acceptable carrier " used herein Should be compatible with the binding molecule of the present invention, it can be blended without composition is greatly lowered in general Effect.

Can be carbohydrate as the specific example of some of pharmaceutically acceptable carrier or its component substances, such as lactose, Portugal Grape sugar and sucrose；Starch, such as cornstarch and potato starch；Cellulose and its derivates, such as sodium carboxymethylcellulose, ethyl Cellulose and methylcellulose；Tragacanth powder；Malt；Gelatin；Talcum；Kollag, such as stearic acid and stearic acid Magnesium；Calcium sulfate；Vegetable oil, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cupu oil；Polyalcohol, such as the third two Alcohol, glycerine, D-sorbite, mannitol and polyethylene glycol；Alginic acid；Emulsifier, such asWetting agent, such as lauryl Sodium sulphate；Colorant；Flavoring agent；Tablet agent, stabilizer；Antioxidant；Preservative；Apirogen water；Isotonic salting liquid；And phosphorus Phthalate buffer etc..

Various dosage forms can be made, and can be by doctor according to patient category, year in the pharmaceutical composition of the present invention as needed Age, weight and substantially the factors such as disease condition, administering mode determine that the dosage beneficial to patient is administered.Administering mode example Injection or other therapeutic modalities such as may be used.

The binding molecule of the present invention can be used in the form of unsegregated or separation.In addition, the combination point of the present invention Son can be used alone or should in the mixture of the binding molecule (or its variant or segment) comprising at least one present invention With.In other words, the binding molecule can with combination application, such as comprising two or more kind the present invention binding molecule, The pharmaceutical composition of its variant or segment.For example, it can be combined in a treatment with different but complementary activity binding molecules To reach desired prevention, treatment or diagnostic effect in scheme, but can also be by the binding molecule with identical activity It combines in a therapeutic scheme to reach desired prevention, treatment or diagnostic effect.

The binding molecule or pharmaceutical composition of the present invention can be examined before for human body in suitable animal model system It surveys.This animal model system includes but not limited to mouse, monkey.

The suitable dosage range of the binding molecule of the present invention can be for example 0.001-100mg/kg weight, preferably 0.01- 15mg/kg weight.It once injects in addition, can for example give, give multiple separate doses at any time or according to treatment Emergency and can reduce in proportion or incremental dose.The molecule and composition of the present invention is preferably sterile.Cause these The method of molecule and composition sterile is known in the art.It can be with for other molecules for diagnosing, prevent and/or treating The dosage regimen similar to the binding molecule of the present invention is given.If individually giving other molecules, the present invention can given One or more binding molecules or pharmaceutical composition before, simultaneously or after give patient.Accurate administration for people patient Scheme is usually picked out during clinical trial.

Binding molecule of the present invention can be placed in appropriate packaging, and medicine box is made, in order to which clinician makes With.Preferably, it also may include illustrating how the operation instructions of administration in the medicine box.

The invention also includes the methods alleviated or treat EGFR overexpression related diseases, and the method includes giving patient At least one monoclonal antibody of a effective amount of present invention.It, can also be by the monoclonal of the present invention when for antineoplaston Antibody and other antitumor medicaments or therapy (such as chemotheraping preparation or radiotheraping method) use in conjunction.

Immunoconjugates

On the other hand, the present invention includes immunoconjugates, i.e., comprising at least one binding protein described herein and into one Step includes at least one functional molecular (molecule of such as detectable part/substance).The antibody and the functional molecular It can be by being covalently attached, being coupled, adhering to, the modes such as being crosslinked and form to stop and close object.The immunoconjugates of the present invention may include one Above label.The label can also by the binding protein of covalent bond and the present invention directly in conjunction with/it is conjugated.It is alternatively, described Label can be combined/be conjugated with the binding protein by one or more connection compounds.Label and protein-bonded conjugated skill Art is well known to those skilled in the art.The label of the immunoconjugates of the present invention can also be therapeutic agent.

The immunoconjugates may include：The antibody and detectable marker of the present invention.The detectable marker Including but not limited to：Fluorescent marker, chromogenic label；Such as：Enzyme, prothetic group, fluorescent material, luminescent material, bioluminescence material Material, radioactive material, positron emitting metal and on-radiation paramagnetic metal ion.Also it may include more than one mark Remember object.In order to detect and/or analyze and/or diagnostic purpose for labelled antibody label dependent on use particular detection/point Analysis/diagnostic techniques and/or method are such as immunohistochemical staining (tissue) sample, flow cytometry.For ability Detection/analysis/diagnostic techniques known to domain and/or method suitably label are well known to those skilled in the art.

In addition, the human conjugated protein or immunoconjugates of the present invention can also be attached on solid support, especially For the in vitroimmunoassay or purifying of EGFR albumen or its segment.This solid support can be porous or non-porous , plane or nonplanar.The binding protein of the present invention can merge to purify with flag sequence.The flag sequence Example include but not limited to six histidine marks, hemagglutinin (HA) label, myc label or flag label.It is alternatively, a kind of Antibody can form antibody heteroconjugate (heteroconjugate) with another antibody conjugate.

Detection reagent and kit

Based on binding molecule of the present invention, it can prepare easily and fast and accurately detect in sample to be tested The reagent or kit of EGFR levels.

