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

Abstract

The invention belongs to the technical field of biomedicine. The invention discloses an anti-human EGFR and Notch multispecific antibody C-T Crossmab, which can combine with EGFR, Notch2 and Notch3. The C-T Crossmab of the present invention includes two heavy chain polypeptides and two light chain polypeptides, and the present invention also provides a nucleotide sequence encoding the heavy and light chain polypeptides, and a recombinant expression vector comprising the nucleotide sequences, and transformed host cells. The multispecific antibody of the present invention can be used for the treatment of tumor diseases.

Description

Anti-human EGFR and Notch multispecific antibody, its preparation method and use

technical field

The invention belongs to the technical field of biomedicine, and more specifically, the invention discloses a bispecific antibody against human EGFR, Notch2 and Notch3, its preparation method and its application in the preparation of antitumor drugs.

Background technique

Surgical treatment, radiotherapy and chemotherapy, supplemented by new targeted treatment schemes are the basic strategies for the treatment of malignant tumors in recent years, and important progress has been made in clinical practice. However, the recurrence, metastasis and therapeutic resistance of malignant tumors are still difficult problems for clinical and scientific researchers. In 2006, at the Symposium on Cancer Stem Cells held by the American Association for Cancer Research, the participating scholars defined cancer stem cells as a cancer cell with self-renewal characteristics, the ability to drive the formation of a variety of heterogeneous tumor differentiation cell groups, and stem cell characteristics. (Clarke et al., Cancer stem cells—perspectives on current status and future directions: AACR Workshop on cancer stem cells., 2006, Cancer research, 66:9339-9344). With its unique theoretical advantages and abundant research supporting evidence, the theory of cancer stem cells has been increasingly recognized and accepted by scholars at home and abroad. Although traditional treatment methods have a good therapeutic and killing effect on tumor solid cells, it is difficult to inhibit tumor stem cells that only account for 0.2% to 10% of the tumor cell population and have a large molecular phenotype difference from tumor solid cells. It is of great significance to the treatment of malignant tumors to study the important role and mechanism of tumor stem cells in the generation, development and metastasis of tumors, as well as to develop new anti-tumor treatment strategies and preparations based on the theory of tumor stem cells.

The theory of cancer stem cells holds that malignant tumors originate from a group of "stem cell-like" tumor cells, which are called cancer stem cells (cancer stem cells, CSCs or cancer-initiating cells, CICs). In the late 1990s, researchers first isolated tumor stem cells from leukemia cells and elaborated a hierarchical tissue model of tumor heterogeneity driven by tumor stem cells (Dick, Human acute myeloid leukemia is organized as ahierarchy that originates from a primitive hematopoietic cell., 1997, Nature Med, 3:730-737). Subsequently, researchers have also confirmed the existence of tumor stem cells and similar tumor heterogeneity in a variety of solid tumors, including breast cancer, colon cancer, brain tumor, ovarian cancer, lung cancer, prostate cancer and pancreatic cancer (Brooks et al. ., Therapeutic implications of cellular heterogeneity and plasticity in breast cancer., 2015, Cell stem cell, 17:260-271). These evidences all indicate that there are tumor cells with stem cell properties in most hematological and solid tumors.

A number of studies have shown that cancer stem cells play an important role in the occurrence and development of malignant tumors; maintenance of tumor cell heterogeneity; tumor evolution; and tumor therapeutic resistance. Its biological function has the following characteristics:

1) Very strong tumorigenicity. Tumor cells directly isolated from cancer patients with malignant tumors are difficult to form clones in vitro or transplanted into immune-deficient mouse models for cloning into tumors, but only a small amount of CSCs can be cloned into tumors in immune-deficient mouse models , and the tumor graft has similar phenotypic heterogeneity to the primary tumor (Al-Hajj et al., Prospective identification of tumorigenic breast cancer cells., 2003, Proceedings of the National Academy of Sciences, 100:3983-3988; Dick, Humanacute myeloid leukemia is organized as a hierarchy that originates from aprimitive hematopoietic cell., 1997, Nature Med, 3:730-737; Ginestier et al., ALDH1 is a marker of normal and malignant human mammalian stem cells and apredictor of poor clinical outcome., 2007, Cell stem cell, 1:555-567).

