CN110028584B - Bispecific antibodies against EGFR and MET proteins - Google Patents

Abstract

Bispecific antibodies directed to EGFR and MET are disclosed. The core fucose-containing ratio of the bispecific antibody is not higher than 4.5% through glycosylation modification, so that the ADCC activity is improved; by modifying the amino acid sequence of the Fc segment, the ADCC activity of the bispecific antibody is enhanced, or the affinity with FcRn is enhanced so as to prolong the half life. The bispecific antibody prepared by the invention has simple preparation, stable structure and better tumor inhibition effect, thereby having good market prospect.

Description

Bispecific antibodies against EGFR and MET proteins

Technical Field

The invention belongs to the field of biomedicine, and relates to a bispecific antibody and a preparation method thereof, and in addition, the invention also relates to application of the bispecific antibody.

Background

Since the first antibody drug was approved in 1986, the antibody drugs have been developed vigorously for over 30 years, and because the antibody drugs have the obvious advantages of high targeting property, specificity and the like, the treatment field is gradually expanded from the traditional treatment fields of cancers and autoimmune diseases to the treatment fields of anti-infection, metabolic diseases and cardiovascular diseases, and the like, and the antibody drugs have remarkable curative effects, and create great economic benefits while improving the life quality of patients.

Bispecific antibodies (BsAb) are artificial antibodies that contain two specific antigen binding sites and can bridge between target cells and functional molecules (cells) to produce targeted effector functions. BsAb has wide application prospect in biomedicine, especially in the immunotherapy of tumor. Killing tumor cells through BsAb mediated cytotoxicity is a hotspot of current immunotherapy application research, and is mainly characterized in that BsAb can be simultaneously combined with tumor-associated antigens and target molecules on effector cells to directly trigger the specific killing of the effector cells on the tumor cells. BsAb can be prepared by chemical engineering, cell engineering, genetic engineering and other methods. The biggest advantage of constructing BsAb by genetic engineering is that the antibody can be engineered, such as constructing humanized BsAb, small molecule BsAb, multivalent BsAb, etc. The bivalent BsAb can be designed and constructed by adopting a genetic engineering technology, and the construction mode comprises a Bispecific miniantibody (Bispecific miniantibody), a Diabody (Diabody), a single-chain Bispecific antibody (Sc-BsAb) and the like. The bivalent BsAb has the advantages of small molecular weight, good tumor tissue permeability, easy construction and expression and the like, but because the bivalent BsAb only has one binding functional domain for each specific antigen, the bivalent BsAb has low affinity for target molecules and poor guiding effect. In order to improve the stability and therapeutic potential of BsAb, construction modes of tetravalent BsAbs have appeared in recent years, such as dimeric bispecific minibodies (dimeric minibodies), tetravalent bispecific antibodies (tandabs), IgG-like bispecific antibodies, etc., which have the characteristics of dual specificity and are bivalent to each target antigen, thereby having higher affinity and stronger effector function to target cells, enhanced stability in human serum, and significantly prolonged retention time in circulation. However, since the tetravalent BsAb is also divalent to effector cells (T lymphocytes, NK cells, etc.), clinical application can cause cross-linking of effector cell surface receptors in circulating blood, which leads to extensive activation of effector cells, release of a large amount of cytokines (e.g., TNF α, etc.), and cause unacceptable toxic reactions. The results of animal and clinical trials over the last decade have shown that BsAb with greater clinical utility should have the properties of (1) having to be able to bind tumor-associated antigens with high selectivity and affinity; (2) in the circulation, BsAb should have relatively low affinity for effector cells or cytotoxic trigger molecules, so as to limit side reactions caused by systemic effector cell activation, while BsAb must be able to effectively induce effector cell cytotoxicity when bound to tumor cells expressing target antigens; (3) BsAb should be humanized antibody, thereby maximally reducing human anti-mouse immune response, and making repeated administration not limited; (4) BsAb molecules should be of moderate size, and not only can penetrate into tumor tissues, but also can ensure proper circulation and retention time. At present, bivalent bispecific or trivalent trispecific antibodies are constructed by using human IgG1 heavy chain first constant region (CH1) and kappa chain constant region (CL) domains as heterodimerization domains, and have only one binding domain for each specific antigen, and have low affinity for target molecules and poor targeting effect. Therefore, BsAbs with more reasonable structure and stronger functional effect still need to be developed.