As used herein, term " sample to be tested " covers various samples type, blood including biological origin and its Its humoral sample, solid tissue sample such as tissue biopsy sample either tissue culture or derived from cell therein or Its offspring.The term is additionally included in obtain after the sample that has been handled by any mode, such as handled with reagent, dissolve or Person is enriched with certain ingredient such as protein or polynucleotides.

Therefore, the present invention provides a kind of for detecting the detection kit of EGFR levels in sample to be tested, the kit In the immunoconjugates that form of the EGFR binding molecules containing the present invention or EGFR binding molecules and detectable marker.

After EGFR binding molecules provided by the invention are obtained, can easily it prepare for specific detection The detection kit of EGFR levels.

In order to be more convenient when detecting, in addition to containing the binding molecule of the present invention or containing EGFR knots in the kit It closes other than the immunoconjugates that molecule is formed with detectable marker, other detection reagents or auxiliary reagent, institute can also be included The auxiliary reagent stated is, for example, some conventional use of reagents in ELISA kit, the characteristic of these reagents and they match Method processed is well-known to those skilled in the art, such as color developing agent, marker, secondary antibody, antiantibody, sensitizer.This field Personnel should be understood that the detection kit of various change form is included in the present invention, as long as this hair is utilized wherein Reagent of the bright binding molecule as identification EGFR.

In addition, it also may include operation instructions in the kit, for illustrating the user of reagent wherein loaded Method.

After binding molecule and/or kit provided by the invention is obtained, panimmunity correlation technique can be utilized EGFR or its content in sample are detected, so as to learn that the donor of sample to be tested whether there is EGFR overexpression related diseases.

With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.Test method without specific conditions in the following example, usually according to routine Condition (such as can refer to usually according to normal condition such as《Molecular cloning：Lab guide》Described in condition or according to system Make the condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.Unless otherwise defined, Wen Zhong Used all professional and scientific terms have the same meanings as commonly understood by one of ordinary skill in the art.In addition, it is any with it is recorded The similar or impartial method of content and material all can be applied in the present invention.Preferred implement methods and materials described herein are only It presents a demonstration and is used.

The preparation of embodiment 1, EGFR mouse resource monoclonal antibodies

First, the preparation of EGFR monoclonal antibody hybridoma cell strain

1st, immunogene

Immunogene is EGFR extracellular regions overall length (EGFR-ECD), passes through CHO/dhfr^-Cell (being purchased from ATCC) surely turns table It reaches, purifying cells culture supernatant obtains.

2nd, Balb/c mouse are immunized

Balb/c mouse are purchased from Shanghai Si Laike experimental animals Co., Ltd, all immune equal with Balb/c mouse The inbred mice disease-free, healthy for 3 week old, female, standardization, meets US FDA standard.

3rd, Balb/c mouse immunization protocols

It is immune for the first time：By 50 μ g (250 μ l) antigens and 250 μ lMF59 adjuvant mixings, 500 μ l solution are configured to, are injected In the subcutaneous multiple spot of Balb/c mouse and vola.Second immune：Away from first time immunization interval after three weeks, it is carried out with method with dosage Second immune.Third and fourth time immune：After two weeks away from second immunization interval, with method with dosage carry out third and fourth time it is immune. Four immunized mice tail vein bloods measure serum antibody titer using conventional enzyme-linked immunosorbent assay (ELISA method), Treat serum titer>10⁵More than when, go to cell fusion.First three day of cell fusion carries out booster immunization：Mouse tail vein is noted Penetrate 20 μ g of antigen (100 μ l).

4th, cell fusion

Sp2/0-Ag14 murine myeloma cells are purchased from ATCC companies of the U.S..

(1) fusion the previous day changes liquid, and Sp2/0-Ag14 myeloma cell is made to keep good growth conditions.

(2) splenocyte：Immune mouse, bloodletting are taken, neck sudden death of breaking impregnates 3-4min in 75% alcohol.It is taken under aseptic condition Go out mouse spleen, be put into 15ml centrifuge tubes, add in a little serum-free RPM 1640 culture mediums, gently blown and beaten with pipette, ground It is broken, until uniform without tissue caking, cell.Then, Mouse spleen cells are washed with serum-free RPM 1640 culture mediums Three times, it counts spare.

(3) take the logarithm the Sp2/0-Ag14 myeloma cell in growth period, and serum-free RPM 1640 culture mediums are washed three times, It counts spare.

(4) by Mouse spleen cells and Sp2/0-Ag14 myeloma cell with 10：1 ratio mixing, 1500rpm centrifugations 7min.Supernatant is washed away, prepares fusion.

1ml PEG (1450), jog 90sec are slowly added into (5) one minutes；Again in 2.5min it is interior addition 5ml without blood Clear RPM 1640 culture mediums finally add 5ml serum-free mediums and terminate reaction, after standing 5min, 1280rpm centrifugation 8min, It discards supernatant, adds in routine RPM 1640 culture mediums (containing 10% fetal calf serum), be prepared into cell suspension.

(6) by above-mentioned cell suspension with every hole 2 × 10⁴The density kind of a cell enters 96 orifice plates, per 200 μ l of hole, is placed in 37 DEG C, 5%CO₂It is incubated in cell incubator.After culture for 24 hours, the conventional RPM 1640 culture mediums containing HAT (25 ×) are replaced, in 37 DEG C, 5%CO₂Continue to be incubated in cell incubator.It is sieved after cultivating 14d with the supernatant of each clone cell of ELISA method detection Select the positive colony of EGFR antibody.

