CN108752478A - Human anti-human EGFR and Notch2/3 multi-specificity antibody, preparation method and the usage - Google Patents

Abstract

The present invention relates to biomedicine technical field, the more targeting antibodies of specifically a kind of Human anti-human EGFR, Notch2/3, preparation method and its application in preparation of anti-tumor drugs.Double targeting antibodies disclosed by the invention, on the architecture basics for retaining whole antibody, tri- kinds of targeting EGFR, Notch2/3 cell membrane surface receptors, you can to block EGFR, also Notch2 and Notch3 can be blocked, and with anti-tumor biological more better than independent targeting EGFR, Notch2/3.

Description

Human anti-human EGFR and Notch2/3 multi-specificity antibody, preparation method and Purposes

Technical field

The present invention relates to biomedicine technical fields, specifically, being a kind of more targets of Human anti-human EGFR, Notch2/3 To antibody, preparation method and its application in preparation of anti-tumor drugs.

Background technology

Based on operative treatment, radiotherapy and chemotherapy, it is in recent years to disliking to be aided with novel targeted therapy scheme The elementary tactics of property oncotherapy, and important progress is obtained in clinical practice.But the Preventive of malignant tumour and Therapeutic tolerance is still the clinical problem with researcher of puzzlement always.It 2006, is held in american association of cancer research Tumor stem cell seminar on, participant scholar define tumor stem cell be it is a kind of there is self-renewing characteristic, driving of having the ability Formed the Tumor Differentiation cell mass of a variety of heterogeneities, with stem cell properties cancer cell (Wosikowski et al., 1997, Altered gene expression in drug-resistant human breast cancer cells,3:2405- 2414).Cancer stem-cell hypothesis is with its unique theory advantage and the research supporting evidence enriched constantly, increasingly by the country The approval and receiving of outer scholar.Although traditional therapy has preferable treatment fragmentation effect to tumor entity cell, to only It accounts for tumor cell group 0.2%~10% and with tumor entity cell there is the tumor stem cell difficulty of bigger molecule phenotypic difference to have Inhibiting effect.Study tumor stem cell tumour Emergence and Development and transfer important function and its mechanism, and exploitation it is novel Antineoplaston strategy based on cancer stem-cell hypothesis and preparation, have a very important significance the treatment of malignant tumour.

Cancer stem-cell hypothesis thinks：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 are separated to what tumor stem cell and elaborating was driven by tumor stem cell from leukaemia cell first Tumor Heterogeneity laminated tissue model (Galizia et al., 2006, Prognostic significance of epidermal growth factor receptor expression in colon cancer patients undergoing curative surgery,13:823-835).Then, researcher also demonstrates swollen in a variety of solid tumors The presence of tumor stem cell and similar Tumor Heterogeneity tissue characteristic, including breast cancer, colon cancer, brain tumor, oophoroma, lung Cancer, prostate cancer and cancer of pancreas (Mendelsohn and Baselga, 2003, Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer,21:2787- 2799).These evidences show there is the tumour cell with stem cell properties in most of neoplastic hematologic disorder and solid tumor.

Multinomial studies have shown that tumor stem cell is in malignant tumour generation, development；Tumour cell heterogeneity maintains；Tumour into Change；It plays an important role during being resistant to 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 It clones or migrates to and carry out clone's tumor formation in immunodeficient mice model, and only need a small amount of CSC can be in immune deficiency Tumor formation is cloned in mouse model, and tumour transplatation object has phenotype heterogeneity (the Baselga and similar with primary tumors Arteaga,2005,Critical update and emerging trends in epidermal growth factor receptor targeting in cancer,23:2445-2459；Galizia et al.,2006,Prognostic significance of epidermal growth factor receptor expression in colon cancer patients undergoing curative surgery,13:823-835；Pao et al.,2005,Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain,2:e73)。

2) complicated heterogeneity and bioremodelable.The source of CSC is possible and its complicated, 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 (Riely the et al., 2006, Clinical course of patients of my updating ability with non–small cell lung cancer and epidermal growth factor receptor exon 19and exon 21mutations treated with gefitinib or erlotinib,12:839-844)；Or come Derived from by the induction oncogene property mutation of the external microenvironment factor normal tissue stem cell (Cunningham et al., 2004, Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory metastatic colorectal cancer,351:337-345).In addition, some reports in recent years also confirm that CSC has It is dry thin can to go out two kinds of not isophenic breast cancer such as in breast carcinoma stem cell with major diagnostic for extremely complex bioremodelable Born of the same parents group：A kind of breast cancer CSC groups of the active proliferation of Epithelial, expression stem cell markers ALDH；With a kind of interstitial sample, phase To inactive but strong wetting capacity breast cancer CSC groups, surface marker expression is CD44+/CD24-.This two crowds of CSC Respective Epithelial or interstitial sample tumor entity Cell Progeny can be generated, at the same can discharge secretion signal enhancing CSC from My updating ability.And the apparent something lost that cytokine signaling, chemotactic factor (CF) signal, transcriptional control mediate in by tumor microenvironment It passes and learns under change, two groups of cells can mutually convert (Hubbard, 2005, EGF receptor inhibition:attacks on multiple fronts,7:287-288).In addition, research is also reported in liver cancer there are CSC groups different, such as CD133+ CSC(Hu et al.,2013,Comparison of the Inhibition Mechanisms of Adalimumab and Infliximab in Treating Tumor Necrosis Factorα-Associated Diseases from a Molecular View,288:27059-27067) and the liver cancer CSC of CD90+/CD44+ (Li et al., 2008, Structural basis for EGF receptor inhibition by the therapeutic antibody IMC- 11F8,16:216-227) there are different stem cell properties.The marker of stem cell also tends to have in different tumor types Difference, such as in cancer of pancreas, the surface marker of CSC be considered as CD24+ (Li et al., 2005, Structural basis for inhibition of the epidermal growth factor receptor by cetuximab,7: 301-311), then it is CD24- and for mammary gland CSC.In addition, the content and ratio of CSC are often different in different malignant tumours, And in the different phase of malignant tumour, complicated variation can also occur for the ratio of CSC.Such as in colon cancer, CD133+ high expression CSC ratios are about 1.8%~24.5% (Mukohara et al., 2005, Differential effects of gefitinib and cetuximab on non–small-cell lung cancers bearing epidermal growth factor receptor mutations,97:1185-1194), and after receiving chemotherapy, the ratio of colon cancer CSC (Yen et al., 2015, Targeting Notch signaling with a Notch2/ can then be improved Notch3antagonist(tarextumab)inhibits tumor growth and decreases tumor- initiating cell frequency,21:2084-2095).And in melanoma, express CD133+ cell surface markers CSC ratio between 1%~20% (Chinnaiyan et al., 2005, Mechanisms of enhanced radiation response following epidermal growth factor receptor signaling inhibition by erlotinib(Tarceva),65:3328-3335).These results of study prompt CSC to have extremely Complicated heterogeneity and bioremodelable.

