CN106800599A

CN106800599A - Anti-human EGFR and Notch multi-specificity antibodies, Preparation Method And The Use

Info

Publication number: CN106800599A
Application number: CN201611102039.XA
Authority: CN
Inventors: 胡适; 雷长海; 傅文燕; 沈亚峰; 李天�
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2016-12-05
Filing date: 2016-12-05
Publication date: 2017-06-06
Anticipated expiration: 2036-12-05
Also published as: CN106800599B

Abstract

The invention belongs to biomedicine technical field, the invention discloses a kind of anti-human EGFR and Notch multi-specificity antibodies C T Crossmab, energy and EGFR, Notch2, and Notch3 are combined.C T Crossmab of the invention include two heavy chain polypeptides and two light chains polypeptide, present invention also offers the nucleotide sequence for encoding the heavy light chain polypeptide, and the recombinant expression carrier including the nucleotide sequence, and the host cell for converting.Multi-specificity antibody of the invention can be used for the treatment of tumor disease.

Description

Anti-human EGFR and Notch multi-specificity antibodies, Preparation Method And The Use

Technical field

The invention belongs to biomedicine technical field, more specifically, the invention discloses a class anti-human EGFR, Notch2 and Notch3 bispecific antibodies, its preparation method and its application in antineoplastic is prepared.

Background technology

Based on operative treatment, radiotherapy and chemotherapy, be aided with new targeted therapy scheme be in recent years to dislike The elementary tactics of property oncotherapy, and important progress has been obtained in clinical practice.But the Preventive of malignant tumour and Therapeutic tolerance is still the problem for perplexing clinical and researcher always.2006, held in american association of cancer research Tumor stem cell seminar on, participant scholar defines tumor stem cell to be had self-renewing characteristic, has the ability to drive for a kind of Form various heterogeneous Tumor Differentiation cell mass, cancer cell (Clarke et al., Cancer with stem cell properties stem cells—perspectives on current status and future directions:AACR Workshop on cancer stem cells., 2006, Cancer research, 66:9339-9344).Tumor stem cell Theory with its unique theory advantage and the research supporting evidence enriched constantly, increasingly by domestic and foreign scholars approval and connect Receive.Although traditional therapy has preferably treatment fragmentation effect to tumor entity cell, to only accounting for tumor cell group 0.2% ~10% and the tumor stem cell difficulty that has bigger molecule phenotypic difference with tumor entity cell has inhibitory action.Research tumour Stem cell is in tumour Emergence and Development and the important function and its mechanism of transfer, and develops new based on tumor stem cell The antineoplaston strategy and preparation said, the treatment tool to malignant tumour are of great significance.

Cancer stem-cell hypothesis think：Malignant tumour originates from the tumour cell of a group " stem-like cell ", this group of cell quilts Referred to as tumor stem cell (cancer stem cells, CSCs or cancer-initiating cells, CICs).Last century The end of the nineties, researcher is separated to tumor stem cell from leukaemia and elaborates what is driven by tumor stem cell first Tumor Heterogeneity laminated tissue model (Dick, Human acute myeloid leukemia is organized as a Hierarchy that originates from a primitive hematopoietic cell., 1997, Nature Med,3:730-737).Then, researcher also demonstrate that the presence of tumor stem cell and swelling for being similar in various solid tumors Knurl heterogeneity tissue characteristic, including breast cancer, colon cancer, brain tumor, oophoroma, lung cancer, prostate cancer and cancer of pancreas (Brooks et al.,Therapeutic implications of cellular heterogeneity and plasticity in Breast cancer., 2015, Cell stem cell, 17:260-271).These evidences show in most of neoplastic hematologic disorder With in solid tumor exist with stem cell properties tumour cell.

Multinomial research shows that tumor stem cell is in malignant tumour generation, development；Tumour cell is heterogeneous to be maintained；Tumour is entered Change；Played an important role during being tolerated with tumor therapeutic.Its biological function has the characteristics that：

1) extremely strong oncogenicity.The tumour cell being directly separated from the cancer stove of malignant tumor patient is difficult to be formed in vitro Clone or migrate to and cloned into immunodeficient mice model knurl, and only need to a small amount of CSC just can be in immune deficiency Knurl is cloned into mouse model, and tumour transplatation thing has phenotype heterogeneity (the Al-Hajj et similar with primary tumors Al., Prospective identification of tumorigenic breast cancer cells., 2003, Proceedings of the National Academy of Sciences,100:3983-3988；Dick,Human acute myeloid leukemia is organized as a hierarchy that originates from a Primitive hematopoietic cell., 1997, Nature Med, 3:730-737；Ginestier et al., ALDH1is a marker of normal and malignant human mammary stem cells and a Predictor of poor clinical outcome., 2007, Cell stem cell, 1:555-567).