Disclosure of Invention

It is an object of the present invention to provide a bispecific antibody that binds EGFR and MET.

The second object of the present invention is to provide a method for producing the bispecific antibody.

It is a further object of the present invention to provide the use of the bispecific antibodies described above.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to one aspect of the invention, there is provided a bispecific antibody comprising a light-heavy chain pair that specifically binds to an EGFR protein, and a light-heavy chain pair or a fusion peptide that specifically binds to a MET protein, said fusion peptide comprising a ScFv.

As a specific embodiment of the present invention, the bispecific antibody comprises a light-heavy chain pair specifically binding to EGFR, and, a light-heavy chain pair specifically binding to MET or a fusion peptide specifically binding to MET, the fusion peptide comprising ScFv, Fc-fragment having CH2 domain and CH3 domain, CH2 domain being located between ScFv fragment and CH3 domain, in other words, the ScFv fragment is linked to CH2 terminus of the Fc-fragment, the ScFv fragment is linked to CH2 terminus of the Fc-fragment by Linker.

The Fc fragment useful in the present invention is a human or humanized Fc fragment. In a particular aspect, the Fc segment of the heavy chain comprises a human IgG Fc fragment. In certain aspects, the Fc portion of the heavy chain and/or the Fc portion of the fusion peptide described above can comprise one or more substitutions that form a knob-hole pair therebetween, as compared to the wild-type antibody fragment. Pestle-mortar configurations are known in the art. See, for example, Ridgway et al, "Knob-endo-holes' Engineering of antibiotics CH3 domains for latent chain leather chemosynthesis," Protein Engineering9(7):617-21 (1996).

Linkers useful for linking the ScFv to the Fc of the present invention include, but are not limited to, the following amino acid sequences: GTCPPCP, DKKTCPPCP, GGGTHTCPPCP, EKDKKTCPPCP, EPKSCDKTHTCPPCP, EPKSSDKTHTCPPCP, AAEPKSSDKTHTCPPCP, GAAAEPKSCDKTHTCPPCPAP, VDKTHTCPPCP, GGGGGVGDTCTCPP, GGGGGGGSGGSGGGGSAESKYGPPCPPCP. In a specific embodiment of the invention, the amino acid sequence for ScFv to Fc linkage is DKTHTCPPCP.

The amino acid sequences for the linkage of the light chain variable region and the heavy chain variable region in the ScFv that can be used in the present invention include, but are not limited to, the following amino acid sequences: (G4S) n (n 1-4), GGGGSGGG, GSTSGGGSGGGSGGGGSS, GSTSGSGKPGSSEGSTKG, VEGGSGGSGGSGGSGGVD, GGGGGG, (EAAAK) n (n 1-3). In a specific embodiment of the present invention, the amino acid sequence for linking the variable region of the light chain and the variable region of the heavy chain in the ScFv is (G4S) 3.

In a specific embodiment of the invention, the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is represented by SEQ ID No.4 or SEQ ID No.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is selected from any one of the amino acid sequences represented by SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is represented by SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is represented by SEQ ID No.6, and the amino acid sequence of the ScFv is represented by SEQ ID No. 8.

In a specific embodiment of the invention, the amino acid sequence of the light chain constant region in the light-heavy chain pair that specifically binds EGFR is shown in SEQ ID No.9, and when the amino acid sequence of the heavy chain constant region in the light-heavy chain pair that specifically binds EGFR is shown in SEQ ID No.11, the amino acid sequence of the heavy chain constant region in the light-heavy chain pair that specifically binds MET is shown in SEQ ID No. 12; when the amino acid sequence of the heavy chain constant region in the light-heavy chain pair that specifically binds EGFR is selected from any one of the sequences shown in SEQ ID No.12, SEQ ID No.15, SEQ ID No.17, the amino acid sequence of the heavy chain constant region in the light-heavy chain pair that specifically binds MET is selected from any one of the sequences shown in SEQ ID No.11, SEQ ID No.16, SEQ ID No. 18; the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown in SEQ ID NO. 9.