2) Complex heterogeneity and biological plasticity. The source of CSC may be extremely complex, which may be derived from differentiated tumor cells that have acquired self-renewal ability during the process of epithelial-to-mesenchymal transition (EMT) (Espinoza and Miele, Deadly crosstalk: Notchsignaling at the intersection of EMT and cancer stem cells., 2013, Cancerletters, 341:41-45); or derived from normal tissue stem cells induced by foreign microenvironmental factors oncogene mutations (Reya et al., Stem cells, cancer, and cancer stem cells., 2001, nature, 414:105-111). In addition, some reports in recent years have also confirmed that CSCs have very complex biological plasticity. For example, in breast cancer stem cells, two breast cancer stem cell populations with different phenotypes can be identified: an epithelioid breast cancer CSC population with active proliferation , expressing the stem cell marker ALDH; and a mesenchymal-like, relatively inactive but highly invasive breast cancer CSC population, whose surface marker expression is CD44+/CD24-. These two groups of CSCs can produce their own epithelial-like or mesenchymal-like tumor solid cell progeny, and can release secretory signals to enhance the self-renewal ability of CSCs. And under the epigenetic changes mediated by cytokine signaling, chemokine signaling, and transcriptional regulation in the tumor microenvironment, the two groups of cells can transform into each other (Liu et al., Breast cancer stem cells transition between epithelial and mesenchymal states reflective of their normal counterparts., 2014, Stem cell reports, 2:78-91). In addition, studies have also reported that there are different CSC populations in liver cancer, such as CD133+ CSCs (Ma et al., CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. 2008, Oncogene, 27: 1749-1758 ) and CD90+/CD44+ liver cancer CSCs (Yang et al., Significance of CD90+cancer stem cells in human livercancer., 2008, Cancer cell, 13:153-166) have different stem cell characteristics. The markers of stem cells in different tumor types are also often different. For example, in pancreatic cancer, the surface marker of CSC is considered to be CD24+ (Li et al., Structural basis for inhibition of the epidermal growth factor receptor bycetuximab., 2007, Cancer research, 67:1030-1037), and for breast CSC, CD24-. In addition, the content and ratio of CSCs in different malignant tumors are often different, and in different stages of malignant tumors, the ratio of CSCs will also undergo complex changes. For example, in colon cancer, the ratio of CD133+ highly expressed CSCs is about 1.8% to 24.5% (O'Brien et al., Ahuman colon cancer cell capable of initiating tumor growth inimmuneficient mice., 2007, Nature, 445:106-110), After receiving chemotherapy, the rate of colon cancer CSC will increase (Dylla et al., Colorectal cancer stem cells are enriched inxenogeneic tumors following chemotherapy., 2008, PloS one, 3:e2428). In melanoma, the ratio of CSCs expressing CD133+ cell surface markers is between 1% and 20% (Quintana et al., Efficienttumour formation by single human melanoma cells., 2008, Nature, 456:593-598). These findings all suggest that CSC has extremely complex heterogeneity and biological plasticity.

3) Poor treatment sensitivity. Several studies in recent years have shown that CSC is resistant to many treatments currently being used in the clinic, including a variety of chemotherapy drugs and radiotherapy. For example, it has been reported that human acute myeloid leukemia CD34+/CD38-progenitor cells have decreased sensitivity tochemotherapy and Fas-mediated apoptosis (Costello et al., Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity tochemotherapy and Fas -induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities., 2000, Cancer Research, 60:4403-4411); and high expression of telomerase, maintaining the normal structure of chromosomes and inhibiting cell apoptosis; high expression of multiple anti-apoptosis Death genes, such as bcl-2 gene, nuclear transcription factor κB (NF-κB) gene, mutant p53 gene and c-myc gene, etc. CD44+/CD24- breast cancer CSCs are not sensitive to radiation therapy (Phillips et al., 2006, Journal of the National Cancer Institute, 98:1777-1785); CD133+ glioma stem cells have strong DNA damage repair ability and are resistant to radiation Insensitive to therapy (Bao et al., Glioma stem cells promote radioresistance by preferential activation of the DNA damage response., 2006, nature, 444:756-760); Dylla et al also reported colon cancer CSC xenogeneic tumor model in nude mice after chemotherapy The proportion of middle cells is increased, and it is not sensitive to chemotherapy (Dylla et al., Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy., 2008, PloS one, 3: e2428). Moreover, both cancer stem cells and normal stem cells highly express ATP-binding cassette transporters (ATP-binding cassette transporters, ABC), such as ABCB1 protein and ABCG2 protein. The former encodes P-glycoprotein, and the latter is obtained from mitoxantrone-resistant cells. identified. ABCB1 and ABCG2-mediated efflux prevent dyes such as Hoechst 33342 and rhodamine 123 from staining stem cells, confirming that ABC proteins are key to the chemotherapy-insensitive properties of stem cells (Zhou et al., The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype., 2001, Nature medicine, 7:1028-1034). CSCs have a variety of biological properties similar to normal stem cells, such as relatively inactive proliferation, insensitivity to drugs and toxins, high expression of various ABC proteins, strong nucleic acid repair capabilities, and apoptosis resistance (Dean et al., Human Acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell., 2005, Nature Reviews Cancer, 5:275-284). In addition, CSCs, similar to normal stem cells, form tumor stem cell niches together with the surrounding microenvironment. The niches are relatively hypoxic and can provide CSCs with a large number of biological signals to maintain homeostasis, and have a barrier effect on external stimuli. Conventional cytotoxic therapy has little effect on stem cells in the cell niche (LaBarge, The difficulty of targeting cancer stem cell niches., 2010, Clinical cancer research, 16:3121-3129). Therefore, CSCs can survive chemotherapy and radiotherapy, and repopulate the tumor, eventually leading to tumor recurrence and therapeutic resistance.

