CN106800599B

CN106800599B - Anti-human EGFR and Notch multispecific antibody, preparation method and application thereof

Info

Publication number: CN106800599B
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: 2021-03-23
Anticipated expiration: 2036-12-05
Also published as: CN106800599A

Abstract

The invention belongs to the technical field of biological medicines, and discloses an anti-human EGFR and Notch multispecific antibody C-T Crossmab which can be combined with EGFR, Notch2 and Notch 3. The C-T Crossmab of the invention comprises two heavy chain polypeptides and two light chain polypeptides, the invention also provides a nucleotide sequence for coding the heavy chain polypeptides, a recombinant expression vector comprising the nucleotide sequence, and a transformed host cell. The multispecific antibodies of the present invention are useful in the treatment of neoplastic disease.

Description

Anti-human EGFR and Notch multispecific antibody, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly discloses anti-human EGFR, Notch2 and Notch3 bispecific antibodies, a preparation method thereof and application thereof in preparation of antitumor drugs.

Background

Surgical treatment, radiotherapy and chemotherapy are the main and the auxiliary new target treatment schemes are the basic strategy for treating malignant tumors in recent years, and have made important progress in clinical practice. The recurrence, metastasis and therapeutic tolerance of malignant tumors remain difficult problems that plague clinical and scientific researchers. In 2006, at the Cancer stem cell Workshop held by the American Cancer research Association, the trainee defined the tumor stem cell as a tumor differentiation cell population with self-renewal property, ability to drive the formation of multiple heterogeneity, Cancer cell with stem cell property (Clarke et al, Cancer stem cells-dependent on current status and future directives: AACR Workshop on Cancer stem cells, 2006, Cancer research 933, 66: 9- -. The theory of tumor stem cells is more and more accepted by scholars at home and abroad according to the unique theoretical advantages and the continuously abundant research support evidence. Although the traditional treatment method has better treatment and killing effects on tumor solid cells, the traditional treatment method has an inhibiting effect on tumor stem cells which only account for 0.2-10% of tumor cell groups and have larger molecular phenotype difference with the tumor solid cells. The research on the important action and mechanism of the tumor stem cells in the generation, development and metastasis of tumors and the development of novel anti-tumor treatment strategies and preparations based on the tumor stem cell theory have very important significance on the treatment of malignant tumors.

The tumor stem cell theory considers that: malignant tumors originate from a population of "stem cell-like" tumor cells, which are called tumor stem cells (CSCs or cancer-inducing cells, CICs). At the end of the last 90 s, researchers first isolated tumor stem cells from leukemia cells and described a heterogeneous hierarchical tissue model of tumors driven by tumor stem cells (Dick, Human breast muscle oil leukemia is organized as a hierarchy of tumor tissues from a proprietary biochemical cell, 1997, Nature Med,3: 730-737). Subsequently, researchers have also demonstrated the presence of tumor stem cells and similar tumor heterogeneous tissue characteristics in a variety of solid tumors, including breast, colon, brain, ovarian, lung, prostate, and pancreatic cancers (Brooks et al, Therapeutic indications of cellular specificity and specificity in tissue cancer, 2015, Cell,17: 260-271). These evidences all indicate the presence of tumor cells with stem cell properties in most hematologic and solid tumors.

Multiple studies show that tumor stem cells are generated and developed in malignant tumors; maintenance of tumor cell heterogeneity; tumor evolution; and in the course of therapeutic tolerance of tumors. The biological function of the medicine has the following characteristics:

1) extremely strong tumorigenicity. Tumor cells isolated directly from the foci of malignant patients are difficult to clone in vitro or to transplant into immunodeficient mouse models for clonogenic neoplasia, whereas only small amounts of CSC are required and tumor grafts have a phenotypic heterogeneity similar to that of the primary tumor foci (Al-Hajj et Al, scientific identification of genetic research cells, 2003, Proceedings of the National Academy of Sciences,100:3983, Dick, Human cardiac myoid tissue of organic as a high temperature of biological cells, 1997, Nature, 3: 730-.

