CN108752478B - Fully human anti-human EGFR and Notch2/3 multispecific antibody, preparation method and application thereof - Google Patents

Abstract

The invention relates to the technical field of biological medicines, in particular to a fully human anti-human EGFR and Notch2/3 multi-targeting antibody, a preparation method thereof and application thereof in preparing anti-tumor medicines. The double-targeting antibody disclosed by the invention targets three cell membrane surface receptors of EGFR and Notch2/3 on the basis of retaining the structure of a whole antibody, can block EGFR, can block Notch2 and Notch3, and has better anti-tumor biological activity than that of singly targeting EGFR and Notch 2/3.

Description

Fully human anti-human EGFR and Notch2/3 multispecific antibody, preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a fully human anti-human EGFR and Notch2/3 multi-targeting antibody, a preparation method thereof and application thereof in preparing anti-tumor medicines.

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 tumor stem cell workshop held by the American cancer research Association, the trainee defined the tumor stem cell as a tumor differentiated cell population with self-renewal properties, ability to drive the formation of multiple heterogeneity, cancer cell with stem cell properties (Wosikowski et al, 1997, alternative gene expression in drug-resistant human breast cancer cells,3: 2405-. 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 elucidated a heterogeneous hierarchical tissue model of tumors driven by tumor stem cells (Galizia et al, 2006, viral signature of epidemic growth factor receptor expression in tumor cells underlying surgery supply, 13: 823-835). 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 (Mendelsohn and Baselga,2003, Status of epithelial growth factor receptors in the biology and treatment of cancer,21: 2787-. 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 directly isolated from the foci of malignant tumor patients are difficult to clone in vitro or transplanted into immunodeficient mouse models for clonogenic tumors, whereas only a small number of CSCs are required and tumor grafts can be cloned in immunodeficient mouse models with phenotypic heterogeneity similar to the primary tumor foci (Baselga and Areaga, 2005, Critical update and injection trs in epidemic growth factor recipient targeting in cancer,23: 2445. times. 249; Galizia et al, 2006, diagnostic signalling signature of epidemic growth factor receptor expression in tumor targeting genes, 13: 835. times. Pao 823. et al, 2005, acquisition of infection specific antigens, 73. EGFR. fig..