4) Strong ability to promote metastasis and invasion. Due to the high heterogeneity of tumor tissue, most tumor solid cells do not have the ability to invade and metastasize, while CSCs have very strong ability to invade and metastasize. For example, it has been reported that breast cancer CSCs are highly related to the important EMT process in the process of breast cancer metastasis, and EMT-like CSCs are often located at the infiltrating edge of solid tumors, and can easily enter the blood circulation and form micrometastases in the distal end (Mani et al. al., The epithelial-mesenchymal transition generates cells with properties of stem cells., 2008, Cell, 133:704-715). At this time, CSC can be down-regulated by some microenvironmental factors (such as ID1-mediated TWIST (Stankic et al., TGF-β-Id1 signaling opposes Twist1 and promotes metastatic colonization via a mesenchymal-to-epithelial transition., 2013, Cell reports, 5: 1228-1242)) into highly proliferative epithelial CSCs, and finally complete the process of breast cancer metastasis. In pancreatic cancer, CSCs expressing CD133+/CXCR4+ cell surface markers are also often located at the edge of tumor invasion, easily enter the blood, and they highly express chemokine receptors. Under the action of CXCR4 chemokine, this type of CSC can easily transfer to the liver (Hermann et al., Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer., 2007, Cell stem cell, 1:313 -323). The above studies all suggest that CSC plays a very important role in the invasion and metastasis of malignant tumors.

In summary, CSC has strong tumorigenicity, complex heterogeneity and bioplasticity, strong metastasis and invasion ability, and poor treatment sensitivity. After the tumor tissue undergoes conventional cytotoxic treatment, such as radiotherapy and chemotherapy and biological targeted therapy, the remaining highly tumorigenic CSCs begin to enhance their proliferative activity and begin to expand and drive the growth of tumor tissue, which manifests as clinical tumor recurrence. On the basis of the above theories and studies, many researchers believe that therapeutic strategies targeting CSC cell populations will supplement the deficiencies of existing therapeutic strategies and can significantly improve the level of tumor treatment (Schaue and McBride, Opportunities and challenges of radiotherapy for treating cancer., 2015, Nature Reviews Clinical Oncology, 12:527-540; Takebe et al., Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update., 2015, Nature Reviews Clinical Oncology).