2) Complex heterogeneity and bioplasticity. The source of CSCs may be extremely complex, possibly derived from differentiated tumor cells that acquire self-renewal capacity during epithelial-to-mesenchymal transition (EMT) (Epinoza and Miele, deep cross: Notch signaling at the interaction of EMT and Cancer stem cells, 2013, Cancer letters,341: 41-45); or normal tissue Stem cells derived from oncogene mutations induced by foreign microenvironment factors (Reya et al, Stem cells, cancer, and cancer Stem cells, 2001, nature,414: 105-111). In addition, several recent reports have also demonstrated that CSCs have very complex bioplasticity, such as two distinct phenotypic breast cancer stem cell populations can be identified primarily among breast cancer stem cells: an epithelioid actively proliferating breast cancer CSC population expressing a stem cell marker ALDH; and a mesenchymal, relatively inactive, but heavily invasive, population of breast cancer CSCs whose surface marker expression is CD44+/CD 24-. The two groups of CSCs can generate respective progenies of epithelial-like or mesenchymal-like tumor solid cells, and can release secretion signals to enhance the self-renewal capacity of the CSCs. And the two groups of cells can be transformed into each other under epigenetic changes mediated by cytokine signals, chemokine signals, transcriptional regulation in the tumor microenvironment (Liu et al, Breast cancer cells transfer between epithelial and mesenchymal Stem reflective of the same normal counts, 2014, Stem cell reports,2: 78-91). In addition, studies have also reported that different CSC populations exist in liver Cancer, such as CSC with CD133+ (Ma et al, CD133& plus; HCC Cancer cells control chemotherapy by prediction expression of the Akt/PKB Survival pathway 2008, Oncogene,27: 1749. sup. 1758) and CSC with CD90+/CD44+ (Yang et al, Significant of CD90+ Cancer cells in human lever. 2008, Cancer cells, 13: 153. sup. 166) with different stem cell characteristics. The markers of stem cells also tend to differ among different tumor types, as in pancreatic Cancer, the surface marker of CSCs is considered to be CD24+ (Li et al, Structural basis for inhibition of the epitopic growth factor receptor by cetuximab, 2007, Cancer research,67:1030 1037), and for breast CSCs, CD 24-. In addition, the content and rate of CSCs often vary from malignancy to malignancy, and the rate of CSCs also varies in a complex fashion at different stages of malignancy. For example, in colon cancer, the rate of CD133+ highly expressed CSCs is about 1.8% to 24.5% (O' Brien et al, A human colon cancer cell mapping of initial cancer growth in immunological cancer, 2007, Nature,445: 106-. Whereas in melanoma, the rate of CSCs expressing CD133+ cell surface markers was between 1% and 20% (Quintana et al, effective tumor formation by single human melanoma cells, 2008, Nature,456: 593-. These findings all suggest that CSCs have extremely complex heterogeneity and bioplasticity.

3) The treatment sensitivity is poor. Several studies in recent years have shown that CSCs are resistant to many of the therapeutic approaches currently in clinical use, including a variety of chemotherapeutic drugs and radiation therapy. Human acute myeloid leukemia CD34+/CD38-progenitor cells have been reported to be insensitive to chemotherapy killing and Fas-mediated apoptosis (Costello et al, Human acute myeloid leukemia CD34+/CD 38-promoter cells have had detailed sensitive sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunological sensitivity, and amplified dendritic cell transformation protocols, 2000, Cancer Research,60: 4403-4411); the telomerase is highly expressed, and the normal structure of the chromosome is maintained, so that the apoptosis of the cell is inhibited; high expression of multiple anti-apoptosis genes, such as bcl-2 gene, nuclear transcription factor kappa B (NF-kappa B) gene, mutant p53 gene and c-myc gene. CD44+/CD 24-Breast Cancer CSC is not sensitive to radiotherapy (Phillips et al, 2006, Journal of the National Cancer Institute,98: 1777-; CD133+ Glioma stem cells have strong DNA damage repair capability and are not sensitive to radiotherapy (Bao et al, Glioma stem cells promoter radioresistance by pressurized actuation of the DNA damageresponse, 2006, nature,444: 756-760); dylla et al also reported that colon cancer CSCs had an increased cell rate in a nude mouse xenograft model following chemotherapy and were not susceptible to chemotherapy (Dylla et al, Colorganic cancer cells are involved in xenogenic tumors around chemotherapy, 2008, PloS one,3: e 2428). And both tumor stem cells and normal stem cells highly express ATP-binding cassette transporters (ABC), such as ABCB1 protein, which encodes P-glycoprotein, and ABCG2 protein, which is identified from mitoxantrone-resistant cells. ABCB 1and ABCG2 mediated efflux renders dyes such as Hoechst 33342 and rhodamine 123 unable to stain stem cells, confirming that ABC protein is critical to The chemo-insensitive Nature of stem cells (Zhou et al, The ABC transporter Bcr 1/ABCG2is expressed in a wide variety of stem cells and is a molecular detector of The side-position phenotyping, 2001, Nature media, 7: 1028-. CSCs have a variety of biological attributes similar to normal stem cells, such as relative proliferative inactivity, drug and toxin insensitivity, high expression of a variety of ABC proteins, strong nucleic acid repair capacity, and apoptosis resistance (Dean et al, Human acid myelid leukemia organic as a high temperature resources from a private hematopathic cell, 2005, Nature Reviews Cancer,5: 275-. In addition, CSCs, like normal stem cells, form a tumor stem cell niche with the surrounding microenvironment, which is relatively hypoxic and provides a number of steady-state-sustaining biological signals to CSCs, providing a barrier to external stimuli. Conventional cytotoxic treatments have minimal effect on stem cells located in The cell niche (LaBarge, The differentiation of targeting cancer cell niches, 2010, Clinical cancer research,16: 3121-. Thus, CSCs can survive chemotherapy and re-proliferate tumors, eventually leading to tumor recurrence and therapeutic tolerance.