In a specific embodiment of the invention, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown in SEQ ID No.9, and when the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown in SEQ ID No.11, the amino acid sequence of the Fc fragment in the fusion peptide specifically binding to MET is a sequence formed by CH2 plus CH3 domain in the sequence shown in SEQ ID No. 12. When the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is selected from any one of the sequences shown as SEQ ID No.12, SEQ ID No.15 and SEQ ID No.17, the amino acid sequence of the Fc fragment in the fusion peptide specifically binding to MET is selected from the sequence formed by CH2 plus CH3 domain in the sequence shown as SEQ ID No.11, the sequence formed by CH2 plus CH3 domain in the sequence shown as SEQ ID No.16, the sequence formed by CH2 plus CH3 domain in the sequence shown as SEQ ID No.18, and the sequence formed by CH2 plus CH3 domain in the sequence shown as SEQ ID No. 13.

In a particular embodiment of the invention, the structure of the bispecific antibody comprises:

(1) the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.4, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.1, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 11; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 12.

(2) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.4, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.1, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 11.

(3) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.4, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.2, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 11.

(4) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 11.

(5) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 15; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 16.

(6) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 17; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.7, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.6, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID No. 18.

(7) The amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the amino acid sequence of ScFv is shown in SEQ ID NO.8, and the amino acid sequence of Linker + Fc segment is shown in SEQ ID NO. 13.

As an alternative embodiment of the invention, the bispecific antibody comprises two light-heavy chain pairs specifically binding to EGFR, and one fusion peptide specifically binding to MET, said fusion peptide comprising ScFv, said fusion peptide being linked via a linker to the C-terminus of the heavy chain in the light-heavy chain pair specifically binding to EGFR.

Still further, the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID No.4 or SEQ ID No.5, the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is selected from any one of the amino acid sequences shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, the amino acid sequence of the ScFv is shown as SEQ ID No.8, and the amino acid sequence of the linker is selected from one of the following sequences: GGGGS, GGGGSGGGS, GGGGSGGG, GGGGSGGGSGGGGS, GGGGSGGGSGGGGSGGGS, GSTSGGGSGGGSGGGGSS, GSTSGSGKPGSSEGSTKG, VEGGSGGSGGSGGSGGVD, GGGGGGGG, GGGGGG, (EAAAK) n (n is 1-3).

Still further, the amino acid sequences of the heavy chain constant regions in the light-heavy chain pair that specifically bind EGFR are as follows: any one of amino acid sequences shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 18.

Still further, the structure of the above bispecific antibody of the present invention may be:

(1) the amino acid sequences of the heavy chain constant regions in the two light-heavy chain pairs which specifically bind to EGFR are shown as SEQ ID No. 10; or

(2) When the amino acid sequence of the heavy chain constant region in one of the light-heavy chain pairs that specifically binds EGFR is set forth in SEQ ID No.11, the amino acid sequence of the heavy chain constant region in the other light-heavy chain pair that specifically binds EGFR is set forth in SEQ ID No. 12; or

(3) When the amino acid sequence of the heavy chain constant region in one of said light-heavy chain pairs that specifically binds EGFR is selected from any one of the sequences shown in SEQ ID No.12, SEQ ID No.15, SEQ ID No.17, the amino acid sequence of the heavy chain constant region in the other of said light-heavy chain pairs that specifically binds EGFR is selected from any one of the sequences shown in SEQ ID No.11, SEQ ID No.16, SEQ ID No. 18.

In a specific embodiment of the invention, the amino acid sequence of the linker is GGGGSGGGSGGGGS.

In specific embodiments of the invention, examples of such bispecific antibodies of the invention are: the amino acid sequence of the light chain variable region in one light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the amino acid sequence of the heavy chain variable region in one light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in one light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in one light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region in the other light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, the sequence of the heavy chain variable region in the other light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.3, the amino acid sequence of the light chain constant region in the other light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.9, and the amino acid sequence of the heavy chain constant region in the other light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 12; the fusion peptide is connected with the C end of a heavy chain constant region with the amino acid sequence shown as SEQ ID NO.11, the fusion peptide and the heavy chain constant region form a fusion heavy chain constant region, and the sequence of the fusion heavy chain constant region is shown as SEQ ID NO. 14.