3) therapeutic sensitivity is poor.In recent years some are studies have shown that treatments of the CSC to being used in many currently clinics Method is resistant to, including Treated with Chemotherapeutic Drugs object and radiotherapy.Such as have been reported that display human acute myeloid leukemia CD34+/CD38- ancestrals Apoptotic effect insensitive (Costello the et al., 2000, Human acute that cell mediates 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,60:4403-4411)；And high expression Telomerase, maintain dye The normal configuration of colour solid is to inhibit Apoptosis；Height expresses a variety of anti-apoptotic genes expressions, 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- breast cancer CSC is insensitive to radiotherapy (Arasada et al.,2014,EGFR blockade enriches for lung cancer stem–like cells through Notch3-dependent signaling,74:5572-5584)；The glioma stem cells of CD133+ have very strong DNA damage repair ability, (Hu et al., 2015, Four-in-one antibodies haves insensitive to radiotherapy superior cancer inhibitory activity against EGFR,HER2,HER3,and VEGF through disruption of HER/MET crosstalk,75:159-170)；Dylla etc. also reports colon cancer CSC after chemotherapy Cells ratio increases in bare mouse different species tumor model, (Yen et al., 2015, Targeting Notchs insensitive to chemotherapy signaling with a Notch2/Notch3antagonist(tarextumab)inhibits tumor growth and decreases tumor-initiating cell frequency,21:2084-2095).And tumor stem cell is done with normal The all high expression ATP binding cassette transporters (ATP-binding cassette transporters, ABC) of cell, 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 The outer row effect that ABCG2 is mediated prevents the dyestuffs such as Hoechst 33342 and rhodamine 123 from dyeing stem cell, it was confirmed that ABC albumen is the key that stem cell to the insensitive characteristic of chemotherapy (Ferguson et al., 2003, EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization, 11:507-517).CSC has a various biological attribute similar with normal stem cell, such as opposite proliferation is inactive, to drug and Toxin is insensitive, a variety of ABC albumen of high expression, nucleic acid repair ability are strong and apoptosis resistivity (Tortora et al., 1999,Cooperative inhibitory effect of novel mixed backbone oligonucleotide targeting protein kinase A in combination with docetaxel and anti-epidermal growth factor-receptor antibody on human breast cancer cell growth,5:875- 881).In addition, CSC is similar with normal stem cell, it is collectively formed tumor stem cell tabernacle with the microenvironment of surrounding, relatively hypoxia in tabernacle And a large amount of bio signal for maintaining stable state can be provided to CSC, there is barrier action to environmental stimuli.Conventional cytotoxicity is controlled Treat influences minimum (Petty et al., 2011, High cyclin D3 expression to the stem cell being in cell tabernacle confers erlotinib resistance in aerodigestive tract cancer,74:384-391).Therefore, CSC can be survived during chemicotherapy, and multiply tumour again, finally so that tumor recurrence and therapeutic resistance to By.

4) it is strong to promote transfer invasive ability.Due to the height heterogeneity of tumor tissues, most of tumor entity cell is not The ability of invasion and transfer, and CSC then has very strong invasion and transfer ability.Such as have been reported that display, breast cancer CSC and breast Important EMT processes are highly relevant in gland cancer transfer process, and the CSC of EMT samples, which is often positioned, is that entity tumor infiltrates edge, and And it easily enters blood circulation and forms micro metastasis (Fu et al., 2014, Insights into distal end HER2signaling from step-by-step optimization of anti-HER2antibodies,6:978- 990).At this moment, CSC again can some microenvironment factors (TWIST that such as ID1 is mediated lower (Perosa et al., 2003, Beta2-microglobulin-free HLA class I heavy chain epitope mimicry by monoclonal antibody HC-10-specific peptide,171:It is changed into high value-added work under 1918-1926) acting on The epithelium state CSC of property, is finally completed Metastasis in Breast Cancer process.In cancer of pancreas, CD133+/CXCR4+ cell surface markers are expressed CSC be also usually located in tumor-infiltrated edge, be easily accessible blood, and its height expression chemokine receptors.Become in CXCR4 Under the action of changing the factor, such CSC is easy to be transferred in liver (Garner et al., 2013, An antibody that locks HER3 in the inactive conformation inhibits tumor growth driven by HER2 or neuregulin,73:6024-6035).The studies above prompts CSC to be played very in the infiltration and transfer of malignant tumour Important role.