4) Strong invasion promoting ability. Due to the high heterogeneity of tumor tissues, most of the tumor solid cells have no invasion and metastasis abilities, while CSCs have very strong invasion and metastasis abilities. As reported, breast cancer CSCs and important EMT processes in breast cancer metastasis are highly correlated, EMT-like CSCs tend to localize at The margins of solid tumor infiltration and readily enter The blood circulation and form micrometastases with properties of stem cells at The distal end (Mani et al, The epithelial-mesenchymal transition genes cells with 2008, Cell,133:704 cells 715). At this time, the CSC can be converted into a high proliferation activity epithelial CSC under the action of some microenvironment factors (such as ID1 mediated TWIST down-regulation (Stankic et al, TGF-beta-Id 1 signalling epitopes TWIST1and promoters metabolic collagen peptide a metabolic-to-epithelial transfer 2013, Cell reports,5:1228-1242)), and finally the breast cancer metastasis process is completed. In pancreatic cancer, CSCs expressing CD133+/CXCR4+ cell surface markers are also often localized at the margins of tumor infiltration, enter the blood easily, and they highly express chemokine receptors. Under the action of CXCR4 chemokines, such CSCs readily metastasize to the liver (Hermann et al, disorders stresses of cancer cells determining growth and metabolic activity in human scientific cancer, 2007, Cell stem cells, 1: 313-. The above studies all suggest that CSCs play a very important role in the infiltration and metastasis of malignant tumors.

In conclusion, CSCs have a strong tumorigenicity, complex heterogeneity and bioplasticity, strong metastatic invasiveness, and poor treatment sensitivity. After conventional cytotoxic therapy, such as chemotherapy and biological targeted therapy, of tumor tissue, the residual highly tumorigenic CSCs begin to enhance proliferative activity and begin to expand and drive tumor tissue growth, manifesting as clinical tumor recurrence. Based on the above theories and studies, many researchers believe that a therapeutic strategy that targets CSC cell populations would complement the deficiencies of existing therapeutic strategies and significantly increase tumor treatment levels (Schaue and McBride, Opportunities and changes of radiotherapeutics for therapeutic cancer, 2015, Nature Reviews Clinical Oncology,12: 527. 540; Takebe et al, Targeting Notch, Hedgehog, and Wnt pathways in cancer cells: Clinical update, 2015, Nature Reviews Clinical Oncology).

It has been clarified from current studies that there are dysregulated signaling pathways in CSCs, which are critical to the regulation of CSCs, including Wnt signaling pathway, Notch signaling pathway, and Hedgehog signaling pathway (Takebe et al, Targeting Notch, Hedgehog, and Wnt pathway in cancer cells: Clinical update, 2015, Nature Reviews Clinical analysis). In 1916, Notch gene was found in Drosophila for the first time, and its mutant can Notch the wing edge of Drosophila, so it is named. The complete Notch signaling pathway includes transmembrane protein receptors, transmembrane proteins, transcription factors and downstream target genes, among which human cells share 4 Notch receptors (Notch 1-4) and 5 ligands (Delta-like-1, Delta-like-3, Delta-like-4, Jagged-1 and Jagged-2) (Bailey et al, Cancer patients with degraded by modified genes: Sonic hedgehog, Notch, and bone morpholino proteins, 2007, Journal of cellular biology, 102: 829-. The Notch gene has a very significant tumorigenic effect, which is reflected in activating mutations in a variety of human tumors, including lymphomas, breast cancers, lung cancers, head and neck tumors, pancreatic cancers, colon cancers, jaw osteosarcomas, and glioblastomas, and extends to members of all levels of the Notch signaling pathway (Weng and Aster, Multiple nucleotides for Notch in cancer: context is imaging, 2004, Current opinion in genetics & development,14: 48-54). In many tumors, the Notch signaling pathway has been shown to be associated with maintenance of the sternness of tumor stem cells. Recently, OncoMed pharmaceutical companies published patient survival and biomarker data for phase ib clinical trials of monoclonal antibody tarextumab targeting the Notch pathway for the treatment of small cell lung cancer. tarextumab is a fully humanized monoclonal antibody, targeting the Notch2/3 receptor. Preclinical studies suggest 2 mechanisms of action, namely down-regulation of Notch pathway signals, anti-tumor stem cell action and influence on the microenvironment of pericytes, stromal cells and tumors.