2) Complex heterogeneity and bioplasticity. The source of CSC may be extremely complex, it may be derived from differentiated tumor cells that acquire self-renewal capacity during epithelial-to-mesenchymal transition (EMT) (Riley et al, 2006, Clinical core of tissues with non-small cell lung cancer and epidermal growth factor receptor ex-19 and ex-21 mutated with gelling or allotinib, 12: 839-844); or from normal tissue stem cells that have been induced by oncogene mutations by foreign microenvironment factors (Cunningham et al, 2004, Cetuximab mongotherpy and Cetuximab plus irinotecan in irinotecan-regenerative genetic cancer,351: 337-345). 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 populations of cells can be transformed into each other under epigenetic changes mediated by cytokine signals, chemokine signals, transcriptional regulation in the tumor microenvironment (Hubbard,2005, EGF receptor inhibition: anchorage on multiple fronts,7: 287-288). In addition, studies have reported that different CSC populations, such as CSC of CD133+ (Hu et al, 2013, composition of the Inhibition Mechanisms of Adalemma and Inhibition in Treating Tumor Necrosis alpha-Associated Diseases from a Molecular View,288:27059-27067) and CSC of CD90+/CD44+ (Li et al, 2008, Structural basis for EGF receptor Inhibition by the therapeutic antibody IMC-11F8,16:216-227) have different stem cell characteristics in liver cancer. Markers for stem cells also tend to differ among different tumor types, such as in pancreatic cancer, where the surface marker for CSCs is considered to be CD24+ (Li et al, 2005, Structural basis for inhibition of the epitopic growth factor receptor by cell 311), 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. As in colon cancer, the CD133+ high expression CSC ratio is about 1.8% to 24.5% (Mukohara et al, 2005, Differential effects of gefitinib and cetuximab on non-small-cell present indicators, 97: 1185. sup. th), while the ratio of colon cancer CSC increases after receiving chemotherapy (Yen et al, 2015, Targeting Notch signaling with a Notch2/Notch3antagonist (tarxtumab) inhibition progress and cleavage-inhibition cell frequency 2095. sup. th: 2084. sup. th). Whereas in melanoma, the rate of CSCs expressing CD133+ cell surface markers is between 1% and 20% (Chinnaiyan et al, 2005, Mechanisms of enhanced radiation response and infecting polypeptide growth factor receptor (Tarceva),65:3328 and 3335). 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, 2000, Human acute myeloid leukemia CD34+/CD 38-promoter cells have had both acquired sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunological specificity, and amplified dendritic cell transformation capsules, 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 radiation therapy (Aralada et al, 2014, EGFR block enriches for Long cancer stem-like cells through Notch3-dependent signaling,74: 5572-; CD133+ glioma stem cells have strong DNA damage repair capacity and are not sensitive to radiotherapy (Hu et al, 2015, Four-in-one antibodies have super cancer inhibition activity against EGFR, HER2, HER3, and VEGF through diagnosis of HER/MET cross 170, 75: 159. fig.; dylla et al also reported that colon cancer CSCs had an increased cell rate in a nude mouse xenograft model after chemotherapy and were insensitive to chemotherapy (Yen et al, 2015, Targeting Notch signaling with a Notch2/Notch3 anticasonist (taraxtumab) inhibition tumor growth and depletion tumor-initiating cell frequency,21: 2084-. 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. ABCB1 and ABCG2 mediated efflux renders dyes such as Hoechst 33342 and rhodamine 123 unable to stain stem cells, confirming that ABC protein is a key to the chemo-insensitive nature of stem cells (Ferguson et al, 2003, EGF activators receptor by removing interaction of human tissue antigen differentiation 517, 11: 507-. CSCs have a variety of biological attributes similar to normal stem cells, such as relative proliferative inactivity, insensitivity to drugs and toxins, high expression of a variety of ABC proteins, strong nucleic acid repair capacity, and resistance to apoptosis (Totorta et al, 1999, Cooperative inhibitory effect of novel mixed bacterial targeting protein kinase A in association with polypeptide and anti-epidermal growth factor-receptor antibody on human breast cancer cell growth,5:875 881). 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 (Petty et al, 2011, High cycle D3 expression control in aerodiagnostic trap cancer 74: 384-391). 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 the 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 at the distal end (Fu et al, 2014, instruments in HER2signaling from step-by-step optimization of anti-HER2antibodies,6: 978. sup. 990). In this case, CSCs can be transformed into high-proliferation-activity epithelial CSCs by some microenvironment factors (such as ID 1-mediated TWIST down-regulation (Perosa et al, 2003, Beta 2-microrogobulin-free HLA class I heart chain epitope by monoclonal antibody HC-10-specific peptide,171:1918-1926), and finally the process of mammary cancer metastasis is completed, in pancreatic cancer, CSCs expressing CD133+/CXCR4+ cell surface markers also often localize at the margins of tumor infiltration, enter the blood easily, and they overexpress chemokine receptors, under the action of CXCR4 chemokines, the CSC is easy to transfer to the liver (Garner et al, 2013, Antntibody at locks HER3 in the active formation inhibition drive by HER2 or neurogenin, 73: 6024-.

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 targeting CSC cell populations would complement the deficiencies of existing therapeutic strategies and significantly increase tumor treatment levels (Bonner et al, 2006, radiotherapeutic plus cetuximab for squarous-cell cancer of the head and the sock, 354: 567-.

It has been clarified from the present studies that there are deregulated signal pathways in CSCs which are critical to the regulation of CSCs, including Wnt signal pathways, Notch signal pathways, and Hedgehog signal pathways, etc. (Shien et al, 2013, Acquired resistance to EGFR inhibition of stem-like properties in cancer cells,73: 3051-3061). 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, 2007, Cancer patients with compromised by quantitative pathways: Sonic hedgehog, Notch, and bone morphogenic proteins,102:829-839), which are highly conserved in evolution, have functions of regulating cell proliferation, differentiation and apoptosis, and involve almost all tissues and organs. The Notch gene has a very significant tumorigenic effect, which is manifested 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,2004, Multiple nucleotides for Notch in cancer: context is every, 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. Can reduce the expression of estrogen receptor and increase the 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. Monoclonal antibodies cetuximab (C225) and panitumumab (vectib), which act on the Epidermal Growth Factor Receptor (EGFR), are the most widely and commonly studied anti-EGFR targeted drugs, and the therapeutic effects thereof are confirmed in various tumor therapies.