In conclusion CSC has, the heterogeneity that oncogenicity is strong, complicated and bioremodelable, transfer invasive ability is strong and controls It is poor to treat sensibility.Tumor tissues are after conventional cytotoxic therapies, after chemicotherapy and Biological target therapy, remaining height Oncogenicity CSC starts to enhance proliferation activity and starts to expand and drive tumor tissue growth, shows as clinical tumor recurrence.Upper On the basis of stating theoretical and research, numerous researchers think, the therapeutic strategy of targeting CSC cell masses will supplement existing treatment plan Deficiency slightly, and can significantly improve oncotherapy level (Bonner et al., 2006, Radiotherapy plus cetuximab for squamous-cell carcinoma of the head and neck,354:567-578；Keysar and Jimeno,2010,More than markers:biological significance of cancer stem cell-defining molecules,9:2450-2457)。

Current research has defined, and there is the signal path of imbalance in CSC, most important to the regulation and control of CSC, including Wnt signal paths, and Notch signal paths and Hedgehog signal paths etc. (Shien et al., 2013, Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell–like properties in cancer cells,73:3051-3061).1916, Notch genes were for the first time in drosophila It is found in vivo, the mutant of the gene can make drosophila wing edge generate notch, hence obtain one's name.Complete Notch signals are logical Road includes transmembrane protein receptor, transmembrane protein, transcription factor and downstream target gene, and wherein human cell shares 4 kinds of Notch receptors (Notch1~4) and 5 kinds of ligands (Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1 and Jagged- 2)(Bailey et al.,2007,Cancer metastasis facilitated by developmental pathways:Sonic hedgehog,Notch,and bone morphogenic proteins,102:829-839), exist It is highly conserved in evolution, have the function of regulating cell proliferation, differentiation and apoptosis, and be almost related in a organized way and organ. Notch genes have very significant oncogenicity effect, this is embodied in a variety of human tumors (including lymthoma, breast cancer, lung Cancer, H/N tumors, cancer of pancreas, colon cancer, osteosarcoma of jaw and glioblastoma) activated mutant, and be expanded to Notch letter Members at different levels (Weng the and Aster, 2004, Multiple niches for Notch in cancer of number access: context is everything,14:48-54).In kinds of tumors, Notch signal paths are proved with tumor stem cell The maintenance of dryness is related.Recently, the monoclonal antibody tarextumab of OncoMed drugmakers announcement targeting Notch accesses is controlled Treat patient's survival and the biomarker data of I b clinical trial phases of Small Cell Lung Cancer.Tarextumab is full-length human Dan Ke Grand antibody targets Notch2/3 receptors.Preclinical study prompt has 2 kinds of mechanism of action, that is, lowers Notch path signals, resists swollen The microenvironment of tumor stem cell role and influence pericyte, stroma cell and 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 are widely present in, the growth through entire cell and breeding, to cell Differentiation has selected important adjustment effect.EGFR families include mainly EGFR (i.e. HER1) and HER2-4.EGFR encodes cross-film egg In vain, signal is activated through receptor tyrosin phosphorylation, by Ras/Raf/MEK/MAPK approach Cascaded amplifications, finally results in The MAPK signals of the phosphorylation of MAPK, modification enter nucleus, promote the phosphorylation of target gene, adjust the expression and work of gene Property.The occurrence and development and prognosis of EGFR signals and tumour are closely related, in kinds of tumors such as lung cancer, colon cancer, breast cancer, forefront 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 receptor increases the resistance to endocrine therapy,.Preclinical study shows that the EGFR of composition activation can be obviously increased 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) and Victibix (panitumumab, Vectibix it is) to study the most extensively to be agreed in kinds of tumors treatment with most common anti-EGFR targeted drugs, curative effect It is fixed.

The interaction mode of EGFR and Notch signals are complicated, can show as acting synergistically, can also show as antagonism Effect, the stage of type and development dependent on tissue.In glioma, Notch raises the expression of EGFR, in differentiated In glioma, the expression of EGFR and Notch are closely related.And in cutaneous squamous cell carcinoma, EGFR signal functions in The upstream of Notch1 signals, and inhibit the expression of signal.It is sent out in the in vitro culture of mammary gland original position duct cell carcinoma with signal Wave collaboration facilitation.These are right research shows that EGFR signals and Notch signals are two bars conduction paths being closely connected There is important adjustment effect in the occurrence and development of tumour.

In conclusion Notch signal paths are the generation for participating in tumour cell EMT, the dimension of tumour CSC cell masses and dryness 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 kind of excessive activation signal path, and occupied an important position in tumor entity tumor targeted therapy.And it is deposited between two signal paths Cross activation at all levels.Therefore, therefore, two kinds of signals can be blocked simultaneously by building one using technique for gene engineering Multi-specificity antibody, EGFR can be blocked and block Notch, and then simultaneously to tumor stem cell and tumor entity cell It is always that those skilled in the art is anxious to be resolved to lead to the problem of inhibiting effect.Before, the present inventor's previous research is Bifunctional antibody drug is prepared using cetuximab and Tarextumab as parental antibody structure, and with very a lot of anti- Tumor stem cell acts on (Hu et al., 2017, Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor-initiating cell frequency,9:Eaag0339), Chinese invention patent application number CN201611102039.X (publication No. CN have been applied for 106800599A), but Cetuximab is a kind of human mouse chimeric antibody, has potential immunogenicity (Galizia, G., et al."Cetuximab,a chimeric human mouse anti-epidermal growth factor receptor monoclonal antibody,in the treatment of human colorectal cancer."Oncogene 26.25(2007):3654.)。

Therefore, EGFR the and Notch targeting antibodies that further exploitation prepares full people source are urgently to be resolved hurrily.

Invention content

The purpose of the present invention is to provide a kind of multi-specificity antibodies that can block multiple target spots simultaneously, you can to block EGFR can also block Notch2 and Notch3.Second object of the present invention is to provide the preparation method of the antibody.The present invention Third purpose be to utilize the antibody, and include medicine for tumor disease of the antibody as active constituent Object.

To achieve the goals above, the present invention uses following technological means：

The first aspect of the present invention provides a kind of anti-EGFR and Notch2/3IgG molecules sample intersection monoclonal antibody (Crossmab) P-T Crossmab, the Crossmab energy and EGFR, Notch2 and Notch3 are combined.

Preferably, the antibody P-T Crossmab are made of four peptide chains, respectively amino acid sequence such as SEQ ID NO：Panitumumab heavy chains knob mutant shown in 19, amino acid sequence such as SEQ ID NO：Shown in 23 Panitumumab light chains, amino acid sequence such as SEQ ID NO：Tarextuimab heavy chains hole mutant, amino shown in 21 Acid sequence such as SEQ ID NO：Tarextumab light chain mutants Tarex-VL-CH1 shown in 25.