In solid tumor cells, Epidermal Growth Factor Receptor (EGFR) signals are another important signal transduction pathway of the body, widely exist in epithelial, mesenchymal and neural tissues, and play an important role in regulating the differentiation and selection of cells throughout the growth and proliferation process of the whole cells. The EGFR family primarily includes EGFR (i.e., HER1) and HER 2-4. EGFR encodes transmembrane protein, the signal of which is activated by receptor tyrosine phosphorylation, and is amplified by Ras/Raf/MEK/MAPK pathway cascade, finally leads to the phosphorylation of MAPK, the modified MAPK signal enters nucleus, promotes the phosphorylation of target genes, and regulates the expression and activity of genes. The EGFR signal is closely related to the occurrence, development and prognosis of tumors, and is expressed in various tumors such as lung cancer, colon cancer, breast cancer, prostate cancer, ovarian cancer, cancer of the wing skin and the like. Highly expressed signals are associated with poor prognosis of breast and lyphotic carcinomas. EGFR expression in breast cancer is associated with tumor proliferation, disease progression and poor prognosis, and its expression can decrease estrogen receptor expression and increase resistance to endocrine therapy. Preclinical studies have shown that constitutively activated EGFR can significantly increase the tumorigenicity of MCF-9 human breast cancer models in vivo. The monoclonal antibody cetuximab (C225) acting on the Epidermal Growth Factor Receptor (EGFR) is one of the most widely studied targeted drugs, and its therapeutic effect is well established in the treatment of various tumors.

The mode of interaction of EGFR with Notch signaling is complex and can be manifested either as a synergistic effect or as an antagonistic effect, depending on the type of tissue and stage of development. In gliomas, Notch upregulates EGFR expression, and in highly differentiated gliomas, EGFR expression correlates strongly with Notch. Whereas in cutaneous squamous cell carcinoma EGFR signaling acts upstream of Notch 1signaling and inhibits the expression of the signal. And the signal plays a synergistic promoting role in the in vitro culture of the breast in-situ ductal carcinoma. These studies indicate that EGFR signaling and Notch signaling are two closely related signaling pathways, and have important regulatory effects on tumor development.

In conclusion, the Notch signaling pathway is a key pathway involved in the generation of tumor cell EMT, the maintenance of tumor CSC cell population and sternness, the proliferation and differentiation thereof, and the regulation of CSC treatment tolerance. EGFR is a main type of over-activation signal pathway in solid tumor cells and plays an important role in the targeted therapy of solid tumor. While there is cross-activation at various levels between the two signal paths.

Therefore, it is a problem to be solved by those skilled in the art to construct a multispecific antibody capable of blocking two signals simultaneously by using genetic engineering technology, which can block both EGFR and Notch, and further simultaneously inhibit tumor stem cells and tumor solid cells.

Disclosure of Invention

The present invention aims to provide a multispecific antibody capable of blocking two signaling pathways simultaneously, namely EGFR, Notch2 and Notch 3. Another object of the present invention is to provide a method for producing the antibody. The third object of the present invention is to utilize the antibody, and a therapeutic agent for tumor diseases comprising the antibody as an active ingredient.

In a first aspect of the invention, there is provided an anti-EGFR and Notch2/3IgG molecule-like cross monoclonal antibody (Crossmab) C-T Crossmab which binds to EGFR, Notch2, and Notch 3.

Preferably, the anti-human EGFR and Notch multispecific antibody of the present invention comprises two heavy chain polypeptides and two light chain polypeptides, wherein the amino acid sequence of the heavy chain polypeptides is as set forth in SEQ ID NO: 1and SEQ ID NO: 2is shown in the specification; the amino acid sequence of the light chain polypeptide is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

The invention also provides a polynucleotide comprising a nucleotide sequence encoding an antibody as described above.