5th, cell screening and subclone

The conventional RPM 1640 culture mediums containing HAT is used to carry out culture screening first, after cultivating 7d, use the normal of HT instead The training of RPM 1640 culture mediums is advised, carries out culture screening again.After cultivating 14d, with ELISA method with the upper of each clone cell Clear liquid screens the positive colony of EGFR antibody.Using limiting dilution assay, cell suspension is diluted to 60/ml, in 96 orifice plates Add 100 μ l (about 6 cells/wells) per hole.2 rows are inoculated with, remaining cell suspension culture solution makees doubling dilution, inoculates 2 rows. It is repeated once.Put 37 DEG C, 5%CO₂It is incubated in cell incubator.Every 2~3 days, 1/2 culture solution is replaced.After cultivating about 10d, The positive hole of single clonal growth is selected to carry out programmed screening and subclone.After being continuously subcloned three times, examined through ELISA method The hybridoma cell strain for being determined as stablizing expression purpose antibody when antibody positive rate is 100% is surveyed, conservation builds library.

6th, the preparation and purification of mouse source monoclonal antibody

The last week injecting fluid paraffin is carried, Balb/c mouse more than 6 week old are only injected intraperitoneally in 0.5ml/, each hybridization Oncocyte injection Balb/c mouse 3.Pneumoretroperitoneum injection 1~2 × 10 in 7 days⁶A hybridoma took ascites every 7~10 days. Antibody purification procedures are carried out with reference to GE protein G-proteins column：

(1) ascites 12000rpm centrifuges 15min decontaminations and upper strata lipid-loweringing alkane, and 5 times of equilibration buffer (pH7.0PBS) is dilute It releases, with 0.45um membrane filtrations.

(2) protein G columns ddH₂10 columns of equilibration buffer pillar are used after O balance 5~10 column volumes of pillar Volume；

(3) according to the cementing carrying capacity loadings for closing 5mg albumen of 1ml protein G, (ascites antibody content is usually：1~ 5mg/ml)

(4) 10 column volume of pillar is balanced with equilibration buffer liquid；

(5) with 10 column volumes of elution (2.7 glycine-HCl of 0.1M pH), it is in charge of collection, elution albumen is fast The ratio that speed is added in 150ul 1M Tris-Cl pH 9.0 in every 1ml is neutralized.

(6) SDS-PAGE analyzes the amount and purity of antibody elution.

Embodiment 2, mouse monoclonal antibody affinity measure

Using proteon^TMXPR36Protein Interaction Array System methods measure EGFR monoclonals and resist The affinity of body.

Measurement result shows that Mab79 affinity KD values are：1.2E-09；Mab87 affinity KD values are： 1.3E-08.

The identification of embodiment 3, anti-EGFR monoclonal antibodies to cell surface EGFR molecules

It is detected using flow cytometry (FACS), indirect IF staining method.

(1) it counts：It takes the logarithm the A431 cells in growth period, adjusts single cell suspension a concentration of 2 × 10⁶/ml。

(2) it washs：1ml single cell suspensions is taken to add in 1.5ml centrifuge tubes, 1000rpm × 3min.Supernatant is abandoned, with 2% PBA (PBS adds 2% fetal calf serum) is washed and cell is resuspended, and 1000rpm × 3min abandons supernatant.

(3) primary antibody is incubated：Anti- EGFR monoclonal antibodies are diluted to 10 μ g/ml with 2%PBA, add in 200 μ l, it is thin gently to blow and beat mixing Born of the same parents, 4 DEG C of ice bath 30min.Do blank control and mouse IgG monoclonal antibody Isotype controls simultaneously.1000 rpm × 3min, abandon supernatant.

(4) fluorescence secondary antibody is incubated：It washed once with the 2%PBA of precooling, add in the suitably diluted FITC labels goat-antis of PBA Mostly anti-(1 μ g/106 cells) the 200 μ l of mouse IgG.Mixing cell is gently blown and beaten, 4 DEG C are protected from light 30 min of ice bath.

(5) it is washed 2 times with the 2%PBA of precooling.Cell is resuspended in 200 μ l PBS, gently mixing, puts streaming pipe In, it is protected from light, is detected with flow cytometer detection instrument.

The results are shown in Figure 1 for flow cytometer detection.

Embodiment 4, EGFR mouse source antibody combination EGFR extracellular region epitopes determine

In order to determine the epitope of antibody, the present inventor according to four structural domains of EGFR extracellular regions, it is extracellular be distinguished into I, IIth, III, IV, I+II, I+II+III, II+III, III+IV, nine sections of EGFR ECD, respectively structure and hGH amalgamation and expressions PCDNA3.1 (+/-) carrier for expression of eukaryon.This nine expression vectors are subjected to transient transfection expression respectively, with the anti-hGH monoclonal antibodies of mouse Wrapper sheet, detection structure domain expression；With the mouse monoclonal antibody packet elisa plate of screening, detection mouse monoclonal antibody and EGFR extracellular region structural domains Combination.It the results are shown in Table 1.

1 MAb 79 of table is combined antigenic domains measurement result with MAb 87

The results show that anti-Mab79 and Mab87 are targeted in III+IV region of EGFR ECD.