Current research has made it clear that there are dysregulated signaling pathways in CSC, which are critical to the regulation of CSC, including Wnt signaling pathway, Notch signaling pathway, and Hedgehog signaling pathway (Takebe et al., Targeting Notch, Hedgehog, and Wnt Pathways in cancer stem cells: clinical update., 2015, Nature Reviews Clinical Oncology). In 1916, the Notch gene was first discovered in fruit flies. The mutation of this gene can cause a gap in the edge of the wing of the fruit fly, hence the name. The complete Notch signaling pathway includes transmembrane protein receptors, transmembrane proteins, transcription factors and downstream target genes, among which there are four kinds of Notch receptors (Notch1-4) and five kinds of ligands (Delta-like-1, Delta-like-1, Delta-like-1) in human cells. -like-3, Delta-like-4, Jagged-1 and Jagged-2) (Bailey et al., Cancer metastasis facilitated by developmental pathways: Sonic hedgehog, Notch, and bone morphogenic proteins., 2007, Journal of cellular biochemistry, 102:829-839), which is highly conserved in evolution, has the function of regulating cell proliferation, differentiation and apoptosis, and involves almost all tissues and organs. The Notch gene has a very significant tumorigenic role, which is reflected in the activation of a variety of human tumors, including lymphoma, breast cancer, lung cancer, head and neck cancer, pancreatic cancer, colon cancer, osteosarcoma of the jaw and glioblastoma Mutation, and expanded to all levels of members of the Notch signaling pathway (Weng and Aster, Multiple niches for Notch in cancer: context is everything., 2004, Current opinion in genetics & development, 14:48-54). In a variety of tumors, the Notch signaling pathway has been shown to be related to the maintenance of stemness of tumor stem cells. Recently, OncoMed Pharmaceuticals announced the patient survival and biomarker data of the phase Ib clinical trial of tarextumab, a monoclonal antibody targeting the Notch pathway, in the treatment of small cell lung cancer. Tarextumab is a fully humanized monoclonal antibody targeting the Notch2/3 receptor. Preclinical studies suggest that there are 2 mechanisms of action, namely down-regulation of Notch pathway signaling, anti-tumor stem cell effects and effects on pericytes, stromal cells and the microenvironment of tumors.

In solid tumor cells, the epidermal growth factor receptor (EGFR) signal is another important signal transduction pathway of the body, which widely exists in epithelial, mesenchymal and neural tissues, and runs through the whole process of cell growth and proliferation. Differentiation selection plays an important regulatory role. The EGFR family mainly includes EGFR (ie HER1) and HER2-4. EGFR encodes a transmembrane protein, and its signal is activated by receptor tyrosine phosphorylation, cascade amplification through the Ras/Raf/MEK/MAPK pathway, and finally leads to the phosphorylation of MAPK, and the modified MAPK signal enters the nucleus to promote the activation of target genes Phosphorylation, regulates gene expression and activity. EGFR signaling 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 and bladder cancer. Highly expressed signals are associated with poor prognosis in breast and bladder cancers. EGFR expression in breast cancer correlates with tumor proliferation, disease progression, and poor prognosis. The expression of estrogen receptor can reduce the expression of estrogen and increase the resistance to endocrine therapy. Preclinical studies have shown that constitutively activated EGFR can significantly increase the tumorigenicity of the MCF-9 human breast cancer model in vivo. Cetuximab (C225), a monoclonal antibody that acts on the epidermal growth factor receptor (EGFR), is one of the most widely studied targeted drugs, and its efficacy has been confirmed in the treatment of various tumors.

The interaction between EGFR and Notch signaling is complex and can be synergistic or antagonistic, depending on the type of tissue and the stage of development. In glioma, Notch up-regulates the expression of EGFR, and in well-differentiated glioma, the expression of EGFR is closely related to Notch. In cutaneous squamous cell carcinoma, EGFR signaling acts upstream of Notch1 signaling and represses the expression of the signal. Synergistic facilitation with signaling in in vitro culture of breast ductal cell carcinoma in situ. These studies have shown that EGFR signaling and Notch signaling are two closely related signal transduction pathways, which play an important role in regulating the occurrence and development of tumors.

In summary, the Notch signaling pathway is a key pathway involved in the occurrence of tumor cell EMT, the maintenance of tumor CSC cell population and stemness, proliferation and differentiation, and the regulation of CSC treatment resistance. EGFR is the main overactivated signaling pathway in tumor solid cells, and plays an important role in the targeted therapy of tumor solid tumors. There are cross-activations at various levels between the two signaling pathways.

Therefore, using genetic engineering technology to construct a multispecific antibody that can block two signals at the same time, which can not only block EGFR, but also block Notch, and then have an inhibitory effect on tumor stem cells and tumor solid cells at the same time has always been a challenge in this field. Problems that technicians urgently need to solve.

Contents of the invention

The purpose of the present invention is to provide a multi-specific antibody that can simultaneously block two signal pathways, that is, it can block EGFR, and it can also block Notch2 and Notch3. Another object of the present invention is to provide a preparation method of 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.

The first aspect of the present invention provides an anti-EGFR and Notch2/3IgG molecule-like cross monoclonal antibody (Crossmab) C-T Crossmab, the Crossmab can bind to EGFR, Notch2, and Notch3.

Preferably, an 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 sequences of the heavy chain polypeptides are as SEQ ID NO: 1 and SEQ ID NO: 2; the amino acid sequence of the light chain polypeptide is shown in SEQ ID NO: 3 and SEQ ID NO: 4.