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 Notch1 signaling 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. Previous research results of the present inventors have been to construct bifunctional antibody drugs using Cetuximab and Tarextumab as parent antibodies, and have very good anti-tumor stem cell effects (Hu et al, 2017, Antagonism of EGFR and Notch limits to EGFR inhibitors and irradiation by characterization cell frequency,9: eaag0339), which has been applied for chinese patent application No. cn111201602039. x (publication No. CN 106800599a), but Cetuximab is a human murine chimeric antibody, and has potential immunogenicity (galidia, g., et al, "Cetuximab, a chimeric human use anti-epitopic growth factor epitope, in the same man et al, 2007, in the same animal family, 2007).

Therefore, further development and preparation of fully human EGFR and Notch targeting antibodies are urgently needed.

Disclosure of Invention

The present invention aims to provide a multispecific antibody capable of blocking multiple targets simultaneously, i.e., EGFR, and Notch2 and Notch 3. The second 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 order to achieve the purpose, the invention adopts the following technical means:

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

Preferably, the antibody P-T Crossmab consists of four peptide chains, and the amino acid sequences are shown as SEQ ID NO: 19, and the amino acid sequence of the panitumumab heavy chain knob mutant is shown as SEQ ID NO: 23, and the amino acid sequence of the panitumumab light chain is shown as SEQ ID NO: 21, and the amino acid sequence of the Tarextuimab heavy chain hole mutant is shown as SEQ ID NO: 25 of the Tarextumab light chain mutant Tarex-VL-CH 1.

In a second aspect of the invention, there is provided a polynucleotide comprising a nucleotide sequence encoding an antibody as described above.

In the present invention, any suitable DNA encoding EGFR, Notch2, and Notch3 is suitable for use in the present invention, and such constructs 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.

Preferably, the polynucleotide comprises a sequence as set forth in SEQ ID NO: 20, and a nucleotide sequence as set forth in SEQ ID NO: 24, and a nucleotide sequence as set forth in SEQ ID NO: 22, and a nucleotide sequence encoding the Tarextumab light chain mutant Tarex-VL-CH1 as set forth in SEQ ID NO: 26.

In a third aspect of the invention, there is provided a recombinant expression vector comprising a polynucleotide as described above.

In the present invention, any suitable vector may be used, and may be any one of pcDNA3.1, pEE6.4, pEE12.4, pDHFR, and the expression vector includes a fusion DNA sequence linked to appropriate transcription and translation regulatory sequences.

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

In a fourth aspect of the invention, there is provided a host cell transformed with the recombinant expression vector as described above, said host cell being a eukaryotic cell.

In the present invention, a mammalian or insect host cell culture system can be used for the expression of P-T Crossmab of the present invention, and COS, CHO,293, NS0, sf9, sf21 and the like can be applied to the present invention.

Preferably, the host cell is a mammalian cell. In a preferred embodiment of the present invention, the host cell is a Chinese hamster ovary CHO cell.

In a fifth aspect of the invention, there is provided a method for preparing an antibody P-T crosssmab as described above, comprising the steps of:

a) culturing the host cell described above under conditions suitable to allow expression of said antibody P-T Crossmab, thereby expressing antibody P-T Crossmab;

b) isolating or purifying the antibody P-T Crossmab.

The preparation method of the P-T Crossmab disclosed by the invention comprises the steps of culturing the host cell under the expression condition so as to express the P-T Crossmab, and separating or purifying the P-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 P-T Crossmmab disclosed in the present invention can be isolated and purified by affinity chromatography, and the P-T Crossmmab 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-described multispecific antibody P-T cross mab disclosed herein is obtained by the following method:

1) the heavy and light chain genes of the multispecific antibody are synthesized by the whole gene. Sequences of The entire genes refer to The sequence of monoclonal antibodies, panitumumab (Sickmier et al, 2016, The panitumumab egr complex associated with mechanism of proteins center induced resistance,11: e0163366) and The Crossmab heavy-chain light-chain design scheme (Konishi et al, 2007, Gamma-gene expression vectors Notch3activation and regeneration in human lung reactors, 67:8051-8057) and (Gu et al, 2012, Notch signals in The endothionium and reactor "stem-like" cells: open units for reactor heat, 4: 1).