Preferably, the polynucleotide comprises a sequence as set forth in SEQ ID NO:5 and SEQ ID NO: 6; and a light chain polypeptide as set forth in SEQ ID NO: 7 and SEQ ID NO: 8.

The present invention also provides a recombinant expression vector comprising a polynucleotide as described above.

Preferably, the recombinant expression vector is selected from pcDNA3.1, pDR1 or pDFFR and the like. In a preferred embodiment of the invention, the expression vector is specifically pcDNA3.1.

The invention also provides a host cell transformed with the vector.

Preferably, the host cell is a eukaryotic cell; more preferably, the cell is selected from Chinese hamster ovary CHO cell, NS0 myeloma, COS or SP2/0 cell, etc.

In the present invention, any suitable DNA encoding EGFR, Notch2, and Notch3 is suitable for use in the present invention, and such structures include those isolated from tissue or cellular mRNA, synthesized from the entire gene based on sequences in published databases, or obtained from other cDNA libraries.

In the present invention, any suitable vector may be used, which may be one of pDR1, pCDNA3.1, pDFHFF, and the expression vector includes a fusion DNA sequence linked to appropriate transcription and translation regulatory sequences.

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

The host cell to be used is a prokaryotic cell containing the above-mentioned vector, and may be one of DH5a, BL21(DE3) and TG 1.

In a second aspect of the present invention, there is provided a method for producing the above antibody (C-T Crossmab), which comprises the steps of:

(i) culturing a host cell according to the above under conditions suitable to allow expression of said antibody; and

(ii) recovering the expressed antibody.

(ii) further isolating or purifying and recovering the expressed antibody.

The preparation method of the C-T Crossmab disclosed by the invention comprises the steps of culturing the host cell under the expression condition so as to express the C-T Crossmab, and separating or purifying the C-T Crossmab.

Using the above method, the bispecific antibody can be purified to a substantially homogeneous material, e.g., as a single band on SDS-PAGE electrophoresis.

The C-T Crossmab disclosed in the present invention can be separated and purified by affinity chromatography, and the C-T Crossmab polypeptide bound to the affinity column can be eluted by conventional methods such as high salt buffer, pH change, etc. depending on the characteristics of the affinity column used.

In a preferred embodiment of the present invention, the above-mentioned multispecific antibody C-T Crossmab disclosed in the present invention is obtained by the following method: total Gene Synthesis of the heavy and light chain genes of multispecific antibodies, the sequence of the total gene refers to the sequence of the monoclonal antibody cetuximab (U.S. Pat. No. 6,17866B 1, Li et al, Structural basic for inhibition of the epitopic growth factor receptor by cetuximab, 2005, Cancer Cell,7: 301. sub.311), the sequence of tarextumab (U.S. Pat. No. 20130323266A 1, Yen, Wan-Ching, entitled Targeting Nothing signalling with a Notch2/Notch3 antagonist (tarextextextext) inhibitor growth and deletion of molecular Cell, the sequence of epidermal growth factor receptor, VEGF trap Cell frequency, epidermal growth factor receptor growth factor, epidermal growth factor receptor growth factor receptor, epidermal growth factor, epidermal growth factor receptor growth factor, epidermal growth factor, epidermal growth factor, VEGF growth factor, epidermal growth factor, epidermal growth factor growth, advances in Bispecific Antibodies Engineering: Novel Concepts for immunotheriages, 2015, J Blood Disorders Transf,6: 2).

The invention synthesizes heavy chain gene and light chain gene of multi-specificity antibody by using whole gene, and respectively loads the above-mentioned two heavy chain genes and two light chain genes into eukaryotic expression vectors pcDNA3.1 and pcDNA3.1Zeo. The above plasmids were used together to transfect CHO-K1 cells by the liposome method and cell clones stably expressing bispecific antibody were selected using selection medium containing 500. mu.g/ml G418 and 300. mu.g/ml Zeocin the multispecific antibody C-T Crossmab was purified from the supernatant of the cell culture by affinity chromatography using a ProteinA column.

In a third aspect of the invention, the application of the antibody (C-T Crossmab) in preparing an anti-tumor medicament is provided.

The medicine takes the C-T Crossmab of the invention as an active ingredient.

The invention also provides a pharmaceutical composition comprising C-T Crossmab, and at least one pharmaceutically acceptable carrier, diluent or excipient.

The application of the pharmaceutical composition in preparing antitumor drugs.

The application also comprises the combined use with other antitumor drugs.

The invention carries out affinity detection on the C-T Crossmab, and finds that the C-T Crossmab well retains the affinity of cetuximab and tarextumab at the same time.