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

Preferably, the polynucleotide comprises the nucleotide sequences encoding heavy chain polypeptides as shown in SEQ ID NO: 5 and SEQ ID NO: 6; and encoding light chain polypeptides such as SEQ ID NO: 7 and Nucleotide sequence shown in SEQ ID NO:8.

The present invention also provides a recombinant expression vector, which includes the above-mentioned polynucleotide.

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

The present invention also provides a host cell transformed with the above-mentioned vector.

Preferably, the host cells are eukaryotic cells; more preferably, they are selected from Chinese hamster ovary CHO cells, NSO myeloma, COS or SP2/0 cells and the like.

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

In the present invention, any suitable vector can be used, which can be one of pDR1, pCDNA3.1, pDHFF, and the expression vector includes a fusion DNA sequence connected with appropriate transcription and translation regulatory sequences.

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

Available host cells are prokaryotic cells containing the above vectors, which can be one of DH5a, BL21(DE3), and TG1.

The second aspect of the present invention provides a method for preparing the above-mentioned antibody (C-T Crossmab), which includes the following steps:

(i) cultivating a host cell as described above under conditions suitable to allow expression of said antibody; and

(ii) Recovering the expressed antibody.

Step (ii), further separating or purifying and recovering the expressed antibody.

The preparation method of the C-T Crossmab disclosed in the present invention is to cultivate the above-mentioned host cells under expression conditions, so as to express the C-T Crossmab, and to isolate or purify the C-T Crossmab.

Using the above method, the bispecific antibody can be purified into a substantially uniform substance, for example, a single band on SDS-PAGE electrophoresis.

The method of affinity chromatography can be used to separate and purify the C-T Crossmab disclosed in the present invention. According to the characteristics of the affinity column used, conventional methods such as high salt buffer, changing pH, etc. can be used to elute the C-T Crossmab bound to the affinity column. C-T Crossmab polypeptide on .

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: the heavy and light chain genes of the multispecific antibody are synthesized from the whole gene, and the sequence of the whole gene refers to the monoclonal antibody The sequence of cetuximab (US Patent US6217866B1, Li et al., Structural basis for inhibition of the epidermal growth factor receptor by cetuximab., 2005, CancerCell, 7:301-311), the sequence of tarextumab (US Patent US20130323266 A1, Yen, Wan- Ching, et al.., titled Targeting Notch signaling with a Notch2/Notch3 antagonist (tarextumab) inhibits tumor growth and decreases tumor-initiating cell frequency., clinical Cancer Research 21.9 (2015): 2084-2095) and Crossmab heavy chain light chain design scheme ( Hu et al., Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk., 2015, Cancerresearch, 75:159-170) and (Spasevska et al. , Advances in Bispecific Antibodies Engineering: Novel Concepts for Immunotherapies., 2015, J Blood Disorders Transf, 6:2).

The present invention uses whole genes to synthesize heavy chain genes and light chain genes of multispecific antibodies, and loads the above two heavy chain genes and two light chain genes into eukaryotic expression vectors pcDNA3.1 and pcDNA3.1Zeo respectively. The above plasmids were transfected into CHO-K1 cells by liposome method, and cell clones stably expressing bispecific antibodies were screened with selection medium containing 500 μg/ml G418 and 300 μg/ml Zeocin. Protein A column was used to pass affinity chromatography The multispecific antibody C-T Crossmab was purified from cell culture supernatants.

The third aspect of the present invention provides the application of the antibody (C-T Crossmab) in the preparation of antitumor drugs.

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

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

Use of the pharmaceutical composition in the preparation of antitumor drugs.

The above uses also include combined use with other antitumor drugs.

The present invention detects the affinity of the above-mentioned C-T Crossmab, and finds that the C-T Crossmab well retains the affinity of cetuximab and tarextumab at the same time.

Therefore, we carried out the next experiment, including the experiments of inhibiting the proliferation of tumor cells, inhibiting tumors in vivo, and inhibiting the expression of tumor EMT genes. The experimental results showed that the C-T Crossmab disclosed in the present invention has the functions of cetuximab and tarextumab at the same time. In addition, the multispecific antibody C-T Crossmab can retain the structure of the traditional monoclonal antibody to the greatest extent like traditional IgG molecules. Because of the presence of the Fc fragment, it can be purified by ordinary Protein A column affinity chromatography, which is beneficial to large Scale production purification. Experiments have shown that at the same dose, the multispecific antibody C-TCrossmab has similar or stronger anti-tumor therapeutic effects than the combined application of cetuximab and tarextumab.