To further determine that the epitope of antibody, by point mutation technology, by the III of the expression plasmid of EGFR extracellular region overall lengths After completing mutation, the expression plasmid being mutated is expressed by way of transient transfection for the several amino acid mutations in+IV region, Later with the method for ELISA detection mouse monoclonal antibody Mab87, Mab79, Cetuximab (western appropriate former times) and Nimotuzumab (Tai Xin It is raw) with having the EGFR extracellular region combination situations of mutation.Finally determine the epitope of 4 monoclonal antibodies all relevant with ligand binding The IIIth structural domains of EGFR, but specific epitope have it is dramatically different.The anti-Mab87 epitope sites of the present invention：N389、N420、 T422；Anti- Mab79 epitope sites：F 352、R 353、G 354、F 357、H 359、L 363、W 386；Western appropriate former times bound site Point：S 418、K 443、I 467、S 468、G 471、N 473、S474；Nimotuzomab binding site：I 467、S 468、G 471、 N 473、S474。

It will be apparent that the anti-Mab87 of the present invention and Mab79, particularly Mab87, targeted epitope site is different from business The monoclonal antibody of production.

Embodiment 5, EGFR monoclonal antibodies are outer to act on A431 cell inhibitory effects

A431 cells (department of human head and neck epidermal carcinoma cell lines, purchased from Cell Bank of Chinese Academy of Sciences) in logarithmic phase growth, It is counted after pancreatin digestion, cell density is adjusted to 20000cells/ml with the DF/12 culture mediums containing 1%FBS, with 200ul/ The amount in hole is taped against in 96 orifice plates, 37 DEG C, 5%CO₂Culture 24 hours.6 row, 8 row, 48 holes among 96 orifice plates are chosen later, point Into 4 groups, every group of 6 repetitions.It is corresponding to add in different number monoclonal antibodies and Cetuximab, the final concentration of 5ug/ml of antibody.37 DEG C, 5%CO₂Culture 4 to 5 days, later with the (purchase of Cell Counting Kit-8 (CCK-8) cell Proliferation-toxicity detection kit From colleague's chemistry) cell activity is detected, cell activity and OD450 reading positive correlations, Mab79, Mab87 and western appropriate former times can press down A431 cell growths processed, are as a result shown in Fig. 2.

To the inhibiting effect of tumour growth in embodiment 6, EGFR monoclonal antibodies

The DMEM/F12 culture solution culture A431 cells of 10% fetal calf serum, with 0.25% pancreas after cell quantity is enough Enzyme (0.02%EDTA) digests, and the DMEM/F12 culture solutions of serum-free are washed one time, then be resuspended to the serum-free of appropriate volume In DMEM/F12 culture solutions, by 2 × 10⁶The nude mice dorsal sc of the A431 cell inoculations of/0.1ml.It can see after about 7 days bright When aobvious tumour is grown, nude mice is randomly divided into group, every group 7, Mab79, Mab87 antibody respectively by 1mg/ dosage only into Row intraperitoneal injection treatment, also sets up antibody combined application group (the wherein Mab79 of Mab79 and Mab87:Mab87 is 1:1；It is namely every Respectively only injected with 0.5mg/ both during secondary injection), it is compared with incoherent homotype mouse source monoclonal antibody TT8D9, was treated every three days Once and gross tumor volume is measured, treatment continues 20 day time.Nude mouse tumor volume size：V=0.5ab², a is that tumor entity is long Diameter, b are the short diameter of tumor entity.

The results show that Mab79 and Mab87 can effectively inhibit the growth of A431 transplantation tumors, but the inhibition of Mab79 Significant effect is better than Mab87, and multiphase pass, affinity are high related compared with this tumor suppression associated epitope site targeted with Mab79； Meanwhile the significant effect of Mab79 and Mab87 use in conjunction is more preferably, the two use in conjunction reaches significant synergy effect Fruit refers to Fig. 3.

Embodiment 7, the clone of EGFR monoclonal antibody variable region encoding sequences and identification

To screen gained EGFR hybridoma cell strains cDNA in embodiment 1 as template, using round pcr, EGFR mouse are cloned Source monoclonal antibody variable region gene；Through sequencing, choose without sequence of the mutation without terminator codon, work(is cloned using 5 ' RACE technologies It can property V_LAnd V_HGene.

First, the clone of EGFR mouse source monoclonal antibody variable region encoding sequences

(1) total serum IgE of EGFR monoclonal antibodies is extracted from hybridoma cell strain

It is extracted with Shanghai Fei Jie biotech firms FAST1000 kits.

(1) 4 × 10 are taken⁵Hybridoma, 1000rpm × 3min abandon supernatant.It washed once with PBS.Cell is resuspended In 100 μ lPBS, it is put into centrifuge tube.

(2) RB1 liquid 1ml are added in, fully reverse mixing is placed at room temperature for 5min up to being completely dissolved.

(3) 500 μ l of RB2 liquid are added in, fully reverse mixing 1min.Liquid after mixing is sucked or directly poured into inner sleeve 1min is centrifuged in pipe.

(4) liquid in outer tube is discarded, 500 μ l washing lotions are added in inner sleeve, centrifuges 1min, is repeated primary.

(5) inner sleeve is taken out, liquid in outer tube is discarded, still recovers inner sleeve, is not added with washing lotion, centrifuges 1min.

(6) inner sleeve is moved into new centrifuge tube, adds in 40 μ l of eluent in film center, be stored at room temperature 1min, obtain Total serum IgE.