According to another aspect of the present invention, there is provided a nucleic acid molecule encoding the bispecific antibody as defined above, the nucleic acid molecule having the sequence shown in SEQ ID NO. 19-35.

According to a further aspect of the present invention there is provided a recombinant vector comprising a nucleic acid molecule having the sequence shown in SEQ ID NO. 19-35.

Types of vectors used for recombinant vectors include bacterial plasmids, cosmids, phagemids, yeast plasmids, plant cell viruses, animal viruses, and various other viral vectors. Vectors among recombinant vectors suitable for use in the present invention include, but are not limited to: vectors for expression in bacteria (prokaryotic expression vectors), vectors for expression in yeast (e.g., pichia vectors, hansenula vectors, etc.), baculovirus vectors for expression in insect cells, vectors for expression in mammalian cells (vaccinia vectors, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, etc.), plant virus vectors for expression in plants, and various vectors for expression in mammalian mammary glands. In general, any plasmid and vector can be used as long as they can stably replicate in a host cell.

According to a further aspect of the invention there is provided a host cell comprising a nucleic acid molecule having the sequence shown in SEQ ID NO.19 to 35 or a recombinant vector as hereinbefore described.

Suitable host cells for use in the present invention are derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for suspension culture may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (e.g., 293 or 293T cells as described in Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK); mouse support cells (such as TM4 cells as described in, for example, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV 1); VERO cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK; Bufarro rat liver cells (BRL 3A); human lung cells (W138); human liver cells (HepG 2); mouse mammary tumor cells (MMT 060562); TRI cells, as described, for example, in Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982); MRC5 cells and FS4 cells other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc.Natl.Acad.Sci.USA77:4216(1980)), and myeloma cell lines such as Y0, NS0 and Sp 2/0. reviews on certain mammalian host cell lines suitable for antibody production are provided, for example, Yazaki and Wu, Methods biological, Vol.B.K.C.255, Hu. 2003, Vol.J. (Prewa, Vol.S.248).

The host cell of the present invention as described above can be obtained by transformation or transfection with a recombinant vector. The host cell, after transformation or transfection with a nucleic acid molecule or recombinant expression vector of the invention as described above, constitutes an engineered cell or cell line, which can be used for the production of antibodies. Suitable transformation and transfection methods include, but are not limited to: for bacterial cells, such as calcium chloride, electroporation; for yeast cells, such as electroporation and protoplast fusion; for mammalian cells and the like, such as plastid encapsulation, calcium phosphate co-precipitation, electrofusion, and microinjection.

According to yet another aspect of the present invention, a method for preparing the bispecific antibody as described above, comprising the steps of:

(1) synthesizing DNA sequences shown in SEQ ID NO.19-35 and connecting the DNA sequences to a plasmid vector to construct a recombinant vector;

(2) introducing the recombinant vector obtained in the step (1) into a host cell for expression; preferably, the host cell is a host cell in which the reading frame of the Fut8 gene is subjected to frame shift mutation;

(3) and (5) purifying and assembling.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a bispecific antibody as hereinbefore described.

The pharmaceutical compositions of the present invention may also include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier and the bispecific antibody together constitute a pharmaceutical composition for more stable therapeutic effect, which can ensure the conformation integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while protecting the multifunctional group of the protein from degradation (including but not limited to aggregation, deamination or oxidation).

The term "pharmaceutically acceptable" refers to conditions approved by a regulatory agency of the federal or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Pharmaceutically acceptable carriers are generally non-toxic solid, semi-solid or liquid fillers, diluents, encapsulating materials or formulation aids of any type.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient, or carrier (vehicle) used to administer therapy. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier for intravenous administration of the pharmaceutical compositions. Saline solutions and aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose (malt), rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. If desired, the composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methylparaben, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, and tonicity adjusting agents such as sodium chloride or dextrose may also be used. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The composition can be formulated as a suppository, using conventional binders and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.

According to a further aspect of the invention, there is provided the use of a bispecific antibody as hereinbefore described in the manufacture of a medicament for the treatment of a tumour.