The present invention discloses that the above-mentioned bispecific antibody C-T Crossmab can be combined with pharmaceutically acceptable excipients to form a pharmaceutical preparation composition to exert a more stable therapeutic effect. These preparations can ensure the conformation of the amino acid core sequence of the bispecific antibody disclosed in the present invention Integrity, while protecting the protein's multifunctional groups from degradation (including but not limited to aggregation, deamination, or oxidation). Generally, for liquid formulations, it can be stored at 2°C-8°C for at least one year, and for lyophilized formulations, it is stable for at least six months at 30°C. The preparations here can be suspension, water injection, freeze-dried preparations commonly used in the pharmaceutical field, preferably water injection or freeze-dried preparations. For the water injection or freeze-dried preparations of the above-mentioned C-T Crossmab disclosed in the present invention, pharmaceutically acceptable excipients Including one or a combination of surfactants, solution stabilizers, isotonic regulators and buffers, wherein the surfactants include nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (Tween 20 or 80); Poloxamer (such as poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; tetradecyl, linoleyl or octadecyl sarcosine; Pluronics; The particle tendency of the bifunctional bispecific antibody protein is minimal, and the solution stabilizer can be sugars, including reducing sugars and non-reducing sugars, amino acids include monosodium glutamate or histidine, and alcohols include trihydric alcohols , one of higher sugar alcohols, propylene glycol, polyethylene glycol or a combination thereof, the addition amount of the solution stabilizer should make the final formulation maintain a stable state within the time when those skilled in the art believe that it is stable, and the isotonicity regulator can be It is one of sodium chloride and mannitol, and the buffer can be one of TRIS, histidine buffer and phosphate buffer.

The above preparation is a composition containing C-T Crossmab, and after administration to animals including humans, the antitumor effect is obvious. Specifically, it is effective for the prevention and/or treatment of tumors, and can be used as an antitumor drug.

The tumor referred to in the present invention includes adenocarcinoma, leukemia, lymphoma, melanoma, sarcoma, and the source of tumor tissue includes but not limited to adrenal gland, gallbladder, bone, bone marrow, brain, breast, bile duct, gastrointestinal tract, heart, kidney , liver, lungs, muscles, ovaries, pancreas, parathyroid glands, penis, prostate, skin, salivary glands, spleen, testes, thymus, thyroid, and uterus. In addition to the above-mentioned tumors, it can also be used for tumors of the central nervous system such as glioma, astrocytoma, etc. In addition, eye tumors include basal cell carcinoma, squamous cell carcinoma, melanoma, etc., and endocrine gland tumors. Tumors, neuroendocrine system tumors, gastrointestinal and pancreatic endocrine system tumors, reproductive system tumors, head and neck tumors, etc. I won't list them one by one here.

The antitumor drugs referred to in the present invention refer to drugs that inhibit and/or treat tumors, which may include delaying the development of symptoms associated with tumor growth and/or reducing the severity of these symptoms, and it further includes existing tumor growth associated with Reduction of symptoms and prevention of other symptoms, but also reduce or prevent metastasis.

In the present invention, when C-T Crossmab and its composition are administered to animals including humans, the dosage varies according to the age and body weight of the patient, the characteristics and severity of the disease, and the route of administration. The results of animal experiments can be referred to. And in various situations, the total dosage should not exceed a certain range. Specifically, the dosage for intravenous injection is 1-1800 mg/day.