2) Fusing the Tarextuimab heavy chain variable region with an antibody light chain constant region to construct a Tarex-VH-CL fusion fragment;

3) fusing the variable region of the Tarextuimab light chain with the CH1 region of an antibody to construct a Tarex-VL-CH1 fusion fragment;

4) construction of knob mutants separately for the antibody Fc region: T366W, S354C, hole mutant: T366S, L368A, Y407V, and Y394C.

5) Respectively fusing panitumumab heavy chain variable region with a knob mutant, fusing Tarex-VH-CL with a hole mutant, and loading panitumumab light chain and Tarex-VL-CH1 into an expression vector;

6) co-transformation expression is carried out on the four expression vectors in the step 5), and the double-target antibody P-T Crossmab is obtained through separation and purification.

The sixth aspect of the invention provides an application of the antibody P-T Crossmab in preparing antitumor drugs.

Preferably, the medicament takes the antibody P-T Crossmab as an active ingredient.

In a seventh aspect of the invention, there is provided a composition comprising the antibody P-T Crossmab as described above and a pharmaceutically acceptable carrier or adjuvant.

In an eighth aspect of the present invention, there is provided a use of the composition as described above in the preparation of an anti-tumor drug.

Preferably, the use as described above, further comprises the use in combination with other anti-neoplastic agents.

According to the invention, the affinity detection is carried out on the P-T Crossmab, and the P-T Crossmab is found to completely retain the affinities of panitumumab and tarextumab, so that the next step of experiment including the experiment of inhibiting tumor cell proliferation and inhibiting tumor in vivo and the like is carried out, and the experiment result shows that the P-TCrossmab disclosed by the invention has the functions of panitumumab and tarextumab at the same time. In addition, the multispecific antibody P-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 P-T Crossmab can be purified by using a common Protein A column affinity chromatography, thereby being beneficial to large-scale production and purification. Experiments have shown that at equal doses, the multispecific antibody P-T crosumab has an antitumor therapeutic effect similar to or greater than that of the combined use of panitumumab and tarextumab.

The invention discloses the bispecific antibody P-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 P-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; MONAQUAT^TMEtc. in an amount to minimize the tendency of 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 isotonicity 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 P-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, P-T Crossmab and its composition are administered to animals including human, the dose varies depending on the age and body weight of the patient, the nature and severity of the disease, and the administration route, and the total dose cannot exceed a certain range with reference to the results of animal experiments and various cases. In particular, the dosage of intravenous injection is 1-1800 mg/day.

The P-T Crossmab and the composition thereof disclosed by the invention can also be used for treating tumors by combined administration or combined radiotherapy with other antitumor drugs, wherein the antitumor drugs comprise 1 cytotoxic drug: (1) drugs acting on the chemical structure of DNA: 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. hormones: such as 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-inhibiting drugs such as interferon mainly through the immune function of the body; interleukin-2; thymosin peptides; 4. monoclonal antibodies: such as rituximab (MabThera), herceptin (Trastuzumab), Bevacizumab (Avastin), immunocheck Point blockers, such as anti-PD 1 antibodies, etc.; 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 P-TCrossmab and the composition thereof disclosed by the invention can be combined with one of the anti-tumor drugs or the combination thereof.

The double-targeting antibody disclosed by the invention targets three cell membrane surface receptors of EGFR and Notch2/3 on the basis of retaining the structure of a whole antibody, and has better anti-tumor biological activity than that of singly targeting EGFR and Notch 2/3.

Drawings

FIG. 1 is a schematic diagram of the structure of P-T Crossmab;

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

FIG. 3P-T Crossmab inhibits HCC827 tumorigenesis.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

The following examples and experimental examples further illustrate the present invention and 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, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., Fritsch, e.f. and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2^ndedition，Cold spring Harbor Laboratory Press。

Example 1 construction and expression of novel multifunctional antibody P-T Crossmab

(1) The variable region genes of the heavy and light chains of Panitumumab and tarextumab antibodies were synthesized in their entirety to obtain VH and VL of Panitumumab and VH and VL fragments of tarextumab (amino acid sequences and nucleotide sequences are shown in SEQ ID NOS: 1-8, respectively). The human IgG light chain constant region, heavy chain constant region CH1, and Fc fragment (amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 9-14, respectively) were synthesized.