2) complicated heterogeneity and bioremodelable.The source of CSC is possible and its complicated, and it is probably derived from thin in epithelium Born of the same parents obtain certainly during converting (epithelial-to-mesenchymal transition, EMT) to mesenchymal cell Differentiated tumour cell (Espinoza and Miele, the Deadly crosstalk of my updating ability:Notch Signaling at the intersection of EMT and cancer stem cells., 2013, Cancer letters,341:41-45)；Or from the normal tissue stem cell being mutated by external microenvironment factor induction oncogene property (Reya et al., Stem cells, cancer, and cancer stem cells., 2001, nature, 414:105- 111).In addition, some reports in recent years also confirm that CSC has extremely complex bioremodelable, such as in breast carcinoma stem cell In two kinds of not isophenic breast carcinoma stem cells groups can be gone out with major diagnostic：A kind of breast cancer CSC of the active proliferation of Epithelial Group, expression stem cell markers ALDH；With a kind of interstitial sample, breast cancer CSC groups that relatively inactive but wetting capacity is strong, its Surface marker expression is CD44+/CD24-.This two crowds of CSC can produce respective Epithelial or interstitial sample tumor entity Cell Progeny, while can discharge secretion signal strengthens the self-renewal capacity of CSC.And in by tumor microenvironment cell because Under subsignal, chemotactic factor (CF) signal, the epigenetics of transcriptional control mediation change, two groups of cells can mutually convert (Liu et al.,Breast cancer stem cells transition between epithelial and mesenchymal States reflective of their normal counterparts., 2014, Stem cell reports, 2:78- 91).In addition, there is different CSC crowds, such as CSC of CD133+ (Ma et al., CD133＆plus in being also reported in liver cancer in research； HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway.2008,Oncogene,27:1749-1758) with the liver cancer of CD90+/CD44+ CSC(Yang et al.,Significance of CD90+cancer stem cells in human liver Cancer., 2008, Cancer cell, 13:153-166) there are different stem cell properties.Done in different tumor types thin The mark of born of the same parents is also tended to difference, such as in cancer of pancreas, the surface marker of CSC be considered as CD24+ (Li et al., Structural basis for inhibition of the epidermal growth factor receptor by Cetuximab., 2007, Cancer research, 67:1030-1037), then it is CD24- and for mammary gland CSC.In addition, not Content and ratio with CSC in malignant tumour is often different, and in the different phase of malignant tumour, the ratio of CSC can also occur Complicated change.Such as in colon cancer, CD133+ expression CSC ratios high are about 1.8%~24.5% (O ' Brien et al., A human colon cancer cell capable of initiating tumour growth in Immunodeficient mice., 2007, Nature, 445:106-110), and after chemotherapy is received, the ratio of colon cancer CSC Rate can then improve (Dylla et al., Colorectal cancer stem cells are enriched in Xenogeneic tumors following chemotherapy., 2008, PloS one, 3:e2428).And in melanoma In, express CD133+ cell surface markers CSC ratio between 1%~20% (Quintana et al., Efficient Tumour formation by single human melanoma cells., 2008, Nature, 456:593-598).This A little results of study point out the CSC to have extremely complex heterogeneity and bioremodelable.

3) therapeutic sensitivity is poor.Some researchs in recent years show, treatments of the CSC to being used in many currently clinics Method is tolerated, including Treated with Chemotherapeutic Drugs thing and radiotherapy.Such as have been reported that display human acute myeloid leukemia CD34+/CD38- ancestrals Insensitive (Costello et al., the Human acute of apoptotic effect that cell is mediated to chemotherapy lethal effect and Fas myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis,reduced immunogenicity,and impaired Dendritic cell transformation capacities., 2000, Cancer Research, 60:4403-4411)； And expression Telomerase high, the normal configuration of chromosome is maintained so as to suppress Apoptosis；The various anti-apoptotic genes expressions of height expression, such as Bcl-2 genes, expression of nuclear factor kappa B (NF- κ B) gene, mutant P 53 gene and c-myc genes etc..CD44+/CD24- mammary gland Cancer CSC (Phillips et al., 2006, Journal of the National Cancers insensitive to radiotherapy Institute,98:1777-1785)；The glioma stem cells of CD133+ have very strong DNA damage repair ability, to radiation Insensitive (Bao et al., the Glioma stem cells promote radioresistance by of therapy Preferential activation of the DNA damage response., 2006, nature, 444:756-760)； Cells ratio increases during Dylla etc. also reports bare mouse different species tumor models of the colon cancer CSC after chemotherapy, insensitive to chemotherapy (Dylla et al.,Colorectal cancer stem cells are enriched in xenogeneic tumors Following chemotherapy., 2008, PloS one, 3:e2428).And tumor stem cell and normal stem cell table all high Up to ATP binding cassette transporters (ATP-binding cassette transporters, ABC), such as ABCB1 albumen and ABCG2 Albumen, the former encodes P- glycoprotein, and the latter is to identify to come from mitoxantrone mdr cell.ABCB1 and ABCG2 mediations Outer row's effect prevents Hoechst 33342 and the grade dyestuffs of rhodamine 123 from dyeing stem cell, it was confirmed that ABC albumen is dry Key (Zhou et al., The ABC transporter Bcrp1/ABCG2is of the cell to the insensitive characteristic of chemotherapy expressed in a wide variety of stem cells and is a molecular determinant of The side-population phenotype., 2001, Nature medicine, 7:1028-1034).CSC have and just The similar various biological attribute of normal stem cell, such as relative propagation is inactive, insensitive to medicine and toxin, expression high is various ABC albumen, nucleic acid repair ability be strong and apoptosis resistivity (Dean et al., Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive Hematopoietic cell., 2005, Nature Reviews Cancer, 5:275-284).In addition, CSC is dry thin with normal Born of the same parents are similar to, and tumor stem cell tabernacle is collectively forming with the microenvironment of surrounding, and relatively hypoxia and substantial amounts of maintenance can be provided to CSC in tabernacle The bio signal of stable state, stimulate has barrier action to external world.Conventional cytotoxic therapies are dry thin in cell tabernacle to being in Born of the same parents' influence it is minimum (LaBarge, The difficulty of targeting cancer stem cell niches., 2010, Clinical cancer research,16:3121-3129).Therefore, CSC can be survived during chemicotherapy, And tumour is multiplied again, finally causes tumor recurrence and therapeutic tolerance.