The C-T Crossmab disclosed in the present invention and its composition can also be administered in combination with other antineoplastic drugs or radiotherapy for the treatment of tumors. These antineoplastic drugs include 1. Cytotoxic drugs (1) act on the chemical structure of DNA Drugs: Alkylating agents such as nitrogen mustards, nitrosouries, methylsulfonates; platinum compounds such as cisplatin, carboplatin, and oxalplatin; mitomycin (MMC); (2) affecting nucleic acid Synthetic drugs: dihydrofolate reductase inhibitors such as methotrexate (MTX) and Alimta, etc.; thymidine synthase inhibitors such as fluorouracils (5FU, FT-207, capecitabine), etc.; purine nucleosides Synthase inhibitors such as 6-mercaptopurine (6-MP) and 6-TG; nucleotide reductase inhibitors such as hydroxyurea (HU); DNA polymerase inhibitors such as cytarabine (Ara-C) and (3) Drugs acting on nucleic acid transcription: selectively acting on DNA templates, inhibiting DNA-dependent RNA polymerase, thereby inhibiting RNA synthesis, such as: actinomycin D, daunorubicin, Adriamycin, epirubicin, aclarmycin, mithromycin, etc.; (4) Drugs mainly acting on tubulin synthesis: paclitaxel, taxotere, vinblastine, vinorelbine, podophylline , homoharringtonine; (5) other cytotoxic drugs: asparaginase mainly inhibits protein synthesis; 2, hormone antiestrogens: tamoxifen, droloxifene, exemestane, etc.; aromatase inhibitors : Aminoglutethimide, Lantron, Letrozole, Arimidex, etc.; Antiandrogens: Flutamide RH-LH agonists/antagonists: Noredex, Enerton, etc.; 3. Biological response modifiers: Mainly through the body's immune function to suppress tumor interferon; interleukin-2; thymosin; 4. Monoclonal antibodies: MabThera; Herceptin (Trastuzumab) Bevacizumab (Avastin); Others include some drugs with unknown mechanisms and to be further studied; cell differentiation inducers such as retinoids; apoptosis inducers. The C-T Crossmab disclosed in the present invention and its composition can be used in combination with one of the above antitumor drugs or its combination.

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

Description of drawings

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

Fig. 2. C-T Crossmab of the present invention inhibits HCC827 cell viability experiment result;

Fig. 3. The curve of inhibition of HCC827 tumorigenesis by C-T Crossmab of the present invention.

detailed description

The present invention will be further described below in conjunction with the examples, experimental examples and accompanying 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 plasmids, insert protein-encoding genes into such vectors and plasmids, or introduce plasmids into host cells. Such methods It is well known to those of ordinary skill in the art and is described in numerous publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2 ^nd edition, Cold spring Harbor Laboratory Press

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

Whole gene synthesis of two heavy chain and two light chain gene sequences of C-T Crossmab (sequence shown in SEQ ID NO: 5, 6, 7 and 8, entrusted Jinweizhi Biological Company to synthesize), two heavy chain gene sequences and pcDNA3 .1 The vector is connected, and the two light chain gene sequences are connected with the pcDNA3.1Zeo vector to construct a eukaryotic expression vector.

Inoculate 3×10 ⁵ CHO-K1 cells in a 3.5 cm tissue culture dish, and conduct transfection when the cells are cultured to 80%-85% confluence: take 10 μg of each heavy chain plasmid, 4 μg of each light chain plasmid, and 30 μl Lipofectamine2000 Reagent (Invitrogen Company Products) were dissolved in 800μl serum-free DMEM medium, and left to stand at room temperature for 5 minutes. The above two liquids were mixed and incubated at room temperature for 20 minutes to form DNA-liposome complexes, during which 3ml of serum-free DMEM medium was replaced Serum-containing medium in a petri dish, and then add the formed DNA-liposome complex to the plate, and after 4 hours of incubation in a CO ₂ incubator, add 2ml of DMEM complete medium containing 10% serum, place in CO ₂ Continue to grow in the incubator. After 24 hours of transfection, the cells were replaced with selection medium containing 500 μg/ml G418 and 300 μg/ml Zeocin to screen for resistant clones. The screened high-expression clones were expanded and cultured in serum-free medium, and the bispecific antibody C-TCrossmab was isolated and purified with a Protein A affinity column (product of GE Company). CT Crossmab was dialyzed with PBS, and finally quantified by ultraviolet absorption method. The structure of CT Crossmab is shown in Figure 1, and the amino acid sequence is shown in SEQ ID NO: 1, 2, 3, and 4, which were successfully sequenced by Jinweizhi Biological Company.

Embodiment 2.Biacore analysis

The polyclonal anti-human FC antibody (Jackson ImmunoResearch Company) was coated on the CM5M5 chip (GE Company), and after the detected antibody was captured, the affinity of C-T Crossmab was detected with Biacore T100 (GE Healthcare). The specific detection affinity values are shown in Table 1.

Table 1. Biacore Analysis

The experimental results show that the C-T Crossmab of the present invention well retains the affinity of cetuximab and tarextumab at the same time.