Tumors that can be treated with the antibodies of the invention include, but are not limited to, leukemias (including acute lymphoblastic leukemia (e.g., acute lymphoblastic leukemia, acute myelogenous leukemia (including granulocytic, promyelocytic, myelomonocytic, monocytic, erythroleukemia)) and chronic leukemias (e.g., chronic myelogenous and chronic lymphocytic leukemias)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovioma, lymphoblastoma, lymphoblastosis, and lymphoblastosis, Mesothelioma, ewing's tumor, leiomyosarcoma, rhabdomyoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, papillary cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, liver cancer, cholangiocellular carcinoma, chorioepithelioma, seminoma, embryonic carcinoma, wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, oligodendroglioma, acoustic neuroma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

The antibodies of the invention include modified antibodies, i.e., any type of molecule can be covalently attached to the antibody and the covalent attachment does not prevent the antibody from binding to an epitope. For example, but not limited to, the antibody may be modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a wide variety of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, tunicamycin metabolic synthesis, and the like. In addition, the antibody may comprise one or more non-canonical amino acids.

The antibodies of the invention may bind to a therapeutic agent, prodrug, peptide, protein, enzyme, virus, lipid, biological response modifier, agent, or PEG. The antibody may be linked or fused to a therapeutic agent, which may include a detectable label, such as a radioactive label, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent, which may be a drug or toxin, an ultrasound enhancing agent, a nonradioactive label, combinations thereof and other such art-known ingredients.

The antibody is detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-labeled antigen-binding polypeptide is then determined by detecting the luminescence generated during the course of the chemical reaction. Examples of chemiluminescent labeling compounds are luminol, isoluminol, thermonathic (theromatic) acridinium esters, imidazole, acridinium salts and oxalate esters. The antibody may also be labeled with a fluorescent luminescent metal, such as 152Eu, or other lanthanide. These metals can be attached to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for attaching various groups to antibodies are well known.

In a specific embodiment of the invention, the bispecific antibody of the invention as described above contains a core fucose type proportion of not more than 4.5%.

"EGFR", as used herein, is a 170kDa transmembrane receptor encoded by the c-erbB proto-oncogene and exhibits intrinsic tyrosine kinase activity. Isoforms and variants of EGFR also exist (e.g., variable RNA transcripts, truncated forms, polymorphisms, etc.), including but not limited to those identified by Swissprot database accession numbers P00533-1, P00533-2, P00533-3, and P00533-4. EGFR regulates many cellular processes via tyrosine kinase-mediated signal transduction pathways, including but not limited to activation of signal transduction pathways that control cell proliferation, differentiation, cell survival, programmed cell death, angiogenesis, mitogenesis, and metastasis.

Herein "MET (mesenchymal-epithelial transition factor)" is a protooncogene encoding the protein MET, (also known as c-Met; hepatocyte growth factor receptor HGFR; HGF receptor; scatter factor receptor; SF receptor). MET is a membrane receptor essential for embryonic development and wound healing. Hepatocyte Growth Factor (HGF) is the only known ligand for the MET receptor. MET is normally expressed by cells of epithelial origin, whereas HGF expression is restricted to cells of mesenchymal origin. Upon HGF stimulation, MET induces several biological responses that collectively produce a program known as invasive growth. Aberrant MET activation in cancer is associated with poor prognosis, where abnormally active MET triggers tumor growth, formation of new blood vessels that supply nutrients to the tumor (angiogenesis), and spread of the cancer to other organs (metastasis). MET is down-regulated in many types of human malignancies, including kidney, liver, stomach, breast and brain cancers. Typically, only stem and progenitor cells express MET, which allows these cells to grow invasively in order to produce new tissue in the embryo or to regenerate damaged tissue in adults. However, cancer stem cells are thought to hijack the ability of normal stem cells to express MET, and thus are responsible for the persistence and spread of cancer to other parts of the body.

The term "scFv" or "single-chain variable fragment" as used herein refers to the heavy chain (V) of an immunoglobulin_H) And light chain (V)_L) The variable region of (1). In certain aspects, the regions are linked with a short linker peptide of 10 to about 25 amino acids. The linker may be glycine rich for flexibility, serine or threonine for solubility, and capable of converting V_HIs linked to V at the N-terminus of_LAnd vice versa. The protein retains the original immunoglobulin properties except for the removal of the constant region and the introduction of a linker. ScFv molecules are known in the art and are described in U.S. patent No.5,892,019.