(2) Introducing mutation points into the obtained antibody Fc fragment by adopting an overlap PCR method: T366W, S354C. The PCR product was purified by agarose gel electrophoresis, recovered and cloned into pGEM-T vector, and the correct clone was confirmed after sequencing verification (amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 15-16, respectively). Namely, the Fc fragment knob mutant is obtained.

(3) In addition, the Fc fragment of the antibody is introduced into a mutation point by adopting the overlap PCR method again: T366S, L368A, Y407V and Y394C. The PCR product was purified by agarose gel electrophoresis, recovered and cloned into pGEM-T vector, and the correct clone was confirmed after sequencing verification (amino acid sequence and nucleotide sequence are shown in SEQ ID NO: 17-18, respectively). Namely obtaining the Fc-fragment hole mutant.

(4) And (3) fusing the panitumumab heavy chain variable region obtained in the step (1) and the Fc fragment knob mutant obtained in the step (3) serving as templates by using an overlap PCR technology and filling the two fragments into an expression vector. Namely, a panitumumab heavy chain knob mutant (the amino acid sequence and the nucleotide sequence are respectively shown in SEQ ID NO: 19-20) is constructed.

(5) And (3) taking the Tarextumab heavy chain variable region obtained in the step (1), the light chain constant region and the Fc segment hole mutant obtained in the step (3) as templates, fusing the three fragments by adopting an overlap PCR technology, and loading the three fragments into an expression vector. Thus obtaining Tarextumab VH-CL-Hing-Fc _ hole _ mut fusion fragment (the amino acid sequence and the nucleotide sequence are respectively shown in SEQ ID NO: 21-22).

(6) The panitumumab light chain variable region and light chain constant region CL obtained in the step (1) are used as templates, the fragments are fused by using overlap PCR technology to obtain the panitumumab light chain, and the sequences are optimized for mutation (the amino acid sequence and the nucleotide sequence are respectively shown as SEQ ID NO: 23-24).

(7) And (2) taking the Tarextumab light chain variable region and the IgG heavy chain constant region CH1 obtained in the step (1) as templates, fusing the fragments by using overlap PCR technology to obtain a Tarextumab VL-CH1 fusion fragment, and loading the fusion fragment into an expression vector (the amino acid sequence and the nucleotide sequence are respectively shown as SEQ ID NO: 25-26). And (4), (5), (6) and (7) obtaining four expression vector plasmids of the four peptide chains for coding the P-T Crossmab.

(8) 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 P-T Crossmab was isolated and purified by Protein A affinity column (product of GE). P-T Crossmab was dialyzed against PBS and finally quantified by UV absorption. The structure of P-T Crossmab is shown in FIG. 1.

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 P-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 affinity assay

Example 3: experiment for inhibiting activity of non-small cell lung cancer cells by P-T Crossmab

HCC827 cells (ATCC) in good growth state were collected and adjusted to a cell concentration of 5X 10³Perml, inoculated in 96-well cell culture plates, 200. mu.l/well, cultured at 37 ℃ for 24 hours in a 5% C02 incubator, and then added to the culture medium were EGF at a final concentration of 5nmol and bispecific antibody P-T Crossmab, panitumumab, tarextumab, panitumumab + tarextumab, bispecific antibodyAntibody CT16(Hu et al, 2017, Antigonism of EGFR and Notch limits resistance to EGFR inhibitors and radiation by evaluating tumor-inducing Cell frequency,9: eaag0339), irrelevant human IgG (Rituximab available from Roche Inc.), 3 days later, Cell Viability was detected with CellTiter-Glo luminescence Cell Viability Assay kit (Promega, Madison, Wis.). The results of the experiment are shown in FIG. 2. The experimental results show that the HCC827 cell activity that P-T Crossmumab can inhibit is more remarkable in inhibition effect than panitumumab and tarextumab, the effect of the double antibody group applied in combination with panitumumab and tarextumab is similar or better, and the effect of the bispecific antibody CT16 is similar (the concentration unit of the drug is ng/mu l).