(2) RT-PCR prepares EGFR mouse source monoclonal antibody cDNA

Using total serum IgE as template, Oligo (dT) 18 is primer, and RT-PCR expands EGFR monoclonal antibodies cDNA.

(3) clone of EGFR mouse source monoclonal antibody variable region gene

A, the synthesis of degenerate primer

According to the conservative of signal peptide for antibody and skeleton area gene, following degenerate primer is designed and synthesized (in following primer W=A/T, K=G/T, R=A/G, Y=C/T, M=A/C, S=C/G, N=C/G/T, V=A/C/G)：

(1) light chain upstream variable region degenerate primer：It is designed (5 ' -3 ') according to signal peptide sequence

VLFWD1	GAATTCCCACCATGGAGACAGACACACTCCTGCTAT(SEQ ID NO:33)
		VLFWD2	GAATTCCCACCATGGATTTTCAAGTGCAGATTTTCAG(SEQ ID NO:34)
VLFWD3	GAATTCCCACCATGGAGWCACAKWCTCAGGTCTTTRTA(SEQ ID NO:35)
		VLFWD4	GAATTCCCACCATGKCCCCWRCTCAGYTYCTKGT(SEQ ID NO:36)
VLFWD5	GAATTCCCACCATGAAGTTGCCTGTTAGGCTGTTG(SEQ ID NO:37)

(5 ' -3 ') are designed according to FR1 conserved sequences

MKac-Fwd GAYATTGTGMTSACMCARWCTMCA

(2) light chain downstream degenerate primer (5 ' -3 ')

MKac-Rev GGATACAGTTGGTGCAGCATC

(3) heavy chain upstream degenerate primer：It is designed (5 ' -3 ') according to signal peptide sequence

(4) heavy chain upstream degenerate primer：(5 ' -3 ') are designed according to FR1 conserved sequences

MHFR Fwd1	SARGTNMAGCTGSAGSAGTC(SEQ ID NO:45)
		MHFR Fwd2	SARGTNMAGCTGSAGSAGTCWGG(SEQ ID NO:46)
MHFR Fwd3	CAGGTTACTCTGAAAGWGTST(SEQ ID NO:47)
		MHFR Fwd4	GAGGTCCARCTGCAACARTC(SEQ ID NO:48)
MHFR Fwd5	GAGGTCCAACTVCAGCARCC(SEQ ID NO:49)
		MHFR Fwd6	AGAGTGAASSTGGTGGAATC(SEQ ID NO:50)
MHFR Fwd7	GATGTGAACTTGGAAGTGTC(SEQ ID NO:51)

(5) heavy chain downstream degenerate primer

MHCC-Rev ATAGACAGATGGGGGTGTCGTTTTGGC(SEQ ID NO:52)

B, the clone of variable region gene

Using above-mentioned degenerate primer and EGFR monoclonal antibodies cDNA has been prepared as template, PCR amplification EGFR mouse source monoclonal antibody variable region base Cause.

(1) PCR system and parameter setting are as follows：

PCR parameter settings：95 DEG C, pre-degeneration, 5min；The following cycle of 30 wheels：95 DEG C of denaturation, 0.5min, 65 DEG C of renaturation, 0.5min, 72 DEG C of extensions, 0.5min；72 DEG C of extensions, 10min.

(2) PCR amplification is analyzed as a result, and judging with DNA molecular amount label L D2000 with 1% agarose gel electrophoresis The size of amplified fragments.As a result it shows：There are 5 degenerate primers to amplify chain variable region gene respectively, there are 4 degenerate primers Heavy chain variable region gene is amplified, size is about 330bp or so, and band is single, with light/heavy chain variable region gene segment Theoretical size is basically identical.

Using the monoclonal antibody variable region pcr amplified fragment at the plastic recovery kit recycling 330bp of vast Imtech, and It is connected on pMD18T cloning vectors (purchased from Takara companies), is transformed into DH5 α competent escherichia coli cells, carry out blue Hickie screens, and positive colony is sent to Invitrogen for sequencing verification.

According to NCBI IgBLAST (http://www.ncbi.nlm.nih.gov/) immunoglobulin gene comparison analysis As a result, functional antibodies variable region gene is filtered out, designerantibodies light chain and heavy chain variable region downstream primer：

The sequence at the end of variable region 5 ' is amplified using 5 ' RACE.The final functional variable region gene for obtaining EGFR monoclonal antibodies.

Obtained antibody gene sequences and orresponding amino acid sequence are as follows.Wherein RED sector represents CDR region.

(following dashed part is followed successively by Mab79 heavy chain variable region genes sequence from front to back：SEQ ID NO:1； SEQ ID NO:2；SEQ ID NO:3)：

CAGGTCCAGCTGCAGCAGTCTGGGCCTGAACTGGTGAGGCCTGGGGTCTCAGTGAAGATTTCCTGCAAGGGTTCCGG CTACAAATTCACTGATTATGATATACACTGGGTGAAGCAGAGTCATGCAAAGAGTCTAGAGTGGATTGGAGTTGTTAATACTTTCTCTGATAATATAAACTACAACCAGAAGTTTAAGGGCAAGGCCACAGTGACTGTTGACAAATCCTCCAGT ACCGCCTATATGGAACTTGGCAGATTGACATCTGAGGATTCTGCCATCTATTACTGTGCAAGCGGGAATTGGGACTA CGGCTTTGCTTACTGGGGCCAAGGGACCCTGGTCACTGTCTCTGCA(SEQ ID NO:25)