The term "heavy chain constant region" herein includes amino acid sequences from immunoglobulin heavy chains. A polypeptide comprising a heavy chain constant region comprises at least one of a CH1 domain, a hinge (e.g., upper hinge region, intermediate hinge region, and/or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. A polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain. A polypeptide chain having a CH1 domain and a CH3 domain. A polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain. Or a polypeptide chain having a CH1 domain, at least a portion of a hinge structure, a CH2 domain, and a CH3 domain. Or at least a portion of the hinge structure, the CH2 domain, and the CH3 domain. It will be appreciated by those of ordinary skill in the art that the heavy chain constant regions may be modified such that they differ in amino acid sequence from the naturally occurring immunoglobulin molecule.

The term "light chain constant region" herein includes amino acid sequences from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain and a constant lambda domain.

The term "light chain-heavy chain pair" herein refers to a collection of light and heavy chains that can form a dimer through disulfide bonding between the CL and CH1 domains of the light chain.

The term "CH 2 domain" as used herein includes a portion of a heavy chain molecule that ranges, for example, from about residue 244 to residue 360 of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al, U.S. department of health and public service, "Sequences of Proteins of immunological Interest" (1983). CH2 domain is unique in that it is not closely paired with another domain.

The term "treatment" herein refers to both therapeutic treatment and prophylactic or preventative measures, wherein the prevention or slowing (alleviation) of an undesirable physiological change or disease, such as the development of cancer, is performed in a subject. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (e.g., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "treatment" may also refer to an increase in survival compared to expected survival in the absence of treatment. Those conditions requiring treatment include those already with the disorder or symptom as well as those that are susceptible to or will prevent the disorder or symptom.

Antibody specificity refers to the selective recognition of a particular epitope of an antibody by an antigen. For example, natural antibodies are monospecific. A "bispecific antibody" according to the invention is an antibody having two different antigen binding specificities. If the antibody has more than one specificity, the recognized epitope may be associated with a single antigen or more than one antigen. The antibodies of the invention are specific for two different antigens, namely EGFR as the first antigen and MET as the second antigen.

The terms "knob" and hole (hole) are used herein to enhance the stability and therapeutic ability of antibodies, recombinant genetic engineering of heavy chains to promote their heterodimerization, i.e., disulfide bonds, salt bridges, knob-hole (holes). Pairing of two polypeptides is directed in vitro or in vivo by introducing a knob (knob) into one polypeptide at the interface where the two polypeptides interact and a cavity (hole) into the other polypeptide. For example, a knob-hole structure has been introduced into the Fc: Fc binding interface, the CL: CH1 interface, or the VH/VL interface of an antibody (e.g., US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al, (1997) protein science 6: 781-. The basis for the creation of knobs (knob) and holes (hole) at juxtaposed positions is that the interaction between the knobs and holes will contribute to the formation of heterodimers.

The term "EGFR" is used herein generically with "EGFR protein".

The term "MET" is used herein in general with "MET protein".

The invention has the advantages and beneficial effects that:

compared with the prior art, the bispecific antibody provided by the invention is more stable, easier to control and has better curative effect.

The bispecific antibody aiming at EGFR and MET improves ADCC activity by knocking out fucose, improves ADCC and prolongs half-life period by modifying an amino acid sequence.

Drawings

FIG. 1 shows a plasmid map of pcDNA3.1 vector;

figure 2 shows an ADCC activity assay for a bispecific antibody according to the invention;

FIG. 3 shows a graph of the tumor-inhibiting effect of the bispecific antibody of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 preparation of bispecific antibodies

1. Sequence design

The amino acid sequence composition and encoding nucleic acid sequence of the antibody prepared by the invention are shown in table 1.