Therefore, the next experiment including the experiments of inhibiting tumor cell proliferation, inhibiting tumor in vivo, inhibiting tumor EMT gene expression and the like is carried out, and the experimental result shows that the C-T Crossmab disclosed by the invention has the functions of both Cetuximab and tarextumab. In addition, the multispecific antibody C-T Crossmab can be the same as the traditional IgG molecule, the structure of the traditional monoclonal antibody is furthest reserved, and because of the existence of the Fc fragment, the multispecific antibody C-T Crossmab can be purified by using a common ProteinA column affinity chromatography, thereby being beneficial to large-scale production and purification. Experiments show that the multispecific antibody C-T Crossmab has similar or stronger anti-tumor treatment effect than the combined application of cetuximab and tarextumab under the condition of equal dosage.

The invention discloses the bispecific antibody C-T Crossmab, which can be combined with pharmaceutically acceptable auxiliary materials to form a pharmaceutical preparation composition so as to exert curative effect more stably, and the preparations can ensure the conformation integrity of the amino acid core sequence of the bispecific antibody disclosed by the invention and simultaneously protect the polyfunctional group of protein from degradation (including but not limited to aggregation, deamination or oxidation). In general, it is generally stable for at least one year at 2 ℃ to 8 ℃ for liquid formulations and at least six months at 30 ℃ for lyophilized formulations. The preparation can be suspension, hydro-acupuncture, freeze-drying and the like which are commonly used in the pharmaceutical field, preferably hydro-acupuncture or freeze-drying preparation, and for the hydro-acupuncture or freeze-drying preparation of the C-T Crossmab disclosed by the invention, pharmaceutically acceptable auxiliary materials comprise one or a combination of a surfactant, a solution stabilizer, an isotonic regulator and a buffer solution, wherein the surfactant comprises a non-ionic surfactant such as polyoxyethylene sorbitol fatty acid ester (Tween 20 or Tween 80); poloxamer (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; tetradecyl, oleyl, or octadecyl sarcosine; pluronics; monaquatm, etc. in an amount to minimize the tendency of the bifunctional bispecific antibody protein to granulate, the solution stabilizer may be a saccharide including reducing and non-reducing saccharides, the amino acids include monosodium glutamate or histidine, the alcohols include one of trihydric alcohols, higher sugar alcohols, propylene glycol, polyethylene glycol or a combination thereof, the solution stabilizer may be added in an amount to maintain the finally formed formulation in a stable state for a period of time deemed to be stable by those skilled in the art, the isotonic adjusting agent may be one of sodium chloride, mannitol, and the buffer may be one of TRIS, histidine buffer, phosphate buffer.

The preparation is a composition containing C-T Crossmab, and has obvious anti-tumor effect after being administrated to animals including human. Specifically, the compound is effective for the prevention and/or treatment of tumors and can be used as an antitumor drug.

Tumors of interest in the present invention include adenocarcinomas, leukemias, lymphomas, melanomas, sarcomas, and sources of tumor tissue include, but are not limited to, adrenal glands, gall bladder, bone marrow, brain, breast, bile ducts, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, skin, salivary glands, spleen, testis, thymus, thyroid, and uterus. In addition to the above-mentioned tumors, the present invention can be used for tumors of the central nervous system such as glioblastoma multiforme, astrocytoma, etc., and furthermore, tumors of the eye region including basal cell carcinoma, squamous cell carcinoma, melanoma, etc., tumors of endocrine glands, tumors of neuroendocrine system, tumors of gastrointestinal pancreatic endocrine system, tumors of reproductive system, tumors of head and neck, etc. This is not further enumerated here.

The anti-tumor drug referred to in the present invention means a drug having an effect of inhibiting and/or treating a tumor, and may include a delay in the development of symptoms associated with tumor growth and/or a reduction in the severity of these symptoms, and it further includes a reduction in existing symptoms associated with tumor growth and prevention of the occurrence of other symptoms, and also a reduction or prevention of metastasis.

In the present invention, when C-T Crossmab and its composition are administered to animals including human, the dose to be administered varies depending on the age and body weight of the patient, the nature and severity of the disease, and the route of administration, and it is possible to refer to the results of animal experiments and various cases, and the total dose of administration cannot exceed a certain range. In particular, the dosage of intravenous injection is 1-1800 mg/day.