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

To test the in vivo tumor-inhibiting activity of P-T Crossmab, HCC827 cells were first inoculated subcutaneously in the right flank of BALB/c nude mice, and injected via caudal vein after tumorigenesis, with 5mg/kg of each of the following antibodies: P-T Crossmumab, panitumumab, tarextumab, CT16, irrelevant control human IgG, panitumumab and tarextumab 2.5mg/kg each in combination, were injected 1 time per week for a period of time until the mice had overtempted. 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 results show that: the tumor growth rate was significantly less in the P-T Crossmumab-treated group than in the panitumumab and tarextumab-treated groups (P <0.01 after 35 days, Mann-Whitney test). And the therapeutic effect of P-T Crossmab is due to the combined effect with panitumumab and tarextumab (P <0.01 after 35 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> Feng tide pharmaceutical technology (Shanghai) Co Ltd

<120> fully human anti-human EGFR and Notch2/3 multispecific antibody, preparation method and application thereof

<130> /

<160> 26

<170> PatentIn version 3.3

<210> 1

<211> 119

<212> PRT

<213> Artificial sequence (Artificial)

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 2

<211> 357

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

caagttcagc tgcaagaaag cggacccggt ttagtgaagc ctagcgagac tttatcttta 60

acttgtacag tgtccggagg cagcgtgagc agcggcgatt actactggac ttggattcgt 120

cagagccccg gtaagggttt agagtggatt ggccacatct actacagcgg caacaccaac 180

tacaacccct ctttaaagtc tcgtctgacc atcagcatcg acaccagcaa gacccagttc 240

tctttaaagc tgagcagcgt gaccgctgcc gacaccgcca tctactactg cgtgagggat 300

cgtgtgaccg gcgccttcga catctgggga caaggtactt tagtgaccgt gagctcc 357

<210> 3

<211> 107

<212> PRT

<213> Artificial sequence (Artificial)

<400> 3

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 4

<211> 321

<212> DNA

<213> Artificial sequence (Artificial)

<400> 4

gacatccaga tgacccagag ccctagctct ttaagcgcca gcgtgggcga tagggtgacc 60

atcacttgtc aagcctctca agatatcagc aactatttaa actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctccaatc tggagaccgg cgtgccctct 180

cgttttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagctc tttacagccc 240

gaggacatcg ccacctactt ctgtcagcac ttcgaccatt tacctctggc ctttggccaa 300

ggtaccaagg tggagatcaa g 321

<210> 5

<211> 115

<212> PRT

<213> Artificial sequence (Artificial)

<400> 5

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Val Ser Ser

115

<210> 6

<211> 345

<212> DNA

<213> Artificial sequence (Artificial)

<400> 6

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ccggtggttc tttaaggctg 60

tcttgtgccg cctccggctt caccttctcc tcctccggca tgagctgggt gaggcaagct 120

cccggtaagg gtttagagtg ggtgtccgtg atcgcctcca gcggctccaa cacctactac 180

gccgactccg tgaagggtcg tttcaccatc tcccgggaca actccaagaa cactttatat 240

ttacagatga actctttaag ggccgaggac accgccgtgt actactgcgc ccggtccatc 300

ttctacacca cttggggcca aggtacttta gtgacagtgt cctcc 345

<210> 7

<211> 108

<212> PRT

<213> Artificial sequence (Artificial)

<400> 7

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105

<210> 8

<211> 324

<212> DNA

<213> Artificial sequence (Artificial)

<400> 8

gacatcgtgc tgacccagtc ccccgctact ttatctttat cccccggtga gagggctacc 60

ttatcttgtc gtgcttccca gtccgtgagg tccaattatt tagcttggta ccagcagaag 120

cccggccaag ctcctaggct gctgatctac ggcgcctctt ctcgtgccac tggtgtgccc 180

gctaggttct ccggctccgg ttctggcacc gacttcactt taaccatctc ctctttagag 240

cccgaagact tcgccgtgta ctactgccag cagtactcca acttccccat caccttcggc 300

caaggtacca aggtggagat caag 324

<210> 9

<211> 107

<212> PRT

<213> Artificial sequence (Artificial)

<400> 9

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 10

<211> 321

<212> DNA

<213> Artificial sequence (Artificial)

<400> 10

aggaccgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctacc ccagagaagc caaagtgcag 120

tggaaggtgg acaacgccct gcagagcgga aacagccagg aaagcgtgac agagcaggat 180

tccaaggatt ccacatacag cctgagcagc acactgacac tgtccaaggc cgactacgag 240

aagcacaagg tgtacgcctg cgaagtgaca caccagggac tgtcctcccc tgtgacaaag 300

agcttcaaca gaggagaatg c 321

<210> 11

<211> 103

<212> PRT

<213> Artificial sequence (Artificial)