TABLE 1 amino acid and nucleic acid sequences of bispecific antibodies

2. Expression of antibodies

2.1 Gene Synthesis and vector construction

Respectively synthesizing DNA sequences shown as SEQ ID NO.19-35, wherein the 5' end of the synthesized DNA sequence contains a Kozak sequence (CCCACCATGG) and a signal peptide (METDTLLLWVLLLWVPGSTG); and the 5 'end contains an EcoRI restriction site and the 3' end contains a HindIII restriction site. The synthesized DNA segment and pcDNA3.1(-) vector plasmid (FIG. 1) were digested with EcoRI and HindIII restriction enzymes, subjected to agarose gel electrophoresis and the corresponding digested gene fragments were recovered. Then the purified heavy chain and light chain coding DNA segments are connected to pcDNA3.1(-) expression vector recovered by cutting glue, transformed into Escherichia coli cells, selected clones are subjected to DNA sequencing, and corresponding plasmids are extracted from the clones confirmed to be correct by the sequencing.

2.2 transient transfection expression

Recombinant immunoglobulin variants were expressed by transient transfection of CHO-K1 cells. CHO-K1 cells in logarithmic growth phase at4X 10⁵Inoculating in shake flask at density of one ml, and placing at 37 deg.C and 5% CO₂After culturing for 24 hours at 125r/min by a shaker, transfection is carried out.

Adding 5mL of OPTI-MEM into 200 μ g of plasmid (the proportion of the plasmid is shown in Table 2) per 100mL of CHO-K1 cells, shaking, mixing uniformly, and incubating at room temperature for 5min to obtain a plasmid solution; another 5mL of OPTI-MEM was added to 600. mu.g of PEI,shaking, mixing uniformly and incubating for 5min at room temperature to obtain a PEI solution; mixing the plasmid and PEI solution, shaking, mixing, incubating at room temperature for 20min, adding dropwise the reaction mixture into cells, standing at 37 deg.C and 5% CO₂Culturing at 125r/min in a shaking table, feeding materials on days 2, 4 and 6, and harvesting supernatant on day 8. Cell culture supernatants containing the antibodies were harvested by centrifugation at 14000g for 30 minutes and filtered through sterile filters (0.22 μm) and the supernatants stored at-20 ℃ until purification.

2.3 Low fucose modified antibody expression

Sequence of gRNA targeting FUT8 gene, 3 grnas were designed with reference to the coding region of the full-length gene sequence (XM _003501735.2) of chinese hamster FUT8 published by the National Center for Biotechnology Information (NCBI): 5'-GGGACCTTATTGTTTTATAT-3' (SEQ ID NO.36), 5'-GTTTTATATAGGTGGTCATT-3' (SEQ ID NO.37) and 5'-GTGTACCATGTATTCCTCAA-3' (SEQ ID NO.38) were synthesized by Shanghai Biotechnology engineering services, Inc., and then inserted into sites recognized by the restriction enzyme BbsI of pSpCas9(BB) -2A-Puro (PX459) V2.0 vector, after sequencing verification, single clones were picked and cultured to extract plasmids. CHO-K1 cells in logarithmic growth phase at4X 10⁵Inoculating in shake flask at density of one ml, and placing at 37 deg.C and 5% CO₂After culturing for 24 hours at 125r/min by a shaker, transfection is carried out. Adding 500 mu L of OPTI-MEM into 20 mu g of plasmid per 10ml of CHO-K1 cells, shaking, mixing uniformly, and incubating at room temperature for 5min to obtain plasmid solution; adding 60 mu g of PEI into 500 mu L of OPTI-MEM, shaking, uniformly mixing, and incubating at room temperature for 5min to obtain a PEI solution; mixing the plasmid and PEI solution, shaking, mixing, incubating at room temperature for 20min, adding dropwise the reaction mixture into cells, standing at 37 deg.C and 5% CO₂Culturing at 125r/min in a shaking table, adding puromycin after 24hr, and screening for 14 days to obtain stable strain cells. After extracting genome DNA from the obtained stable strain, cloning the PCR amplified gene segment to a T vector and sequencing, and screening out a monoclonal cell strain with frame shift mutation of a Fut8 gene reading frame, wherein the monoclonal cell strain is recorded as CHO-KF.