The C-T Crossmab and the composition thereof disclosed by the invention can also be used for combined administration or radiotherapy with other anti-tumor drugs, and can be used for treating tumors, wherein the anti-tumor drugs comprise 1 cytotoxic drug (1) drugs acting on DNA chemical structures: alkylating agents such as nitrogen mustards, nitrosoureas, methyl sulfonates; platinum compounds such as cisplatin, carboplatin, and platinic oxalate; mitomycin (MMC); (2) drugs that affect nucleic acid synthesis: dihydrofolate reductase inhibitors such as Methotrexate (MTX) and Alimata, and the like; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine), etc.; purine nucleoside synthase inhibitors such as 6-mercaptopurine (6-MP), 6-TG and the like; ribonucleotide reductase inhibitors such as Hydroxyurea (HU) and the like; DNA polymerase inhibitors such as cytarabine (Ara-C) and Gemz (Gemz); (3) drugs acting on nucleic acid transcription: drugs that act selectively on DNA templates to inhibit DNA-dependent RNA polymerase and thus RNA synthesis such as: actinomycin D, daunorubicin, doxorubicin, epirubicin, aclarubicin, mithramycin, etc.; (4) drugs that act primarily on tubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophylline, homoharringtonine; (5) other cytotoxic agents: asparaginase mainly inhibits protein synthesis; 2. hormonal antiestrogens: tamoxifen, droloxifene, exemestane, and the like; aromatase inhibitors: aminoglutethimide, triton, letrozole, renningde, etc.; anti-androgens: flutamide RH-LH agonists/antagonists: norrad, etalone, and the like; 3. biological response modifier: tumor interferon is mainly inhibited through the immune function of the organism; interleukin-2; thymosin peptides; 4. monoclonal antibodies: rituximab (MabThera); herceptin (Trastuzumab) bevacizumab (avastin); 5. various radiation therapies; 6. other drugs include those whose current mechanism is unknown and yet to be further studied; cell differentiation inducers such as tretinoin; an apoptosis-inducing agent. The C-T Crossmab and the composition thereof disclosed by the invention can be combined with one of the anti-tumor drugs or the combination thereof.

The invention provides a multispecific antibody which can simultaneously block EGFR and two signal pathways of Notch2 and Notch3, and the multispecific antibody can be used as a therapeutic agent of tumor diseases.

Drawings

FIG. 1 is a schematic representation of the structure of C-T Crossmab of the present invention;

FIG. 2 shows the results of the experiment for inhibiting HCC827 cell activity by C-T Crossmab;

FIG. 3 is a curve showing that the C-T Crossmab of the present invention inhibits HCC827 tumorigenesis.

Detailed Description

The present invention will be further described with reference to the following examples, experimental examples and drawings, which should not be construed as limiting the present invention. The examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plastrons, methods of inserting genes encoding proteins into such vectors and plastrons, or methods of introducing plasmids into host cells. A Laboratory Manual, 2^ndedition，Cold spring Harbor Laboratory Press

Example 1 construction and expression of the multifunctional antibody C-T Crossmab of the present invention

Two heavy chain and two light chain gene sequences of the C-T Crossmab are synthesized by the whole gene (the sequences are shown as SEQ ID NO:5, 6, 7 and 8, and are synthesized by the Kingjinzhi organism Co., Ltd.) are connected with the pcDNA3.1 vector, and the two light chain gene sequences are connected with the pcDNA3.1Zeo vector to construct the eukaryotic expression vector.

3X 10 inoculation in 3.5cm tissue culture dish⁵CHO-K1 cells, transfected when cultured to 80% -85% confluence: mu.g each of the heavy chain plasmid 10. mu.g, the light chain plasmid 4. mu.g and 30. mu.l of Lipofectamine2000Reagent (Invitrogen Co.) were dissolved in 800. mu.l of serum-free DMEM medium, allowed to stand at room temperature for 5 minutes, the above 2 liquids were mixed, and incubated at room temperature for 20 minutes to form a DNA-liposome complex, in which the serum-containing medium in the petri dish was replaced with 3ml of serum-free DMEM medium, and then the formed DNA-liposome complex was added to the plate, CO₂After 4 hours of incubator culture, 2ml of DMEM complete medium containing 10% serum is supplemented and placed in CO₂And (5) continuously culturing in an incubator. Cells were selected for resistant clones by changing to selection medium containing 500. mu.g/ml G418 and 300. mu.g/ml Zeocin 24h after transfection. The high expression clones obtained by screening were subjected to amplification culture in a serum-free medium, and the bispecific antibody C-T Crossmab was isolated and purified by Protein A affinity column (product of GE). C-T Crossmab was dialyzed against PBS and finally quantified by UV absorption. The structure of C-T Crossmab is shown in figure 1, the amino acid sequence is shown in SEQ ID NO 1, 2, 3 and 4, and the Kingzhi organism company is entrusted with successful sequencing.