The second aspect of the present invention provides a kind of polynucleotides, and it includes the nucleotides sequences for encoding antibody as described above Row.

In the present invention, the suitable DNA of any coding EGFR, Notch2 and Notch3 are adapted to the present invention, such Structure includes isolated from tissue or cell mRNA, is synthesized from basis with sequence full genome in publishing database, or It is obtained from other cDNA libraries.

Preferably, the polynucleotides, it includes coding panitumumab heavy chain knob mutant such as SEQ ID NO：Nucleotide sequence shown in 20, and coding panitumumab light chains such as SEQ ID NO：Nucleotide sequence shown in 24, With coding Tarextuimab heavy chain hole mutant such as SEQ ID NO：Nucleotide sequence shown in 22, and coding Tarextumab light chain mutants Tarex-VL-CH1 such as SEQ ID NO：Nucleotide sequence shown in 26.

The third aspect of the present invention provides a kind of recombinant expression carrier, and it includes polynucleotides as described above.

In the present invention, any suitable carrier can use, and can be pcDNA3.1, pEE6.4, pEE12.4, pDHFR One of, expression vector includes the fusion dna sequence for being connected with suitable transcription and translation regulatory sequence.

Preferably, the recombinant expression carrier can be selected from pcDNA3.1, pEE6.4, pEE12.4, one of pDHFR. In a preferred embodiment of the present invention, the recombinant expression carrier is pcDNA3.1.

The fourth aspect of the present invention provides a kind of host cell converted with recombinant expression carrier as described above, described Host cell be eukaryocyte.

In the present invention, the table of mammal or insect host cell culture systems P-T Crossmab for use in the present invention It reaches, COS, CHO, 293, NS0, sf9 and sf21 etc. may be applicable to the present invention.

Preferably, the host cell is mammalian cell.In a preferred embodiment of the present invention, described Host cell be Chinese hamster ovary Chinese hamster ovary celI.

The fifth aspect of the present invention provides a kind of method preparing antibody P-T Crossmab as described above, including such as Lower step：

A) in the case where being suitble to allow the antibody P-T Crossmab expression conditions, above-mentioned host cell is cultivated, to table Up to antibody P-T Crossmab；

B) the antibody P-T Crossmab described in isolated or purified.

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

Using the above method, it is substantially uniform substance that can purify bispecific antibody, such as in SDS-PAGE electricity It is single band in swimming.

P-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 can use conventional method such as high-salt buffer, change the methods of PH elution of bound affine P-T Crossmab polypeptides on column.

In a preferred embodiment of the present invention, above-mentioned multi-specificity antibody P-T Crossmab disclosed by the invention It obtains by the following method：

1) the heavy and light chain gene of full genome synthesis multi-specificity antibody.The sequence reference of the full genome sequence of monoclonal antibody Arrange panitumumab (Sickmier et al., 2016, The panitumumab egfr complex reveals a binding mechanism that overcomes cetuximab induced resistance,11:E0163366) and Crossmab heavy chain light chains design scheme (Konishi et al., 2007, Gamma-secretase inhibitor prevents Notch3activation and reduces proliferation in human lung cancers,67: 8051-8057) and (Gu et al., 2012, Notch signals in the endothelium and cancer " stem- like"cells:opportunities for cancer therapy,4:1)。

2) Tarextuimab heavy chain variable regions are constructed into Tarex-VH-CL with antibody light chain constant region and merges segment；

3) Tarextuimab light chain variable regions are constructed into Tarex-VL-CH1 with the areas heavy chain of antibody CH1 and merges segment；

4) other that knob mutant is built to antibody Fc region：T366W, S354C, hole mutant：T366S, L368A, Y407V and Y394C.

5) panitumumab heavy chain variable regions are merged with knob mutant respectively, Tarex-VH-CL and hole mutant Fusion, panitumumab light chains and Tarex-VL-CH1 are packed into expression vector；

6) by above-mentioned steps 5) in four kinds of expression vectors carry out corotation expression, by isolating and purifying to obtain double targeting antibodies P-T Crossmab。

The sixth aspect of the present invention provides a kind of antibody P-T Crossmab as described above in the preparation of antitumor drugs Application.

Preferably, the drug, using the antibody P-T Crossmab as active constituent.

The seventh aspect of the present invention provides a kind of composition, contains antibody P-T Crossmab as described above and medicine Acceptable carrier or auxiliary material on.

The eighth aspect of the present invention provides a kind of purposes of composition as described above in the preparation of antitumor drugs.

Preferably, purposes as described above further includes and other antitumor drugs is used in combination.

The present invention carries out affinity detection to above-mentioned P-T Crossmab, it is found that P-T Crossmab are fully protected simultaneously The affinity of panitumumab and tarextumab are stayed, therefore we carry out next step experiment, including tumour cell is inhibited to increase Grow with the experiments such as internal tumor suppression, the experimental results showed that, P-TCrossmab disclosed by the invention have simultaneously panitumumab and The function of tarextumab.In addition, multi-specificity antibody P-T Crossmab can be maximum as traditional IgG molecules Conventional monoclonal antibody structure is remained to degree, it, can be affine with common Protein A columns because there is the presence of Fc segments Purification by chromatography is conducive to large-scale production and purifies.Experiment shows under equal dose, multi-specificity antibody P-T Crossmab has similar to use in conjunction panitumumab and tarextumab or stronger antineoplaston effect.