4) transfer invasive ability is promoted strong.Due to the height heterogeneity of tumor tissues, most of tumor entity cell does not have Invasion and attack and the ability of transfer, and CSC then has very strong invasion and attack and transfer ability.Such as have been reported that display, breast cancer CSC and breast EMT process height correlations important in gland cancer transfer process, it is entity tumor infiltration edge that the CSC of EMT samples is often positioned, and And easily enter blood circulation and form micro metastasis (Mani et al., The epithelial- in distal end Mesenchymal transition generates cells with properties of stem cells., 2008, Cell,133:704-715).At this moment, CSC again can be in some microenvironment factors (TWIST downwards (Stankic of such as ID1 mediations et al.,TGF-β-Id1signaling opposes Twist1and promotes metastatic colonization Via a mesenchymal-to-epithelial transition., 2013, Cell reports, 5:1228-1242)) make It is changed into the epithelium state CSC of high value-added activity under, is finally completed Metastasis in Breast Cancer process.In cancer of pancreas, expression CD133+/ The CSC of CXCR4+ cell surface markers is also usually positioned at tumor-infiltrated edge, is easily accessible blood, and its height expression chemotactic Factor acceptor.In the presence of CXCR4 chemotactic factor (CF)s, during such CSC is easy to be transferred to liver (Hermann et al., Distinct populations of cancer stem cells determine tumor growth and Metastatic activity in human pancreatic cancer., 2007, Cell stem cell, 1:313- 323).The studies above points out CSC to play very important effect in the infiltration of malignant tumour and transfer.

In sum, CSC has strong, the complicated heterogeneity of oncogenicity and bioremodelable, transfer invasive ability is strong and controls Treat sensitiveness poor.Tumor tissues by after conventional cytotoxic therapies, such as after chemicotherapy and Biological target therapy, the height of remaining Oncogenicity CSC starts enhancing proliferation activity and starts to expand and drive tumor tissue growth, shows as clinical tumor recurrence.Upper State on the basis of theoretical and research, numerous researchers think, the therapeutic strategy for targetting CSC cell masses will supplement existing treatment plan Deficiency slightly, and can significantly improve oncotherapy level (Schaue and McBride, Opportunities and Challenges of radiotherapy for treating cancer., 2015, Nature Reviews Clinical Oncology,12:527-540；Takebe et al.,Targeting Notch,Hedgehog,and Wnt pathways in cancer stem cells:Clinical update., 2015, Nature Reviews Clinical Oncology).

Clearly, there is the signal path of imbalance in current research, its regulation and control to CSC is most important in CSC, including Wnt signal paths, Notch signal paths, and Hedgehog signal paths etc. (Takebe et al., Targeting Notch, Hedgehog,and Wnt pathways in cancer stem cells:Clinical update., 2015, Nature Reviews Clinical Oncology).1916, Notch genes were found in fruit bat body first, the mutation of the gene Body can make fruit bat wing edge produce breach, hence obtain one's name.Complete Notch signal paths include transmembrane protein acceptor, cross-film Albumen, transcription factor and downstream target gene, wherein human cell have 4 kinds of Notch receptors (Notch1~4) and 5 kinds of parts (Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1 and Jagged-2) (Bailey et al., Cancer metastasis facilitated by developmental pathways:Sonic hedgehog,Notch, And bone morphogenic proteins., 2007, Journal of cellular biochemistry, 102:829- 839), it is highly conserved in evolution, with regulating cell propagation, differentiation and apoptosis function, and be almost related in a organized way And organ.Notch genes have very significant oncogenicity effect, and this is embodied in various human tumors (including lymthoma, mammary gland Cancer, lung cancer, H/N tumors, cancer of pancreas, colon cancer, osteosarcoma of jaw and glioblastoma) activated mutant, and be expanded to Members at different levels (Weng and Aster, the Multiple niches for Notch in cancer of Notch signal paths: Context is everything., 2004, Current opinion in genetics＆development, 14:48- 54).In kinds of tumors, Notch signal paths are proved relevant with the maintenance of tumor stem cell dryness.Recently, OncoMed systems Medicine company announces the trouble of I b clinical trial phases of the monoclonal antibody tarextumab treatment ED-SCLCs of targeting Notch paths Person is survived and biomarker data.Tarextumab is full-length human monoclonal antibody, targets Notch2/3 acceptors.It is preclinical Research prompting has 2 kinds of mechanism of action, that is, lower Notch path signals, and resisting tumour stem cells role and influence pericyte, matrix are thin Born of the same parents and the microenvironment of tumour.

And in solid tumor cell, EGF-R ELISA (EGFR) signal is that another signal of interest of body turns Approach is led, epithelium, interstitial and nerve fiber is widely present in, growth and breeding through whole cell, to cell Differentiation has selected important adjustment effect.EGFR families mainly include EGFR (i.e. HER1) and HER2-4.EGFR encodes cross-film egg In vain, its signal is activated through receptor tyrosin phosphorylation, by Ras/Raf/MEK/MAPK approach Cascaded amplifications, finally results in The phosphorylation of MAPK, the MAPK signals of modification enter nucleus, promote the phosphorylation of target gene, the expression and work of regulatory gene Property.EGFR signals are closely related with the generation development and prognosis of tumour, in kinds of tumors such as lung cancer, colon cancer, breast cancer, prostatitis There is expression in gland cancer, oophoroma and wing skin cancer etc..The signal of height expression is related to the poor prognosis of breast cancer and wing moon bright cancer. Expression and tumor proliferation of the EGFR in breast cancer, progression of disease, and poor prognosis correlation one.Expression can reduce estrogen The expression of acceptor, increases the resistance of Endocrine treatment,.Preclinical study shows that the EGFR of composition activation can substantially increase The Tumor formation of internal MCF-9 people's breast cancer model.Act on EGF-R ELISA (epithelial growth factor Receptor, EGFR) monoclonal antibody Cetuximab (cetuximab, C225) be the most commonly used targeted drug of research One of, its curative effect is affirmed in kinds of tumors treatment.