(following dashed part is followed successively by Mab79 heavy chain variable amino acids sequence from front to back：SEQ ID NO: 7； SEQ ID NO:8；SEQ ID NO:9)：

QVQLQQSGPELVRPGVSVKISCKGSGYKFTDYDIHWVKQSHAKSLEWIGVVNTFSDNINYNQKFKGKATVTVDKSSS TAYMELGRLTSEDSAIYYCASGNWDYGFAYWGQGTLVTVSA(SEQ ID NO:26)

(following dashed part is followed successively by Mab79 chain variable region genes sequence from front to back：SEQ ID NO:4； SEQ ID NO:5；SEQ ID NO:6)：

GATGTTTTGATGACCCAAAGTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAGGCCTCCATCTCTTGCACATCTAG TCAGCCCATTATGTATAGTAATGGAAAAATCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCC TG ATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCAC ACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGGTTCACATGGTCCGTGGACGT TCGGTGG AGGCACCAAGCTGGAAATCAAACGG(SEQ ID NO:27)

(following dashed part is followed successively by Mab79 chain variable region amino acids sequence from front to back：SEQ ID NO: 10； SEQ ID NO:11；SEQ ID NO:12)：

DVLMTQSPLSLPVSLGDQASISCTSSQPIMYSNGKIYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFT LKISRVEAEDLGIYYCFQGSHGPWTFGGGTK LEIKR(SEQ ID NO:28)

(following dashed part is followed successively by Mab87 heavy chain variable region genes sequence from front to back：SEQ ID NO: 13； SEQ ID NO:14；SEQ ID NO:15)：

CAGGCGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCGTCACTTGCACTGTCTCTGG GTTTTCATTAACCAGCTATAATGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTAATAT GGGCTGGTGGAATCACAAATTATAATTCGGCTCTCATGTCCAGACTGAGCATCAGGAAAGACAACTCCAAAAGCCAA GTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATGTACTATTGTGCCAGAGTGGGGGGTTACGACGG GGACTGGCTTGCCTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA(SEQ ID NO: 29)

(following dashed part is followed successively by Mab87 heavy chain variable amino acids sequence from front to back：SEQ ID NO: 19； SEQ ID NO:20；SEQ ID NO:21)：

QAQLKESGPGLVAPSQSLSVTCTVSGFSLTSYNVHWVRQPPGKGLEWLGVIWAGGITNYNSALMSRLSIRKDNSKSQ VFLKMNSLQTDDTAMYYCARVGGYDGDWLAYWGQGTLVTVSA(SEQ ID NO:30)

(following dashed part is followed successively by Mab87 chain variable region genes sequence from front to back：SEQ ID NO: 16； SEQ ID NO:17；SEQ ID NO:18)：

GACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAG CCAAAGTGTCAGTACATCTAGTCATAGTTATATGCACTGGTACCAACAGAAACCAGGGCAACCACCCAAACTCCTCA TCAAATATGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTC AATATCCATCCTGTGGAGGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGGAGATTCCGTACACGTTCGG AGGG GGGACCAAGCTGGAAATAAAACGG(SEQ ID NO:31)

(following dashed part is followed successively by Mab87 chain variable region amino acids sequence from front to back：SEQ ID NO: 22； SEQ ID NO:23；SEQ ID NO:24)：

DIVLTQSPASLAVSLGQRATISCRASQSVSTSSHSYMHWYQQKPGQPPKLLIKYASNLESGVPARFSGSGSGTDFTL NIHPVEEEDTATYYCQHSWEIPYTFGGGTK LEIKR(SEQ ID NO:32)

The structure of embodiment 8, EGFR people-mouse chimeric mAb carrier for expression of eukaryon

Using overlapping pcr by light chain V_LThe C of gene and people Ig_LGene is spliced, and forms light chain mosaic gene；Gently The end of chain 5 ' introduces BamHI restriction endonuclease sites, and the end of light chain 3 ' introduces EcoRI restriction endonuclease sites.Similarly, it builds Heavy chain mosaic gene.Above-mentioned light chain/heavy chain gene is inserted into pcDNA3.1 (+/-) expression vector respectively and (is purchased from Invitrogen Company) monoclonal restriction enzyme site, build EGFR Chimeric antibodies expression vector.

(1) upstream and downstream object is designed according to a conventional method, is drawn using round pcr respectively at heavy chain/end of chain variable region gene 5 ' Enter BamHI single endonuclease digestions site, EcoR I single endonuclease digestions site is introduced at heavy chain/end of light chain constant region gene 3 '.BamHI and EcoRI Double digestion heavy chain/light chain mosaic gene, gel extraction target fragment.

(2) processing of pcDNA3.1 (+/-) carrier for expression of eukaryon：BamHI and EcoRI double digestions pcDNA3.1 is (+/-) to be carried Body, gel extraction target fragment (~5400bp).

(3) by heavy chain of antibody/light chain gene in (1) be cloned into respectively in (2) BamHI of pcDNA3.1 (+/-) carrier and EcoRI sites.

(4) DH5 α competent cells are converted with above-mentioned connection product, extracts recombinant plasmid dna in a small amount.Select insertion purpose The positive colony of segment serves extra large Invitrogen companies sequencing identification.