The present invention discloses above-mentioned bispecific antibody P-T Crossmab, can be with pharmaceutically acceptable auxiliary material together For composition pharmaceutical preparations composition to more stably play curative effect, these preparations can ensure that bispecific disclosed by the invention is anti- The conformation integrality of body amino acid core sequence, while the polyfunctional group of protected protein matter also being wanted to prevent its degradation (including but not It is limited to cohesion, deamination or oxidation).Under normal conditions, it for liquid preparation, can usually be preserved at least under the conditions of 2 DEG C -8 DEG C Stablize 1 year, for lyophilized preparation, keeps at least six months stablizing at 30 DEG C.Preparation can be that pharmaceutical field commonly mixes herein The preparations, preferably liquid drugs injection or lyophilized preparation such as outstanding, liquid drugs injection, freeze-drying, for the liquid drugs injection of above-mentioned P-T Crossmab disclosed by the invention Or lyophilized preparation, pharmaceutically acceptable auxiliary material include surfactant, solution stabilizer, isotonic regulator and buffer solution it One or combinations thereof, wherein surfactant includes 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 sarcosine；Pluronics；MONAQUAT^TMDeng 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, advanced sugar alcohol, propylene glycol, polyethylene glycol, The addition of solution stabilizer should make the preparation those skilled in the art eventually formed think to reach in stable time Keep stable state, isotonic regulator can be sodium chloride, one of mannitol, buffer solution can be TRIS, histidine buffering liquid, One of phosphate buffer.

Above-mentioned preparation is the composition for including P-T Crossmab, antitumor after to animal administration including people It is with obvious effects.Specifically, effective to the prevention of tumour and/or treatment, it can be used as antitumor drug.

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, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovum Nest, pancreas, parathyroid gland, penis, prostate, skin, salivary gland, spleen, testis, thymus gland, thyroid gland and uterus.In addition to above-mentioned Tumour outside, it may also be used for tumour of central nervous system such as spongiocyte diversity tumor, astrocytoma etc., eye is swollen in addition Tumor includes basal-cell carcinoma, squamous cell carcinoma, melanoma etc., further include endocrine disrupting effects, neuroendocrine system tumour, Gastrointestinal tract pancreatic endocrine system tumor, genital system and head and neck neoplasm etc..Here it will not enumerate.

The so-called antitumor drug of the present invention, refers to the drug for inhibiting and/or treating tumour, may include with tumour The delay of related symptoms development and/or the reduction of these severity of symptom are grown, it further comprises already present tumour It grows the mitigation of simultaneous phenomenon and prevents the appearance of other symptoms, still reduce or prevent to shift.

For P-T Crossmab and combinations thereof when to animal administration including people, dosage is because of disease in the present invention The age of people and weight, disease traits and seriousness and administration route and it is different, the result of zoopery and various can be referred to Situation, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 1~1800mg/ days.

P-T Crossmab disclosed by the invention and combinations thereof can also be with other antineoplastic administering drug combinations or connection Treatment of the radiotherapy for tumour is closed, these antineoplastics include 1, cytotoxic drug：(1) DNA chemistry knots are acted on The drug of structure：Alkylating agent such as nitrogen mustards, nitrous urine class, pyrovinic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and oxalic acid platinum Deng；Mitomycin (MMC)；(2) drug of nucleic acid synthesis is influenced：Dihydrofolate reductase inhibitor such as methopterin (MTX) and Alimta etc.；Thymus nucleoside synthetase inhibitors such as fluorouracil (5FU, FT-207, capecitabine) etc.；Purine nucleosides closes At enzyme inhibitor such as Ismipur (6-MP) and 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxycarbamide (HU) etc.；DNA Poly enzyme inhibitor such as cytarabine (Ara-C) and gemzar (Gemz) etc.；(3) drug of transcribed nucleic acid is acted on：Selectivity is made For DNA profiling, inhibit DNA dependenc RNA polymerases, to inhibit the drug of RNA synthesis such as：Actinomycin D, daunorubicin, Adriamycin, Epi-ADM, aclacinomycin, mithramycin etc.；(4) drug of tubulin synthesis is mainly acted on：Taxol, Taxotere, vinblastine, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine；(5) other Cytotoxic drugs：L-Asparaginasum is main Inhibit the synthesis of protein；2, steroids：Such as anti-estrogens：Tamoxifen, Droloxifene, Exemestane etc.；Aromatizing enzyme Inhibitor：Aminoglutethimide, Lactel be grand, Letrozole, auspicious Ningde etc.；Antiandrogen：Its ammonia RH-LH agonist/antagonist of fluorine：Promise thunder Moral, enatone etc.；3, biological response modifiers：Tumour class drug, such as interferon are mainly inhibited by body's immunity；It is white thin Born of the same parents' interleukin -2；Thymic；4, monoclonal antibodies：Such as Mabthera (MabThera), Trastuzumab (Trastuzumab), Check point blocking agents, such as anti-PD1 antibody is immunized in Bevacizumab (Avastin)；5, various radiotherapy；6, its It is unknown and need the drug further studied that he includes some current mechanism；Cell-differentiation inducers such as retinoids；Apoptosis lures Lead agent.P-TCrossmab disclosed by the invention and combinations thereof can be with one or a combination set of above-mentioned antitumor drug joint Medication.

Double targeting antibodies disclosed by the invention, on the architecture basics for retaining whole antibody, tri- kinds of targeting EGFR, Notch2/3 Cell membrane surface receptors, and with anti-tumor biological more better than independent targeting EGFR, Notch2/3.

Description of the drawings

Fig. 1 .P-T Crossmab structural schematic diagrams；

Fig. 2 .P-T Crossmab inhibit HCC827 cell activity experimental results；

Fig. 3 .P-T Crossmab inhibit HCC827 tumour formation curves.

Specific implementation mode

It elaborates to specific implementation mode provided by the invention with reference to embodiment.

The present invention is further detailed in following embodiment, experimental example, should not be construed as limiting the invention.It is real The base of albumen will not encoded including detailed descriptions of conventional methods such as the method that those are used for carrier construction and plasmid by applying example Method because of carrier as being inserted into and plasmid or method that plasmid is introduced into host cell.Such method is for this field In be well-known with the personnel of ordinary skill, 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 1. novel and multifunctional antibody P-T Crossmab of embodiment and expression

(1) variable region gene of full genome synthesis panitumumab and tarextumab heavy chain of antibody and light chain, obtains (amino acid sequence and nucleotide sequence are respectively such as SEQ for VH, VL segment of VH, VL and Tarextummab of Panitumumab ID NO：Shown in 1-8).Full genome synthesize human IgG constant region of light chain, heavy chain constant region CH1 and Fc segment (amino acid sequence and Nucleotide sequence is respectively such as SEQ ID NO：Shown in 9-14).