EGFR is complicated with the interaction mode of Notch signals, can show as synergy, it is also possible to show as antagonism Effect, depends on the type of tissue and the stage of development.In glioma, Notch raises the expression of EGFR, in differentiated In glioma, the expression of EGFR is closely related with Notch.And in cutaneous squamous cell carcinoma, EGFR signal functions in The upstream of Notch1 signals, and suppress the expression of signal.Sent out in the in vitro culture of mammary gland original position duct cell carcinoma with signal Wave collaboration facilitation.These researchs show that EGFR signals and Notch signals are two bars conduction paths being closely connected, right There is important adjustment effect in the generation development of tumour.

In sum, Notch signal paths are the dimensions of the generation, tumour CSC cell masses and dryness that participate in tumour cell EMT Hold and its Proliferation, Differentiation and CSC tolerance to treatment regulation and control in critical path.And EGFR is main in tumor entity cell A class excessive activation signal path, and occupy critical role in tumor entity knurl targeted therapy.And between two signal paths There is the cross activation at all levels.

Therefore, building one using technique for gene engineering can simultaneously block two kinds of multi-specificity antibodies of signal, can Blocking EGFR, can block Notch again, and then it is always this to produce inhibitory action to tumor stem cell and tumor entity cell simultaneously The technical staff in field problem anxious to be resolved.

The content of the invention

Two kinds of multi-specificity antibodies of signal path can be simultaneously blocked object of the present invention is to provide one, you can To block EGFR, Notch2 and Notch3 can be also blocked.Preparation method another object of the present invention is to provide the antibody.This 3rd purpose of invention be using the antibody, and comprising the antibody as active component the treatment for tumor disease Agent.

The first aspect of the present invention, there is provided a kind of anti-EGFR and Notch2/3IgG molecules sample intersects monoclonal antibody (Crossmab) C-T Crossmab, the Crossmab energy and EGFR, Notch2, and Notch3 are combined.

Preferably, a kind of anti-human EGFR and Notch multi-specificity antibodies of the invention, it includes two heavy chain polypeptides and two The amino acid sequence of bar light chain polypeptide, wherein heavy chain polypeptide such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；Light chain polypeptide Amino acid sequence such as SEQ ID NO：3 and SEQ ID NO：Shown in 4.

Present invention also offers a kind of polynucleotides, it includes the nucleotide sequence for encoding antibody as described above.

Preferably, described polynucleotides, its include encoding heavy chain polypeptide such as SEQ ID NO：5 and SEQ ID NO：6 Shown nucleotide sequence；And coding light chain polypeptide such as SEQ ID NO：7 and SEQ ID NO：Nucleotides sequence shown in 8 Row.

Present invention also offers a kind of recombinant expression carrier, it includes polynucleotides as described above.

Preferably, described recombinant expression carrier, selected from pcDNA3.1, pDR1 or pDHFR etc..In preferred reality of the invention Apply in example, expression vector is specially pcDNA3.1.

Present invention also offers a kind of host cell converted with above-mentioned carrier.

Preferably, described host cell, is eukaryotic；More excellent, selected from Chinese hamster ovary Chinese hamster ovary celI, NS0 bones Myeloma, COS or SP2/0 cells etc..

In the present invention, the suitable DNA of any coding EGFR, Notch2 and Notch3 is adapted to the present invention, so Structure include it is isolated from tissue or cell mRNA, from basis with publishing database sequence full genome synthesize, Or obtained from other cDNA libraries.

In the present invention, any suitable carrier can be used, and can be pDR1, one of pCDNA3.1, pDHFF, table Include being connected with the fusion dna sequence of suitable transcription and translation regulatory sequence up to carrier.

Mammal or insect host cell culture systems can be used for the expression of C-T Crossmab of the invention, COS, CHO, NS0, sf9 and sf21 etc. may be applicable to the present invention.

Available host cell is the prokaryotic containing above-mentioned carrier, can be one of DH5a, BL21 (DE3), TG1.

The second aspect of the present invention, there is provided the preparation method of above-mentioned antibody (C-T Crossmab), it includes following step Suddenly：

I () is cultivated according to foregoing host cell under conditions of being adapted to allow the antibody expression；With

(ii) antibody of expression is reclaimed.

Step (ii), further isolated or purified reclaims the antibody of expression.

The preparation method of the C-T Crossmab disclosed in the present invention is under expression condition, to cultivate above-mentioned host thin Born of the same parents, so that C-T Crossmab are expressed, the C-T Crossmab described in isolated or purified.

Can be substantially uniform material by bispecific antibody purifying, such as in SDS-PAGE electricity using the above method It is single band in swimming.

C-T Crossmab disclosed by the invention can be isolated and purified using the method for affinity chromatography, according to institute's profit The characteristic of affinity column, it is possible to use the method elution of bound such as conventional method such as high-salt buffer, change PH is affine C-T Crossmab polypeptides on post.