Through digestion and sequencing identification, the recombinant expression carrier of EGFR monoclonal antibodies heavy chain/light chain its sequence that the verification present invention is built Row are correct.

The expression and identification of embodiment 9, EGFR Chimeric antibodies in Chinese hamster ovary celI

Using liposome method by EGFR Chimeric antibodies heavy chain/light chain expression vector cotransfection CHO/dhfr^-Cell (being purchased from ATCC), is compareed with the ghost of untransfected plasmid.After cultivating 48h, 72h, using conventional ELISA methods, marked with HRP Goat anti-human igg antibody detect EGFR Chimeric antibodies expression and chimeric antibody to the specific recognitions of EGFR antigens, Qualification result is as shown in table 3.

The expression of 3 chimeric antibody of table

The results show that the Chinese hamster ovary celI successful expression chimeric antibody of cotransfection expression plasmid carrier, can effectively identify EGFR Antigen, and the Chinese hamster ovary celI culture supernatant of untransfected expression plasmid cannot identify EGFR antigens.Transiently transfect CHO successes Express the Chimeric antibody of energy specific recognition EGFR antigens.

Simultaneously using proteon^TMXPR36Protein Interaction Array System methods measure chimeric antibody Affinity.Measurement result shows that 79 chimeric antibody affinity KD values are：3.29E-10,87 chimeric antibody parent's affinity KD values For：1.25E-09.

All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, people in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>Shanghai Institute of Biological Products Co., Ltd.

<120>Anti-egfr antibodies, its preparation method and its application

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20 25 30

Asp Ile His Trp Val Lys Gln Ser His Ala Lys Ser Leu Glu Trp Ile

35 40 45

Gly Val Val Asn Thr Phe Ser Asp Asn Ile Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Val Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

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

85 90 95

Ala Ser Gly Asn Trp Asp Tyr Gly Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 27

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

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Pro Ile Met Tyr Ser

20 25 30

Asn Gly Lys Ile Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Gly Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg

<210> 29

<211> 357

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 29

caggcgcagc tgaaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60

acttgcactg tctctgggtt ttcattaacc agctataatg tacactgggt tcgccagcct 120

ccaggaaagg gtctggagtg gctgggagta atatgggctg gtggaatcac aaattataat 180

tcggctctca tgtccagact gagcatcagg aaagacaact ccaaaagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatgtact attgtgccag agtggggggt 300

tacgacgggg actggcttgc ctactggggc caagggactc tggtcactgt ctctgca 357

<210> 30

<211> 119

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 30

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

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr

20 25 30

Asn Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ala Gly Gly Ile Thr Asn Tyr Asn Ser Ala Leu Met

50 55 60

Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ala

115

<210> 31

<211> 336

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 31

gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gagggccacc 60

atctcatgca gggccagcca aagtgtcagt acatctagtc atagttatat gcactggtac 120

caacagaaac cagggcaacc acccaaactc ctcatcaaat atgcatccaa cctagaatct 180

ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caatatccat 240

cctgtggagg aggaggatac tgcaacatat tactgtcagc acagttggga gattccgtac 300

acgttcggag gggggaccaa gctggaaata aaacgg 336

<210> 32

<211> 112

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 32

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

1 5 10 15

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

20 25 30

Ser His Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Thr Ala Thr Tyr Tyr Cys Gln His Ser Trp

85 90 95

Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

<210> 34

<211> 36

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 34

gaattcccac catggagaca gacacactcc tgctat 36

<210> 35

<211> 37

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 35

gaattcccac catggatttt caagtgcaga ttttcag 37

<210> 36

<211> 38

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 36

gaattcccac catggagwca cakwctcagg tctttrta 38

<210> 37

<211> 34

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 37

gaattcccac catgkccccw rctcagytyc tkgt 34

<210> 38

<211> 35

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 38

gaattcccac catgaagttg cctgttaggc tgttg 35

<210> 39

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

atgaaatgca gctggrtyat sttctt 26

<210> 39

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 39

atggrcagrc ttacwtyytc attcct 26

<210> 40

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 40

atgatggtgt taagtcttct gtacc 25

<210> 41

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 41

atgaacttyg ggytsagmtt grttt 25

<210> 42

<211> 25

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 42

atgtacttgg gactgagctg tgtat 25

<210> 43

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 43

atgagagtgc tgattctttt gtg 23

<210> 44

<211> 28

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 44

atggattttg ggctgatttt ttttattg 28

<210> 45

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 45

sargtnmagc tgsagsagtc 20

<210> 46

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 46

sargtnmagc tgsagsagtc wgg 23

<210> 47

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 47

caggttactc tgaaagwgts t 21

<210> 48

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 48

gaggtccarc tgcaacartc 20

<210> 49

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 49

gaggtccaac tvcagcarcc 20

<210> 50

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 50

agagtgaass tggtggaatc 20

<210> 51

<211> 20

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 51

gatgtgaact tggaagtgtc 20

<210> 52

<211> 27

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 52

atagacagat gggggtgtcg ttttggc 27

Claims (15)

1. specifically bind the binding protein of EGFR, which is characterized in that the binding protein has light chain variable region and weight chain variable Area, and,

The amino acid sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 7, the amino acid sequence such as SEQ ID of CDR2 NO:Shown in 8, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 9；The amino acid sequence of the CDR1 of its light chain variable region is such as SEQ ID NO:Shown in 10, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 11；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 12；Or

The amino acid sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 19, the amino acid sequence such as SEQ ID of CDR2 NO:Shown in 20, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 21；The amino acid sequence of the CDR1 of its light chain variable region is such as SEQ ID NO:Shown in 22, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 23；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 24.