(2) by the antibody Fc fragment of acquisition, catastrophe point is introduced using the method for overlap PCR：T366W,S354C.PCR Product purifies through agarose gel electrophoresis and recycles and be cloned into pGEM-T carriers, confirms after sequence verification and obtains correct gram It is grand that (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO：Shown in 15-16).Obtain Fc sections of knob mutant.

(3) separately the method for overlap PCR is used to introduce catastrophe point again the Fc segments of antibody：T366S, L368A, Y407V and Y394C.PCR product purifies through agarose gel electrophoresis and recycles and be cloned into pGEM-T carriers, after sequence verification really Recognize and obtains correctly clone (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO：Shown in 17-18).Obtain Fc Section hole mutant.

(4) the Fc segments for obtaining the panitumumab heavy chain variable regions and step (3) that are obtained in above-mentioned steps (1) Knob mutant is template, by above-mentioned two segment composition and is fitted into expression vector using overlap round pcrs.Build Obtaining panitumumab heavy chain knob mutant, (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO：19-20 institutes Show).

(5) Tarextumab heavy chain variable regions, the Fc that chain constant region and step (3) obtain will be obtained in step (1) Section hole mutant is template, by above three segment composition and is fitted into expression vector using overlap round pcrs.Obtain Obtaining Tarextumab VH-CL-Hing-Fc_hole_mut fusions segment, (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO：Shown in 21-22).

(6) it using the panitumumab light chain variable regions obtained in step (1) and constant region of light chain CL as template, utilizes Above-mentioned segment composition is obtained panitumumab light chains, and optimize to the sequence, dashed forward by overlap round pcrs (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO for change：Shown in 23-24).

(7) using the Tarextumab light chain variable regions obtained in step (1) and IgG heavy chain constant region CH1 as template, profit With overlap round pcrs by above-mentioned segment composition, that is, obtains Tarextumab VL-CH1 fusion segments and be packed into expression vector In (amino acid sequence and nucleotide sequence are respectively such as SEQ ID NO：Shown in 25-26).Step (4) (5) (6) (7) is compiled Four kinds of expression vector plasmids of code P-T tetra- peptide chains of Crossmab.

(8) 3 × 10 are inoculated in 3.5cm tissue culture dishes⁵CHO-K1 cells, when cell culture to 80%-85% is merged It is transfected：Take each 10 μ g of heavy chain plasmid, each 4 μ g of light chain plasmids and 30 μ l Lipofectamine2000Reagent (Invitrogen Products) are dissolved in 800 μ l plasma-free DMEM mediums respectively, are stored at room temperature 5 minutes, by above 2 kinds of liquid Mixing is incubated at room temperature 20 minutes so that DNA- liposome complexes are formed, therebetween with the replacement training of the DMEM culture mediums of 3ml serum-frees The serum-containing media in ware is supported, then the DNA- liposome complexes of formation are added in plate, CO₂Incubator culture 4 hours The DMEM complete mediums that 2ml contains 10% serum are added afterwards, are placed in CO₂Continue to cultivate in incubator.Cell changes after transfection carries out for 24 hours Resistance clone is screened containing the Selective agar medium of 500 μ g/ml G418 and 300 μ g/ml Zeocin.The Gao Biaodake that screening is obtained Grand expanded with serum free medium is cultivated, and bispecific antibody P-T is isolated and purified with Protein A affinity columns (GE Products) Crossmab.P-T Crossmab are dialysed with PBS, finally with UV absorption standard measure.The structure of P-T Crossmab As shown in Figure 1.

Embodiment 2.Biacore analyses

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

1. affinity determination of table is analyzed

Embodiment 3：P-T Crossmab inhibit non-small cell lung cancer cell viability experiment

It is 5 × 10 to take the good HCC827 cells (ATCC) of growth conditions, adjustment cell concentration³It is thin to be inoculated in 96 holes by/ml Born of the same parents' culture plate, 200 holes μ l/ are added final concentration of 5nmol's after being cultivated for 24 hours in 37 DEG C, 5%C02 incubators in culture solution Bispecific antibody P-T Crossmab, panitumumab, the tarextumab of EGF and various concentration gradient, Panitumumab+tarextumab, bispecific antibody CT16 (Hu et al., 2017, Antagonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by decreasing tumor- initiating cell frequency,9:Eaag0339), unrelated human IgG (Rituximab is purchased from Roche companies), 3 After it, cell viability with CellTiter-Glo Luminescent Cell Viability Assay kits (Promega, Madison, WI) detection.Experimental result is as shown in Figure 2.The experimental results showed that the suppressible HCC827 of P-T Crossmab are thin Born of the same parents' vigor has more significant inhibition than panitumumab and tarextumab, with panitumumab and tarextumab The double antibody group effect of use in conjunction it is similar or it is more excellent it is similar with bispecific antibody CT16 effects (drug concentration unit be ng/ μl)。

Embodiment 4：Inhibit tumour growth experiment in P-T Crossmab bodies

For antitumor activity in detection P-T Crossmab bodies, HCC827 cells are used first, it is right to be inoculated in BALB/c nude mices It is subcutaneous to coerce side, tail vein injection after tumor formation, 5mg/kg each group following antibodies：P-T Crossmab, panitumumab, Tarextumab, CT16, each 2.5mg/kg connection of two kinds of monoclonal antibodies of unrelated control human IgG, panitumumab and tarextumab Application is closed, injects 1 time weekly, continues to the excessive execution of mouse tumor.The length and width for measuring tumour daily, calculate gross tumor volume.

Tumor growth curve is as shown in Figure 3.The result shows that：The P-T Crossmab treatment group tumors speeds of growth significantly less than Panitumumab and tarextumab treatment groups (after 35 days, P<0.01, Mann-Whitney examines).And P-T Crossmab Therapeutic effect due to panitumumab and tarextumab use in conjunction effect (after 35 days, P<0.01, Mann- Whitney is examined).