In a preferred embodiment of the invention, above-mentioned multi-specificity antibody C-T Crossmab disclosed by the invention are Obtain by the following method：Full genome synthesizes the heavy and light chain gene of multi-specificity antibody, the sequence reference of full genome Dan Ke Sequence (United States Patent (USP) US6217866B1, Li et al., the Structural basis for of grand antibody cetuximab Inhibition of the epidermal growth factor receptor by cetuximab., 2005, Cancer Cell,7:301-311), sequence (United States Patent (USP) the US20130323266 A1, Yen, Wan-Ching, et of tarextumab Al.., autograph Targeting Notch signaling with a Notch2/Notch3antagonist (tarextumab) Inhibits tumor growth and decreases tumor-initiating cell frequency., linical Cancer Research 21.9(2015):2084-2095) and Crossmab heavy chain light chains design (Hu et al., Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk., 2015, Cancer research,75:159-170) with (Spasevska et al., Advances in Bispecific Antibodies Engineering:Novel Concepts for Immunotherapies., 2015, J Blood Disorders Transf,6:2)。

The present invention is the heavy chain gene and light chain gene for synthesizing multi-specificity antibody with full genome, and by above-mentioned two heavy chains Gene, two light chains gene are respectively charged into eukaryotic expression vector pcDNA3.1 and pcDNA3.1Zeo.Above-mentioned plasmid one reinstates lipid Body method transfects CHO-K1 cells, and screens stabilization table with the Selective agar medium containing 500 μ g/ml G418 and 300 μ g/ml Zeocin Cell clone up to bispecific antibody is purified by affinity chromatography using Protein A posts from the supernatant of cell culture Multi-specificity antibody C-T Crossmab.

The third aspect of the present invention, there is provided described antibody (C-T Crossmab) answering in antineoplastic is prepared With.

Described medicine, with C-T Crossmab of the invention as active ingredient.

Present invention also offers a kind of pharmaceutical composition, it includes C-T Crossmab, and at least one pharmaceutically useful load Body, diluent or excipient.

Purposes of the described pharmaceutical composition in antineoplastic is prepared.

Purposes above, also including being used in combination with other antineoplastics.

The present invention carries out affinity detection to above-mentioned C-T Crossmab, it is found that C-T Crossmab are fully protected simultaneously The affinity of cetuximab and tarextumab is stayed.

Therefore, we carry out next step experiment, including suppress tumor cell proliferation, internal tumor suppression and suppress tumour EMT bases Because expression etc. experiment, test result indicate that, C-T Crossmab disclosed by the invention have simultaneously Cetuximab with The function of tarextumab.Additionally, multi-specificity antibody C-T Crossmab can be maximum as traditional IgG molecules Conventional monoclonal antibody structure is remained to degree, because there is the presence of Fc fragments, can be affine with common ProteinA posts Purification by chromatography, beneficial to large-scale production purifying.Experiment shows, under equal dose, multi-specificity antibody C-T Crossmab has the antineoplaston effect similar or stronger to use in conjunction cetuximab and tarextumab.

The present invention discloses above-mentioned bispecific antibody C-T Crossmab, can be with pharmaceutically acceptable auxiliary material together So as to more stably play curative effect, these preparations can ensure that bispecific disclosed by the invention resists to composition pharmaceutical preparations composition The conformation integrality of body amino acid core sequence, while also wanting the polyfunctional group of protected protein matter prevents its degraded (including but not It is limited to cohesion, deamination or oxidation).Under normal circumstances, for liquid preparation, can generally be preserved at least under the conditions of 2 DEG C -8 DEG C Stablize 1 year, for lyophilized formulations, stabilization is kept within least six months at 30 DEG C.Preparation can be mixing that pharmaceutical field is commonly used herein The preparation, preferably liquid drugs injection or lyophilized formulations such as outstanding, liquid drugs injection, lyophilized, for the liquid drugs injection of above-mentioned C-T Crossmab disclosed by the invention Or lyophilized formulations, pharmaceutically acceptable auxiliary material include surfactant, solution stabilizer, isotonic regulator and buffer solution it One or its combination, wherein surfactant include nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters (tween 20 or 80)；Poloxamer (such as poloxamer 188)；Triton；Lauryl sodium sulfate (SDS)；Sodium Laurylsulfate；14 Alkyl, sub- oil base or octadecyl methyl amimoacetic acid；Pluronics；MONAQUATTM etc., its addition should make difunctional bispecific The granulating trend of antibody protein is minimum, and solution stabilizer can be carbohydrate, including reducing sugar and nonreducing sugar, amino acid Class includes monosodium glutamate or histidine, and alcohols includes one or a combination set of trihydroxylic alcohol, senior sugar alcohol, propane diols, polyethylene glycol, The addition of solution stabilizer should make the preparation those skilled in the art for eventually forming think to reach in the stable time Keep stable state, isotonic regulator can one of for sodium chloride, mannitol, buffer solution can for TRIS, histidine buffering liquid, One of phosphate buffer.

Above-mentioned preparation is the composition comprising C-T Crossmab, antitumor after to animal administration including people Effect is obvious.Specifically, to tumour prevention and/or treatment is effective, can be used as antineoplastic.

Signified tumour of the invention, including gland cancer, leukaemia, lymthoma, melanoma, sarcoma, the source of tumor tissues Including but not limited to adrenal gland, gall-bladder, bone, marrow, brain, mammary gland, bile duct, intestines and stomach, heart, kidney, liver, lung, muscle, Ovary, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis, thymus gland, thyroid gland and uterus.Except upper Outside the tumour stated, it may also be used for the tumour of central nervous system such as spongiocyte diversity knurl, astrocytoma etc., eye in addition Tumour also swells including basal-cell carcinoma, squamous cell carcinoma, melanoma etc. including endocrine disrupting effects, neuroendocrine system Knurl, intestines and stomach pancreatic endocrine system tumor, genital system and head and neck neoplasm etc..Here will not enumerate.

Antineoplastic alleged by the present invention, refers to the medicine for suppressing and/or treating tumour, can include with tumour The delay of growth related symptoms development and/or the reduction of these severity of symptom, it further comprises already present tumour Grow the mitigation of simultaneous phenomenon and prevent the appearance of other symptoms, still reduce or prevent transfer.

, when being administered to animal including people, dosage is because of disease for C-T Crossmab and combinations thereof in the present invention The age of people and body weight, disease traits and seriousness, and method of administration and it is different, may be referred to the result of zoopery and a variety of Situation, total dosage is no more than certain limit.The dosage being specifically injected intravenously is 1~1800mg/ days.