2. binding protein as described in claim 1, which is characterized in that the amino acid sequence of its heavy chain variable region such as SEQ ID NO:Shown in 26, the amino acid sequence of light chain variable region is as indicated at 28；Or

The amino acid sequence of its heavy chain variable region such as SEQ ID NO:Shown in 30, the amino acid sequence such as SEQ of light chain variable region ID NO:Shown in 32.

3. binding protein as claimed in claim 1 or 2, which is characterized in that it is monoclonal antibody.

4. encode the protein-bonded polynucleotides of any specific binding EGFR of claims 1 to 3.

5. polynucleotides as claimed in claim 4, which is characterized in that the nucleotide sequence of the CDR1 of its heavy chain variable region is such as SEQ ID NO:Shown in 1, the nucleotide sequence such as SEQ ID NO of CDR2:Shown in 2, the nucleotide sequence such as SEQ ID of CDR3 NO:Shown in 3；The nucleotide sequence of the CDR1 of its light chain variable region such as SEQ ID NO:Shown in 4, the nucleotide sequence of CDR2 is such as SEQ ID NO:Shown in 5；The nucleotide sequence of CDR3 such as SEQ ID NO:Shown in 6；Or

The nucleotide sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 13, the nucleotide sequence such as SEQ ID of CDR2 NO:Shown in 14, the nucleotide sequence such as SEQ ID NO of CDR3:Shown in 15；The nucleotide sequence of the CDR1 of its light chain variable region is such as SEQ ID NO:Shown in 16, the nucleotide sequence such as SEQ ID NO of CDR2:Shown in 17；The nucleotide sequence of CDR3 such as SEQ ID NO:Shown in 18.

6. polynucleotides as claimed in claim 5, which is characterized in that the nucleotide sequence of its heavy chain variable region such as SEQ ID NO:Shown in 25, the nucleotide sequence of light chain variable region is as shown in 27；Or

The nucleotide sequence of its heavy chain variable region such as SEQ ID NO:Shown in 29, the nucleotide sequence such as SEQ of light chain variable region ID NO:Shown in 31.

7. a kind of expression vector, it includes any polynucleotides of claim 4~6.

8. a kind of host cell, it includes be integrated with claim 4~6 in the expression vector or genome described in claim 7 Any polynucleotides.

9. a kind of binding protein composition, which is characterized in that the composition contains Binding Protein 1 and binding protein 2, the knot Hop protein 1 has light chain variable region and heavy chain variable region, and the amino acid sequence of the CDR1 of its heavy chain variable region such as SEQ ID NO:Shown in 7, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 8, the amino acid sequence such as SEQ ID NO of CDR3:Shown in 9； The amino acid sequence of the CDR1 of its light chain variable region such as SEQ ID NO:Shown in 10, the amino acid sequence such as SEQ ID NO of CDR2: Shown in 11；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 12；

The binding protein 2 has light chain variable region and heavy chain variable region, and the amino acid sequence of the CDR1 of its heavy chain variable region is such as SEQ ID NO:Shown in 19, the amino acid sequence such as SEQ ID NO of CDR2:Shown in 20, the amino acid sequence such as SEQ ID of CDR3 NO:Shown in 21；The amino acid sequence of the CDR1 of its light chain variable region such as SEQ ID NO:Shown in 22, the amino acid sequence of CDR2 is such as SEQ ID NO:Shown in 23；The amino acid sequence of CDR3 such as SEQ ID NO:Shown in 24.

10. the combination egg described in the binding protein or claim 9 of any specific binding EGFR of claims 1 to 3 White composition is preparing for the purposes in diagnosing, treat and/or preventing the drug of EGFR overexpression related diseases；Preferably, The EGFR overexpression related diseases are：There are the entity tumors that EGFR is overexpressed.

11. purposes as claimed in claim 10, which is characterized in that the entity tumor includes：Non- alveolar cells lung cancer, knot Intestinal cancer, kidney, oophoroma, head and neck cancer, cancer of the esophagus, prostate cancer, cancer of pancreas, breast cancer, glioma.

12. a kind of pharmaceutical composition, which is characterized in that described pharmaceutical composition contains：A effective amount of claims 1 to 3 is any The binding protein and pharmaceutically acceptable carrier of the specific binding EGFR；Or

Described pharmaceutical composition contains：Binding protein composition described in a effective amount of claim 9 and pharmaceutically acceptable Carrier.

13. a kind of medicine box for being used to treating and/or preventing EGFR overexpression related diseases, which is characterized in that the medicine box includes：

The binding protein of any specific binding EGFR of claims 1 to 3；Or

Binding protein composition described in claim 9；Or

Pharmaceutical composition described in claim 12.

14. immune stop of one kind closes object, which is characterized in that the immune conjunction object that stops includes：

The binding protein of any specific binding EGFR of claims 1 to 3；And

Detectable marker；Preferably, the detectable marker includes：Fluorescent marker, chromogenic label.

15. a kind of detection kit for being used to detect EGFR levels, which is characterized in that the detection kit includes：

The binding protein of any specific binding EGFR of claims 1 to 3；Or

Immune stop described in claim 13 closes object.