The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is described Embodiment, those skilled in the art can also make various equivalent under 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>Feng tide medical sci-teches（Shanghai）Co., Ltd

<120>Human anti-human EGFR and Notch2/3 multi-specificity antibody, preparation method and the usage

<130> /

<160> 26

<170> PatentIn version 3.3

<210> 1

<211> 119

<212> PRT

<213>Artificial sequence (Artificial)

<400> 1

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 2

<211> 357

<212> DNA

<213>Artificial sequence (Artificial)

<400> 2

caagttcagc tgcaagaaag cggacccggt ttagtgaagc ctagcgagac tttatcttta 60

acttgtacag tgtccggagg cagcgtgagc agcggcgatt actactggac ttggattcgt 120

cagagccccg gtaagggttt agagtggatt ggccacatct actacagcgg caacaccaac 180

tacaacccct ctttaaagtc tcgtctgacc atcagcatcg acaccagcaa gacccagttc 240

tctttaaagc tgagcagcgt gaccgctgcc gacaccgcca tctactactg cgtgagggat 300

cgtgtgaccg gcgccttcga catctgggga caaggtactt tagtgaccgt gagctcc 357

<210> 3

<211> 107

<212> PRT

<213>Artificial sequence (Artificial)

<400> 3

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 4

<211> 321

<212> DNA

<213>Artificial sequence (Artificial)

<400> 4

gacatccaga tgacccagag ccctagctct ttaagcgcca gcgtgggcga tagggtgacc 60

atcacttgtc aagcctctca agatatcagc aactatttaa actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctccaatc tggagaccgg cgtgccctct 180

cgttttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagctc tttacagccc 240

gaggacatcg ccacctactt ctgtcagcac ttcgaccatt tacctctggc ctttggccaa 300

ggtaccaagg tggagatcaa g 321

<210> 5

<211> 115

<212> PRT

<213>Artificial sequence (Artificial)

<400> 5

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

115

<210> 6

<211> 345

<212> DNA

<213>Artificial sequence (Artificial)

<400> 6

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ccggtggttc tttaaggctg 60

tcttgtgccg cctccggctt caccttctcc tcctccggca tgagctgggt gaggcaagct 120

cccggtaagg gtttagagtg ggtgtccgtg atcgcctcca gcggctccaa cacctactac 180

gccgactccg tgaagggtcg tttcaccatc tcccgggaca actccaagaa cactttatat 240

ttacagatga actctttaag ggccgaggac accgccgtgt actactgcgc ccggtccatc 300

ttctacacca cttggggcca aggtacttta gtgacagtgt cctcc 345

<210> 7

<211> 108

<212> PRT

<213>Artificial sequence (Artificial)

<400> 7

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

100 105

<210> 8

<211> 324

<212> DNA

<213>Artificial sequence (Artificial)

<400> 8

gacatcgtgc tgacccagtc ccccgctact ttatctttat cccccggtga gagggctacc 60

ttatcttgtc gtgcttccca gtccgtgagg tccaattatt tagcttggta ccagcagaag 120

cccggccaag ctcctaggct gctgatctac ggcgcctctt ctcgtgccac tggtgtgccc 180

gctaggttct ccggctccgg ttctggcacc gacttcactt taaccatctc ctctttagag 240

cccgaagact tcgccgtgta ctactgccag cagtactcca acttccccat caccttcggc 300

caaggtacca aggtggagat caag 324

<210> 9

<211> 107

<212> PRT

<213>Artificial sequence (Artificial)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 10

<211> 321

<212> DNA

<213>Artificial sequence (Artificial)

<400> 10

aggaccgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctacc ccagagaagc caaagtgcag 120

tggaaggtgg acaacgccct gcagagcgga aacagccagg aaagcgtgac agagcaggat 180

tccaaggatt ccacatacag cctgagcagc acactgacac tgtccaaggc cgactacgag 240

aagcacaagg tgtacgcctg cgaagtgaca caccagggac tgtcctcccc tgtgacaaag 300

agcttcaaca gaggagaatg c 321

<210> 11

<211> 103

<212> PRT

<213>Artificial sequence (Artificial)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Lys Val Glu Pro Lys Ser Cys

100

<210> 12

<211> 309

<212> DNA

<213>Artificial sequence (Artificial)

<400> 12

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaacctgt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgt 309

<210> 13

<211> 227

<212> PRT

<213>Artificial sequence (Artificial)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 14

<211> 681

<212> DNA

<213>Artificial sequence (Artificial)

<400> 14

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 15

<211> 227

<212> PRT

<213>Artificial sequence (Artificial)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 16

<211> 681

<212> DNA

<213>Artificial sequence (Artificial)

<400> 16

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gccgggatga gctgaccaag 420

aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 17

<211> 227

<212> PRT

<213>Artificial sequence (Artificial)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 18

<211> 681

<212> DNA

<213>Artificial sequence (Artificial)

<400> 18

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtcctg cgcggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcgt cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 19

<211> 449

<212> PRT

<213>Artificial sequence (Artificial)

<400> 19

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly

20 25 30

Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

85 90 95

Cys Val Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser 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 Lys 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 Asp Glu Leu 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> 20

<211> 1347

<212> DNA

<213>Artificial sequence (Artificial)

<400> 20

caagttcagc tgcaagaaag cggacccggt ttagtgaagc ctagcgagac tttatcttta 60

acttgtacag tgtccggagg cagcgtgagc agcggcgatt actactggac ttggattcgt 120

cagagccccg gtaagggttt agagtggatt ggccacatct actacagcgg caacaccaac 180

tacaacccct ctttaaagtc tcgtctgacc atcagcatcg acaccagcaa gacccagttc 240

tctttaaagc tgagcagcgt gaccgctgcc gacaccgcca tctactactg cgtgagggat 300

cgtgtgaccg gcgccttcga catctgggga caaggtactt tagtgaccgt gagctccgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aacctgtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatgccg ggatgagctg 1080

accaagaacc aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 21

<211> 449

<212> PRT

<213>Artificial sequence (Artificial)