Example 2 Biacore analysis

Polyclonal anti-human FC antibody (Jackson ImmunoResearch) was coated on CM5M5 chip (GE) and after capture of the antibody to be detected, the affinity of C-T Crossmab was measured using Biacore T100(GE Healthcare), and the specific values of the affinity to be detected are shown in Table 1.

TABLE 1 Biacore analysis

The experimental results show that the C-T Crossmab of the invention well retains the affinity of both cetuximab and tarextumab.

Example 3 inhibition of non-Small cell Lung cancer cell viability by C-T Crossmab

Taking HCC827 cells with good growth state (ATCC) adjusted to 5 × 10 cell concentration³Perml, inoculated in 96-well cell culture plates at 200. mu.l/well at 37 ℃ with 5% C0₂After 24h of incubation in an incubator, the culture medium was supplemented with a final concentration of 5nmol EGF and various concentration gradients of bispecific antibody C-T Crossmab, cetuximab, tarextumab, cetuximab + tarextumab, irrelevant human IgG (Rituximab available from Roche), and after 4 days, Cell Viability was measured using CellTiter-Glo luminescence Cell Viability Assay kit (Promega, Madison, Wis.).

The results of the experiment are shown in FIG. 2. The experimental result shows that the C-T Crossmab can inhibit the activity of HCC827 cells, has more obvious inhibiting effect than cetuximab and tarextumab, and has similar or better effect with the double antibody group combined application of cetuximab and tarextumab.

Example 4: C-T Crossmab in vivo tumor growth inhibition experiment

To examine the in vivo tumor-inhibiting activity of C-T Crossmab, the mouse was first inoculated subcutaneously on the right flank of BALB/C nude mice (laboratory animal center of second university of military medical science) with HCC827 cells, and the following antibodies were injected into each group at 10mg/kg in caudal vein after tumorigenesis: C-T Crossmumab, cetuximab, tarextumab, irrelevant control human IgG, 5mg/kg of each of the two monoclonal antibodies cetuximab and tarextumab were combined and injected 1 time per week for a duration of time until the mice were sacrificed due to tumor overdose. The length and width of the tumor were measured daily and the tumor volume was calculated.

The tumor growth curve is shown in figure 3. The experimental results show that: the tumor growth rate was significantly less in the C-T Crossmab treated group than in the cetuximab and tarextumab treated groups (P <0.01 after 70 days, Mann-Whitney test). And the therapeutic effect of C-T Crossmab is superior to that of combined use with cetuximab and tarextumab (P <0.01 after 70 days, Mann-Whitney test).

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

SEQUENCE LISTING

<110> second military medical university of China people liberation army

<120> anti-human EGFR and Notch multispecific antibody, preparation method and application thereof

<130> description, 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

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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

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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. An anti-human EGFR and Notch multispecific antibody comprising two heavy chain polypeptides and two light chain polypeptides, wherein the amino acid sequence of the heavy chain polypeptides is as set forth in SEQ ID NO: 1and SEQ ID NO: 2is shown in the specification; the amino acid sequence of the light chain polypeptide is shown as SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

2. A polynucleotide comprising a nucleotide sequence encoding the antibody of claim 1.

3. The polynucleotide of claim 2, comprising a heavy chain polypeptide as set forth in SEQ ID NO:5 and SEQ ID NO: 6; and a light chain polypeptide as set forth in SEQ ID NO: 7 and SEQ ID NO: 8.

4. A recombinant expression vector comprising the polynucleotide of claim 2 or 3 and an expression vector carrying the polynucleotide.

5. The recombinant expression vector of claim 4, wherein the expression vector is pcDNA3.1, pDR1 or pDFFR.

6. A host cell transformed with the recombinant expression vector of claim 4 or 5.

7. The host cell of claim 6, wherein the cell is a Chinese hamster ovary cell, an NS0 myeloma cell, a COS cell, or an SP2/0 cell.

8. The method for producing an antibody according to claim 1, which comprises the steps of:

(i) culturing a host cell according to claim 6 or 7 under conditions suitable to allow expression of said antibody; and

(ii) recovering the expressed antibody.

9. The use of the antibody of claim 1 in the preparation of an anti-tumor medicament.

10. A pharmaceutical composition comprising the antibody of claim 1, and at least one pharmaceutically acceptable carrier, diluent, or excipient.