C-T Crossmab disclosed by the invention and combinations thereof can also and other antineoplastic administering drug combinations or be put Treatment is penetrated, for the treatment of tumour, these antineoplastics include that 1, cytotoxic drug (1) acts on the medicine of DNA chemical constitutions Thing：Alkylating agent such as nitrogen mustards, nitrous urine class, pyrovinic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and JM-216 etc.；Mitogen Mycin (MMC)；(2) medicine of influence nucleic acid synthesis：Dihydrofolate reductase inhibitor such as methopterin (MTX) and Alimta Deng；Thymus nucleoside synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) etc.；Purine nucleosides synzyme presses down Preparation such as Ismipur (6-MP) and 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxycarbamide (HU) etc.；DNA polymerases Inhibitor such as cytarabine (Ara-C) and gemzar (Gemz) etc.；(3) medicine of transcribed nucleic acid is acted on：Selectively acting is in DNA Template, suppresses DNA dependenc RNA polymerases, so as to suppress the medicine of RNA synthesis such as：Actinomycin D, daunorubicin, adriamycin, Epi-ADM, aclacinomycin, mithramycin etc.；(4) medicine of tubulin synthesis is mainly acted on：Taxol, taxotere, Vinblastine, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine；(5) other Cytotoxic drugs：L-Asparaginasum mainly suppresses egg The synthesis of white matter；2nd, steroids antiestrogenic：TAM, Droloxifene, Exemestane etc.；Arimedex：Ammonia Shandong Meter Te, Lactel are grand, Letrozole, auspicious Ningde etc.；Antiandrogen：Fluorine its ammonia RH-LH agonist/antagonist：Zoladex, enatone Deng；3rd, BRM：It is main that tumour interferon is suppressed by body's immunity；Interleukin 2；Thymic peptide Class；4th, monoclonal antibody：Mabthera (MabThera)；Trastuzumab (Trastuzumab) Bevacizumab (Avastin)；5th, it is each Plant radiotherapy；6th, other include the medicine that some current mechanism are failed to understand and need further research；Cell-differentiation inducers are as tieed up Class A；Cell death inducer.C-T Crossmab disclosed by the invention and combinations thereof can be with above-mentioned antineoplastic One or a combination set of drug combination.

EGFR can be simultaneously blocked the invention provides one, and two kinds of signal paths of Notch2 and Notch3 is how special Property antibody, multi-specificity antibody of the invention can be used for the therapeutic agent of tumor disease.

Brief description of the drawings

Fig. 1 C-T Crossmab structural representations of the present invention；

Fig. 2 C-T Crossmab of the present invention suppress HCC827 cytoactive experimental results；

Fig. 3 C-T Crossmab of the present invention suppress HCC827 tumour formation curves.

Specific embodiment

The present invention is further detailed with reference to embodiment, experimental example and accompanying drawing, be should not be construed as to this hair Bright limitation.Embodiment does not include detailed descriptions of conventional methods, and such as those are used for the method that carrier construction and matter are drawn, and will compile The gene of code albumen be inserted into such carrier and the matter method drawn or plasmid is introduced host cell method as method Be for person having ordinary skill in the art it is well known that and be all described in many publications, including Sambrook, J., Fritsch, E.F.and Maniais, T. (1989) Molecular Cloning：A Laboratory Manual, 2^ndEdition, Cold spring Harbor Laboratory Press

The structure of the multipurpose antibody C-T Crossmab of the present invention of embodiment 1. and expression

Two heavy chains and two light chains gene order (sequence such as SEQ ID NO of full genome synthesis C-T Crossmab:5、 6th, shown in 7 and 8, the synthesis of trust money Wei Zhi biotech firms), two heavy chain gene sequences are connected with pcDNA3.1 carriers, and two are light Chain gene sequence is connected with pcDNA3.1Zeo carriers, is built into carrier for expression of eukaryon.

3 × 10 are inoculated with 3.5cm tissue culture dishes⁵CHO-K1 cells, are carried out when cell culture to 80%-85% is merged Transfection：Take each 10 μ g of heavy chain plasmid, each 4 μ g of light chain plasmids and 30 μ l Lipofectamine2000 Reagent (Invitrogen Products) are dissolved in 800 μ l plasma-free DMEM mediums respectively, are stored at room temperature 5 minutes, 2 kinds of liquid by more than Mixing, incubation at room temperature replaces training so that DNA- liposome complexes are formed in 20 minutes with the DMEM culture mediums of 3ml serum-frees therebetween The serum-containing media in ware is supported, then the DNA- liposome complexes of formation is added in plate, CO₂Incubator culture 4 hours DMEM complete mediums of the 2ml containing 10% serum is added afterwards, is placed in CO₂Continue to cultivate in incubator.Cell is changed after transfection carries out 24h Selective agar medium screening resistance clone containing 500 μ g/ml G418 and 300 μ g/ml Zeocin.The Gao Biaodake for obtaining will be screened It is grand to use serum free medium Amplification Culture, isolate and purify bispecific antibody C-T with Protein A affinity columns (GE Products) Crossmab.C-T Crossmab are dialysed with PBS, finally with UV absorption standard measure.The structure of C-T Crossmab As shown in figure 1, amino acid sequence such as SEQ ID NO:1st, shown in 2,3,4, trust money Wei Zhi biotech firms are sequenced successfully.

Embodiment 2.Biacore is analyzed

Polyclonal anti-human FC antibody (Jackson ImmunoResearch companies) is coated on CM5M5 chips (GE companies) On, after capture is detected antibody, the affinity of C-T Crossmab, tool are detected with Biacore T100 (GE Healthcare) Physical examination is surveyed affinity numerical value and is shown in Table 1.