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

Take well-growing HCC827 cells (ATCC), adjust the cell concentration to 5×10 ³ /ml, inoculate in a 96-well cell culture plate, 200 μl/well, and cultivate in a 37°C, 5% CO ₂ incubator for 24 hours. EGF with a final concentration of 5nmol and bispecific antibodies CT Crossmab, cetuximab, tarextumab, cetuximab+tarextumab, and irrelevant human IgG (Rituximab purchased from Roche Company) with different concentration gradients were added to the culture medium. After 4 days, the cell viability was tested with CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison, WI) detection.

The experimental results are shown in Figure 2. The experimental results show that C-T Crossmab can inhibit the viability of HCC827 cells, and has a more significant inhibitory effect than cetuximab and tarextumab, and the effect of the double antibody group combined with cetuximab and tarextumab is similar or better.

Embodiment 4: C-T Crossmab inhibits tumor growth experiment in vivo

In order to test the anti-tumor activity of C-T Crossmab in vivo, HCC827 cells were first used to inoculate subcutaneously on the right flank of BALB/c nude mice (Experimental Animal Center of Second Military Medical University). After tumor formation, 10 mg/kg of the following antibodies were injected into the tail vein of each group: C-TCrossmab, cetuximab, tarextumab, irrelevant control human IgG, cetuximab and tarextumab were combined at 5 mg/kg each, injected once a week, and the mice were sacrificed until the tumor was too large. The length and width of the tumor were measured every day, and the tumor volume was calculated.

Tumor growth curves are shown in Figure 3. The experimental results showed that the tumor growth rate in the C-T Crossmab treatment group was significantly lower than that in the cetuximab and tarextumab treatment groups (after 70 days, P<0.01, Mann-Whitney test). And the therapeutic effect of C-TCrossmab was better than that of combined application with cetuximab and tarextumab (after 70 days, P<0.01, Mann-Whitney test).

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments, and those skilled in the art can also make various equivalents without violating the spirit of the present invention. These equivalent modifications or replacements are all included within the scope defined by the claims of the present application.

SEQUENCE LISTING

<110> The Second Military Medical University of the Chinese People's Liberation Army

<120> Anti-human EGFR and Notch multispecific antibody, its preparation method and use

<130> specification, claims

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 449

<212> PRT

<213> Artificial sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 2

<211> 449

<212> PRT

<213> Artificial sequence

<400> 2

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser 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 Ser Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 3

<211> 214

<212> PRT

<213> Artificial sequence

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 213

<212> PRT

<213> Artificial sequence

<400> 4

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ile Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ser Ser 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 tgaacccaaa gcccagcaac accaaggtgg acaagcgcgt ggagcccaag 660

agctgcgaca agacccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg cacccccgag 780

gtgacctgcg tggtggtgga cgtgagccac gaggacccccg 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

cccagcgtgttcatcttccc 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 tgaccccacca gggcctgagc agccccgtga ccaagagctt caaccgcggc 660

gagtgcgaca agaccacac ctgccccccc tgccccgccc ccgagctgct gggcggcccc 720

agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagccg cacccccgag 780

gtgacctgcg tggtggtgga cgtgagccac gaggacccccg 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 (10)

1. An anti-human EGFR and Notch multispecific antibody, which comprises two heavy chain polypeptides and two light chain polypeptides, wherein the amino acid sequences of the heavy chain polypeptides are as shown in SEQ ID NO: 1 and SEQ ID NO: 2; The amino acid sequence of the light chain polypeptide is shown in SEQ ID NO:3 and SEQ ID NO:4. 2. A polynucleotide comprising a nucleotide sequence encoding the antibody of claim 1. 3. The polynucleotide according to claim 2, comprising a nucleotide sequence encoding a heavy chain polypeptide such as SEQ ID NO: 5 and SEQ ID NO: 6; and encoding a light chain polypeptide such as SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO:8. 4. A recombinant expression vector comprising the polynucleotide according to claim 2 or 3. 5. The recombinant expression vector according to claim 4, characterized in that, the expression vector is pcDNA3.1, pDR1 or pDHFR. 6. A host cell transformed with the vector according to claim 5 or 6. 7. The host cell according to claim 6, characterized in that, the cell is Chinese hamster ovary cell, NSO myeloma cell, COS cell or SP2/0 cell. 8. The preparation method of antibody as claimed in claim 1, it comprises the steps: (i) cultivating 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. Use of the antibody as claimed in claim 1 in the preparation of antitumor drugs. 10. A pharmaceutical composition comprising the antibody of claim 1, and at least one pharmaceutically acceptable carrier, diluent or excipient.