<400> 21

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 Asp Glu Leu 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> 22

<211> 1347

<212> DNA

<213>Artificial sequence (Artificial)

<400> 22

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ccggtggttc tttaaggctg 60

tcttgtgccg cctccggctt caccttctcc tcctccggca tgagctgggt gaggcaagct 120

cccggtaagg gtttagagtg ggtgtccgtg atcgcctcca gcggctccaa cacctactac 180

gccgactccg tgaagggtcg tttcaccatc tcccgggaca actccaagaa cactttatat 240

ttacagatga actctttaag ggccgaggac accgccgtgt actactgcgc ccggtccatc 300

ttctacacca cttggggcca aggtacttta gtgacagtgt cctccgctag cgtggctgca 360

ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 420

gtgtgcctgc tgaataactt ctaccccaga gaagccaaag tgcagtggaa ggtggacaac 480

gccctgcaga gcggaaacag ccaggaaagc gtgacagagc aggattccaa ggattccaca 540

tacagcctga gcagcacact gacactgtcc aaggccgact acgagaagca caaggtgtac 600

gcctgcgaag tgacacacca gggactgtcc tcccctgtga caaagagctt caacagagga 660

gaatgcgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gtcctgcgcg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctcgtcagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 23

<211> 214

<212> PRT

<213>Artificial sequence (Artificial)

<400> 23

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

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

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu

85 90 95

Ala Phe Gly Gln Gly Thr Lys Val Glu Ile 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> 24

<211> 642

<212> DNA

<213>Artificial sequence (Artificial)

<400> 24

gacatccaga tgacccagag ccctagctct ttaagcgcca gcgtgggcga tagggtgacc 60

atcacttgtc aagcctctca agatatcagc aactatttaa actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctccaatc tggagaccgg cgtgccctct 180

cgttttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagctc tttacagccc 240

gaggacatcg ccacctactt ctgtcagcac ttcgaccatt tacctctggc ctttggccaa 300

ggtaccaagg tggagatcaa gaggaccgtg gccgccccta gcgtgttcat cttcccccct 360

agcgacgagc agctgaagag cggcaccgct agcgttgtgt gcctgctgaa taacttctac 420

cccagagaag ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccag 480

gaaagcgtga cagagcagga ttccaaggat tccacataca gcctgagcag cacactgaca 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac acaccaggga 600

ctgtcctccc ctgtgacaaa gagcttcaac agaggagaat gc 642

<210> 25

<211> 213

<212> PRT

<213>Artificial sequence (Artificial)

<400> 25

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 Ser Ser Lys Ser Thr

115 120 125

Ser Gly Gly 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 Ser Leu Gly Thr Gln Thr Tyr Ile

180 185 190

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

195 200 205

Glu Pro Lys Ser Cys

210

<210> 26

<211> 639

<212> DNA

<213>Artificial sequence (Artificial)

<400> 26

gacatcgtgc tgacccagtc ccccgctact ttatctttat cccccggtga gagggctacc 60

ttatcttgtc gtgcttccca gtccgtgagg tccaattatt tagcttggta ccagcagaag 120

cccggccaag ctcctaggct gctgatctac ggcgcctctt ctcgtgccac tggtgtgccc 180

gctaggttct ccggctccgg ttctggcacc gacttcactt taaccatctc ctctttagag 240

cccgaagact tcgccgtgta ctactgccag cagtactcca acttccccat caccttcggc 300

caaggtacca aggtggagat caagtcctcg gctagcacca agggcccatc ggtcttcccc 360

ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 420

gactacttcc ccgaacctgt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 480

cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 540

gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 600

aacaccaagg tggacaagaa agttgagccc aaatcttgt 639

Claims (10)

1. a kind of anti-EGFR and Notch2/3IgG molecules sample intersects monoclonal antibody P-T Crossmab, which is characterized in that by four Peptide chain composition, respectively amino acid sequence such as SEQ ID NO：Panitumumab heavy chains knob mutant, amino shown in 19 Acid sequence such as SEQ ID NO：Panitumumab light chains shown in 23, amino acid sequence such as SEQ ID NO：Shown in 21 Tarextuimab heavy chain hole mutant, amino acid sequence such as SEQ ID NO：Tarextumab light chain mutants shown in 25 Tarex-VL-CH1。

2. a kind of polynucleotides, which is characterized in that it includes the nucleotide sequences for encoding antibody as described in claim 1.

3. polynucleotides according to claim 2, which is characterized in that it includes coding panitumumab heavy chains knob is prominent Variant such as SEQ ID NO：Nucleotide sequence shown in 20, and coding panitumumab light chains such as SEQ ID NO：24 institutes The nucleotide sequence shown, and coding Tarextuimab heavy chain hole mutant such as SEQ ID NO：Nucleotides sequence shown in 22 Row, and coding Tarextumab light chain mutants Tarex-VL-CH1 such as SEQ ID NO：Nucleotide sequence shown in 26.

4. a kind of recombinant expression carrier, which is characterized in that it includes polynucleotides as claimed in claim 2 or claim 3.

5. recombinant expression carrier according to claim 4, which is characterized in that the recombinant expression carrier is PcDNA3.1, pEE6.4, pEE12.4, pDHFR.

6. a kind of host cell converted with recombinant expression carrier as described in claim 4 or 5, the host cell is true Nucleus.

7. host cell according to claim 6, which is characterized in that the host cell is Chinese hamster ovary CHO Cell.

8. a kind of method preparing antibody P-T Crossmab as described in claim 1, includes the following steps：

A) in the case where being suitble to allow the antibody P-T Crossmab expression conditions, the host cultivated as claimed in claims 6 or 7 is thin Born of the same parents, to express antibody P-T Crossmab；

B) the antibody P-T Crossmab described in isolated or purified.

9. a kind of antibody P-T Crossmab application in preparations of anti-tumor drugs as described in claim 1.

10. a kind of composition contains antibody P-T Crossmab as described in claim 1 and pharmaceutically acceptable carrier Or auxiliary material.