<400> 11

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Lys Val Glu Pro Lys Ser Cys

100

<210> 12

<211> 309

<212> DNA

<213> Artificial sequence (Artificial)

<400> 12

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaacctgt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatcttgt 309

<210> 13

<211> 227

<212> PRT

<213> Artificial sequence (Artificial)

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 14

<211> 681

<212> DNA

<213> Artificial sequence (Artificial)

<400> 14

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 15

<211> 227

<212> PRT

<213> Artificial sequence (Artificial)

<400> 15

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 16

<211> 681

<212> DNA

<213> Artificial sequence (Artificial)

<400> 16

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gccgggatga gctgaccaag 420

aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 17

<211> 227

<212> PRT

<213> Artificial sequence (Artificial)

<400> 17

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 18

<211> 681

<212> DNA

<213> Artificial sequence (Artificial)

<400> 18

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtcctg cgcggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcgt cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa a 681

<210> 19

<211> 449

<212> PRT

<213> Artificial sequence (Artificial)

<400> 19

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 20

<211> 1347

<212> DNA

<213> Artificial sequence (Artificial)

<400> 20

caagttcagc tgcaagaaag cggacccggt ttagtgaagc ctagcgagac tttatcttta 60

acttgtacag tgtccggagg cagcgtgagc agcggcgatt actactggac ttggattcgt 120

cagagccccg gtaagggttt agagtggatt ggccacatct actacagcgg caacaccaac 180

tacaacccct ctttaaagtc tcgtctgacc atcagcatcg acaccagcaa gacccagttc 240

tctttaaagc tgagcagcgt gaccgctgcc gacaccgcca tctactactg cgtgagggat 300

cgtgtgaccg gcgccttcga catctgggga caaggtactt tagtgaccgt gagctccgct 360

agcaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 420

acagcggccc tgggctgcct ggtcaaggac tacttccccg aacctgtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 600

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 660

tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatgccg ggatgagctg 1080

accaagaacc aggtcagcct gtggtgcctg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 21

<211> 449

<212> PRT

<213> Artificial sequence (Artificial)

<400> 21

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Lys

<210> 22

<211> 1347

<212> DNA

<213> Artificial sequence (Artificial)

<400> 22

gaggtgcagc tggtggagtc cggaggagga ctggtgcagc ccggtggttc tttaaggctg 60

tcttgtgccg cctccggctt caccttctcc tcctccggca tgagctgggt gaggcaagct 120

cccggtaagg gtttagagtg ggtgtccgtg atcgcctcca gcggctccaa cacctactac 180

gccgactccg tgaagggtcg tttcaccatc tcccgggaca actccaagaa cactttatat 240

ttacagatga actctttaag ggccgaggac accgccgtgt actactgcgc ccggtccatc 300

ttctacacca cttggggcca aggtacttta gtgacagtgt cctccgctag cgtggctgca 360

ccatctgtct tcatcttccc gccatctgat gagcagttga aatctggaac tgcctctgtt 420

gtgtgcctgc tgaataactt ctaccccaga gaagccaaag tgcagtggaa ggtggacaac 480

gccctgcaga gcggaaacag ccaggaaagc gtgacagagc aggattccaa ggattccaca 540

tacagcctga gcagcacact gacactgtcc aaggccgact acgagaagca caaggtgtac 600

gcctgcgaag tgacacacca gggactgtcc tcccctgtga caaagagctt caacagagga 660

gaatgcgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 720

tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 780

gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 840

gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 900

acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 960

tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1020

gccaaagggc agccccgaga accacaggtg tgcaccctgc ccccatcccg ggatgagctg 1080

accaagaacc aggtcagcct gtcctgcgcg gtcaaaggct tctatcccag cgacatcgcc 1140

gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1200

gactccgacg gctccttctt cctcgtcagc aagctcaccg tggacaagag caggtggcag 1260

caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1320

aagagcctct ccctgtctcc gggtaaa 1347

<210> 23

<211> 214

<212> PRT

<213> Artificial sequence (Artificial)

<400> 23

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 24

<211> 642

<212> DNA

<213> Artificial sequence (Artificial)