Table 1.Biacore is analyzed

Experimental result illustrate, C-T Crossmab of the invention fully remain simultaneously cetuximab with The affinity of tarextumab.

Embodiment 3:C-T Crossmab suppress non-small cell lung cancer cell viability experiment

The good HCC827 cells (ATCC) of growth conditions are taken, adjustment cell concentration is 5 × 10³/ ml, is inoculated in 96 holes thin Born of the same parents' culture plate, 200 μ l/ holes, in 37 DEG C, 5%C0₂After cultivating 24h in incubator, add final concentration of 5nmol's in nutrient solution Bispecific antibody the C-T Crossmab, cetuximab, tarextumab, cetuximab+ of EGF and various concentrations gradient Tarextumab, unrelated human IgG (Rituximab is purchased from Roche companies), after 4 days, cell viability CellTiter- Glo Luminescent Cell Viability Assay kits (Promega, Madison, WI) is detected.

Experimental result is as shown in Figure 2.Test result indicate that, the suppressible HCC827 cell viabilities of C-T Crossmab, There is more significant inhibition than cetuximab and tarextumab, it is double with cetuximab and tarextumab use in conjunction Antibodyome effect is similar or more excellent.

Embodiment 4：Suppress tumour growth experiment in C-T Crossmab bodies

It is antitumor activity in detection C-T Crossmab bodies, HCC827 cells is used first, is inoculated in BALB/c nude mices (the Two army medical university's Experimental Animal Centers) right flank side is subcutaneous, into knurl after tail vein injection 10mg/kg each group following antibodies：C-T Crossmab, cetuximab, tarextumab, two kinds of monoclonal antibodies of unrelated control human IgG, cetuximab and tarextumab Each 5mg/kg use in conjunction, injects 1 time weekly, continues to the excessive execution of mouse tumor.The length and width of measurement tumour, calculate swollen daily Knurl volume.

Tumor growth curve is as shown in Figure 3.Test result indicate that：The C-T Crossmab treatment group tumors speeds of growth are notable Less than cetuximab and tarextumab treatment groups (after 70 days, P<0.01, Mann-Whitney inspection).And C-T The therapeutic effect of Crossmab be better than with cetuximab and tarextumab use in conjunction effect (after 70 days, P<0.01,Mann- Whitney is checked).

Below the preferred embodiment to the invention is illustrated, but the invention be not limited to it is described Embodiment, those of ordinary skill in the art can also make a variety of equivalent on the premise of without prejudice to the invention spirit Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.

SEQUENCE LISTING

<110>Second Military Medical University, PLA

<120>Anti-human EGFR and Notch multi-specificity antibodies, Preparation Method And The Use

<130>Specification, claims

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 449

<212> PRT

<213>Artificial sequence

<400> 1

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

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

20 25 30

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

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

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

65 70 75 80

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

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 2

<211> 449

<212> PRT

<213>Artificial sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser

20 25 30

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

35 40 45

Ser Val Ile Ala Ser Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Ser Ile Phe Tyr Thr Thr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 3

<211> 214

<212> PRT

<213>Artificial sequence

<400> 3

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

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 213

<212> PRT

<213>Artificial sequence

<400> 4

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Ala Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Asn Phe Pro