<400> 24

gacatccaga tgacccagag ccctagctct ttaagcgcca gcgtgggcga tagggtgacc 60

atcacttgtc aagcctctca agatatcagc aactatttaa actggtacca gcagaagccc 120

ggcaaggccc ccaagctgct gatctacgac gcctccaatc tggagaccgg cgtgccctct 180

cgttttagcg gaagcggcag cggcaccgac ttcaccttca ccatcagctc tttacagccc 240

gaggacatcg ccacctactt ctgtcagcac ttcgaccatt tacctctggc ctttggccaa 300

ggtaccaagg tggagatcaa gaggaccgtg gccgccccta gcgtgttcat cttcccccct 360

agcgacgagc agctgaagag cggcaccgct agcgttgtgt gcctgctgaa taacttctac 420

cccagagaag ccaaagtgca gtggaaggtg gacaacgccc tgcagagcgg aaacagccag 480

gaaagcgtga cagagcagga ttccaaggat tccacataca gcctgagcag cacactgaca 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaagtgac acaccaggga 600

ctgtcctccc ctgtgacaaa gagcttcaac agaggagaat gc 642

<210> 25

<211> 213

<212> PRT

<213> Artificial sequence (Artificial)

<400> 25

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Glu Pro Lys Ser Cys

210

<210> 26

<211> 639

<212> DNA

<213> Artificial sequence (Artificial)

<400> 26

gacatcgtgc tgacccagtc ccccgctact ttatctttat cccccggtga gagggctacc 60

ttatcttgtc gtgcttccca gtccgtgagg tccaattatt tagcttggta ccagcagaag 120

cccggccaag ctcctaggct gctgatctac ggcgcctctt ctcgtgccac tggtgtgccc 180

gctaggttct ccggctccgg ttctggcacc gacttcactt taaccatctc ctctttagag 240

cccgaagact tcgccgtgta ctactgccag cagtactcca acttccccat caccttcggc 300

caaggtacca aggtggagat caagtcctcg gctagcacca agggcccatc ggtcttcccc 360

ctggcaccct cctccaagag cacctctggg ggcacagcgg ccctgggctg cctggtcaag 420

gactacttcc ccgaacctgt gacggtgtcg tggaactcag gcgccctgac cagcggcgtg 480

cacaccttcc cggctgtcct acagtcctca ggactctact ccctcagcag cgtggtgacc 540

gtgccctcca gcagcttggg cacccagacc tacatctgca acgtgaatca caagcccagc 600

aacaccaagg tggacaagaa agttgagccc aaatcttgt 639

Claims (10)

1. An anti-EGFR and Notch2/3IgG molecule-like cross monoclonal antibody P-T Crossmab, which is characterized by consisting of four peptide chains, wherein the four peptide chains respectively have amino acid sequences shown as SEQ ID NO: 19, and the amino acid sequence of the panitumumab heavy chain knob mutant is shown as SEQ ID NO: 23, and the amino acid sequence of the panitumumab light chain is shown as SEQ ID NO: 21, and the amino acid sequence of the Tarextuimab heavy chain hole mutant is shown as SEQ ID NO: 25 of the Tarextumab light chain mutant Tarex-VL-CH 1.

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

3. The polynucleotide of claim 2, comprising a sequence as set forth in SEQ ID NO: 20, and a nucleotide sequence as set forth in SEQ ID NO: 24, and a nucleotide sequence as set forth in SEQ ID NO: 22, and a nucleotide sequence encoding the Tarextumab light chain mutant Tarex-VL-CH1 as set forth in SEQ ID NO: 26.

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

5. The recombinant expression vector of claim 4, wherein the recombinant expression vector is pcDNA3.1, pEE6.4, pEE12.4, pDAFR.

6. A host cell transformed with the recombinant expression vector of claim 4 or 5, said host cell being a eukaryotic cell.

7. The host cell of claim 6, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

8. A method for producing the antibody P-T Crossmab of claim 1, comprising the steps of:

a) culturing the host cell of claim 6 or 7 under conditions suitable to allow expression of said antibody P-T crosssmab, thereby expressing antibody P-T crosssmab;

b) isolating or purifying the antibody P-T Crossmab.

9. Use of the antibody P-T crosphab according to claim 1 for the preparation of an anti-tumor medicament.

10. A composition comprising the antibody P-T Crossmab of claim 1 and a pharmaceutically acceptable carrier or adjuvant.

Images