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser Ala Ser

100 105 110

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

115 120 125

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

130 135 140

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

145 150 155 160

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

165 170 175

Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr

180 185 190

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

195 200 205

Glu Arg Lys Cys Cys

210

<210> 5

<211> 1347

<212> DNA

<213>Artificial sequence

<400> 5

caggtgcagc tgaagcagag cggccccggc ctggtgcagc ccagccagag cctgagcatc 60

acctgcaccg tgagcggctt cagcctgacc aactacggcg tgcactgggt gcgccagagc 120

cccggcaagg gcctggagtg gctgggcgtg atctggagcg gcggcaacac cgactacaac 180

acccccttca ccagccgcct gagcatcaac aaggacaaca gcaagagcca ggtgttcttc 240

aagatgaaca gcctgcagag caacgacacc gccatctact actgcgcccg cgccctgacc 300

tactacgact acgagttcgc ctactggggc cagggcaccc tggtgaccgt gagcgccgcc 360

agcaccaagg gccccagcgt gttccccctg gcccccagca gcaagagcac cagcggcggc 420

accgccgccc tgggctgcct ggtgaaggac tacttccccg agcccgtgac cgtgagctgg 480

aacagcggcg ccctgaccag cggcgtgcac accttccccg ccgtgctgca gagcagcggc 540

ctgtacagcc tgagcagcgt ggtgaccgtg cccagcagca gcctgggcac ccagacctac 600

atctgcaacg tgaaccacaa gcccagcaac accaaggtgg acaagcgcgt ggagcccaag 660

agctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg cacccccgag 780

gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840

gtggacggcg tggaggtgca caacgccaag accaagcccc gcgaggagca gtacaacagc 900

acctaccgcg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020

gccaagggcc agccccgcga gccccaggtg tacaccctgc ccccctgccg cgaggagatg 1080

accaagaacc aggtgagcct gtggtgcctg gtgaagggct tctaccccag cgacatcgcc 1140

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200

gacagcgacg gcagcttctt cctgtacagc aagctgaccg tggacaagag ccgctggcag 1260

cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320

aagagcctga gcctgagccc cggcaag 1347

<210> 6

<211> 1347

<212> DNA

<213>Artificial sequence

<400> 6

gaggtgcagc tggtggagag cggcggcggc ctggtgcagc ccggcggcag cctgcgcctg 60

agctgcgccg ccagcggctt caccttcagc agcagcggca tgagctgggt gcgccaggcc 120

cccggcaagg gcctggagtg ggtgagcgtg atcgccagca gcggcagcaa cacctactac 180

gccgacagcg tgaagggccg cttcaccatc agccgcgaca acagcaagaa caccctgtac 240

ctgcagatga acagcctgcg cgccgaggac accgccgtgt actactgcgc ccgcagcatc 300

ttctacacca cctggggcca gggcaccctg gtgaccgtga gcagcgccag cgtggccgcc 360

cccagcgtgt tcatcttccc ccccagcgac gagcagctga agagcggcac cgccagcgtg 420

gtgtgcctgc tgaacaactt ctacccccgc gaggccaagg tgcagtggaa ggtggacaac 480

gccctgcaga gcggcaacag ccaggagagc gtgaccgagc aggacagcaa ggacagcacc 540

tacagcctga gcagcaccct gaccctgagc aaggccgact acgagaagca caaggtgtac 600

gcctgcgagg tgacccacca gggcctgagc agccccgtga ccaagagctt caaccgcggc 660

gagtgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg cacccccgag 780

gtgacctgcg tggtggtgga cgtgagccac gaggaccccg aggtgaagtt caactggtac 840

gtggacggcg tggaggtgca caacgccaag accaagcccc gcgaggagca gtacaacagc 900

acctaccgcg tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 960

tacaagtgca aggtgagcaa caaggccctg cccgccccca tcgagaagac catcagcaag 1020

gccaagggcc agccccgcga gccccaggtg tgcaccctgc cccccagccg cgaggagatg 1080

accaagaacc aggtgagcct gagctgcgcc gtgaagggct tctaccccag cgacatcgcc 1140

gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccccgtgctg 1200

gacagcgacg gcagcttctt cctggtgagc aagctgaccg tggacaagag ccgctggcag 1260

cagggcaacg tgttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag 1320

aagagcctga gcctgagccc cggcaag 1347

<210> 7

<211> 642

<212> DNA

<213>Artificial sequence

<400> 7

gacatcctgc tgacccagag ccccgtgatc ctgagcgtga gccccggcga gcgcgtgagc 60

ttcagctgcc gcgccagcca gagcatcggc accaacatcc actggtacca gcagcgcacc 120

aacggcagcc cccgcctgct gatcaagtac gccagcgaga gcatcagcgg catccccagc 180

cgcttcagcg gcagcggcag cggcaccgac ttcaccctga gcatcaacag cgtggagagc 240

gaggacatcg ccgactacta ctgccagcag aacaacaact ggcccaccac cttcggcgcc 300

ggcaccaagc tggagctgaa gcgcaccgtg gccgccccca gcgtgttcat cttccccccc 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagagcgg caacagccag 480

gagagcgtga ccgagcagga cagcaaggac agcacctaca gcctgagcag caccctgacc 540

ctgagcaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac ccaccagggc 600

ctgagcagcc ccgtgaccaa gagcttcaac cgcggcgagt gc 642

<210> 8

<211> 639

<212> DNA

<213>Artificial sequence

<400> 8

gacatcgtgc tgacccagag ccccgccacc ctgagcctga gccccggcga gcgcgccacc 60

ctgagctgcc gcgccagcca gagcgtgcgc agcaactacc tggcctggta ccagcagaag 120

cccggccagg ccccccgcct gctgatctac ggcgccagca gccgcgccac cggcgtgccc 180

gcccgcttca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240

cccgaggact tcgccgtgta ctactgccag cagtacagca acttccccat caccttcggc 300

cagggcacca aggtggagat caagagcagc gccagcacca agggccccag cgtgttcccc 360

ctggccccct gcagccgcag caccagcgag agcaccgccg ccctgggctg cctggtgaag 420

gactacttcc ccgagcccgt gaccgtgagc tggaacagcg gcgccctgac cagcggcgtg 480

cacaccttcc ccgccgtgct gcagagcagc ggcctgtaca gcctgagcag cgtggtgacc 540

gtgcccagca gcaacttcgg cacccagacc tacacctgca acgtggacca caagcccagc 600

aacaccaagg tggacaagac cgtggagcgc aagtgctgc 639

Claims

1. a kind of anti-human EGFR and Notch multi-specificity antibodies, it includes two heavy chain polypeptides and two light chains polypeptide, wherein heavy The amino acid sequence of chain polypeptide such as SEQ ID NO：1 and SEQ ID NO：Shown in 2；The amino acid sequence of light chain polypeptide such as SEQ ID NO：3 and SEQ ID NO：Shown in 4.

2. polynucleotides, it includes the nucleotide sequence for encoding antibody as claimed in claim 1.

3. polynucleotides according to claim 2, its include encoding heavy chain polypeptide such as SEQ ID NO：5 and SEQ ID NO：Nucleotide sequence shown in 6；And coding light chain polypeptide such as SEQ ID NO：7 and SEQ ID NO：Nucleotides shown in 8 Sequence.

4. recombinant expression carrier, it includes polynucleotides as claimed in claim 2 or claim 3.

5. recombinant expression carrier according to claim 4, it is characterised in that described expression vector be pcDNA3.1, PDR1 or pDHFR.

6. the host cell for being converted with the carrier as described in claim 5 or 6.

7. host cell according to claim 6, it is characterised in that described cell is Chinese hamster ovary cell, NS0 Myeloma cell, COS cells or SP2/0 cells.

8. the preparation method of antibody as claimed in claim 1, it comprises the following steps：

I () under conditions of being adapted to allow the antibody expression, culture is according to the host cell such as claim 6 or 7；With

(ii) antibody of expression is reclaimed.

9. application of the antibody as claimed in claim 1 in antineoplastic is prepared.

10. pharmaceutical composition, it includes antibody as claimed in claim 1, and at least one pharmaceutically useful carrier, diluent or Excipient.