CHO-KF cells in logarithmic growth phase at4X 10⁵Inoculating in shake flask at density of one ml, and placing at 37 deg.C and 5% CO₂After culturing for 24 hours at 125r/min by a shaker, transfection is carried out. Per 100ml CHO-KF fineAdding 5mL of OPTI-MEM into 200 μ g of plasmid (the proportion of the plasmid is shown in Table 2), shaking, mixing uniformly, and incubating at room temperature for 5min to obtain a plasmid solution; adding another 5mL of OPTI-MEM into 600 mu g of PEI, shaking, uniformly mixing, and incubating at room temperature for 5min to obtain a PEI solution; mixing the plasmid and PEI solution, shaking, mixing, incubating at room temperature for 20min, adding dropwise the reaction mixture into cells, standing at 37 deg.C and 5% CO₂Culturing at 125r/min in a shaking table, feeding materials on days 2, 4 and 6, and harvesting supernatant on day 8. Cell culture supernatants containing the antibodies were harvested by centrifugation at 14000g for 30 minutes and filtered through sterile filters (0.22 μm) and the supernatants stored at-20 ℃ until purification.

TABLE 2 transfection protocol

2.4 purification and Assembly

(1) Purification of

After the completion of the incubation, the supernatant was harvested, sterile-filtered, and applied to a Protein A affinity chromatography column (MabSelect Sure) equilibrated with PBS buffer (pH7.4), the column was further washed with 500mM phosphate buffer pH 6.0, and then eluted with citric acid buffer pH2.8 to obtain an antibody or a half-antibody, and the solution was adjusted to pH 5.8 with 1M Tris pH 8.5 and stored at 4 ℃.

(2) Assembly

Mixing the half antibodies according to the molar ratio of 1:1 of the pestle-mortar structure, adjusting the pH value of the citric acid buffer solution to 7.9-8.2, adding a proper amount of beta-mercaptoethanol reducing agent for reduction, stirring at a constant speed at room temperature for assembly for 8-12 hours, removing the reducing agent through ultrafiltration after the reduction is finished, and stopping the reaction.

Example 2 Mass Spectrometry identification of bispecific antibodies

The experimental method comprises the following steps:

respectively taking 400 mu g of each antibody, adding 10 mu L G7PNGaseF enzyme digestion buffer solution, 10 mu L of 10% NP40, 2.5 mu L PNGaseF and additionally ultrapure water, enabling the final volume to reach 100 mu L, uniformly mixing, wrapping the mixture by using a sealing film, and placing the mixture in a 37 ℃ water bath kettle for warm bath overnight. The N-sugar cleaved antibody samples were desalted through a C4 reverse phase chromatography column and analyzed by AB Sciex TripleTOF 5600(AB Sciex) mass spectrometry, and data were analyzed by Analyst TF software.

As a result:

the molecular weight of the purified and assembled antibody after N-glycosyl modification by glycosidase removal is shown in table 3, and the difference between the purified and assembled antibody and the theoretical value is within the error range of the instrument, which is shown in table 3, indicating that the antibody is successfully expressed, purified and assembled according to the design.

TABLE 3 Mass Spectrometry molecular weight determination

Example 3 determination of the purity of bispecific antibodies

(1) Size exclusion high performance liquid chromatography

At 200mM KH₂PO₄250mM KCl, pH 6.0,column temperature 25 ℃, sample size: the bispecific antibody and control antibody samples were analyzed for monomer purity and proportion of the oligomers by size exclusion high performance liquid chromatography at a flow rate of 0.6ml/min, run isocratic for 25 min.

(2) Sodium dodecyl sulfate capillary electrophoresis (CE-SDS)

And (3) quantitatively determining the purity of the recombinant monoclonal antibody product by adopting a sodium dodecyl sulfate capillary electrophoresis (CE-SDS) ultraviolet detection method according to the molecular weight under a non-reduction condition. A100. mu.g sample was added to 5. mu.l of a 0.8M aqueous iodoacetamide solution, vortexed, mixed, incubated at 70 ℃ for 5 minutes, cooled to room temperature, and centrifuged at 6000 rpm for 1 minute. From the sample tubes, 75. mu.l of each sample was taken out into a sample bottle, and immediately analyzed by adding to a capillary electrophoresis apparatus, and the bispecific antibody and control antibody samples were analyzed for monomer purity and half antibody ratio by sodium dodecyl sulfate capillary electrophoresis (CE-SDS).

(3) Results

The purity of the purified and assembled antibody determined by size exclusion high performance liquid chromatography and sodium dodecyl sulfate capillary electrophoresis (CE-SDS) is shown in Table 4 below, and it can be seen from Table 4 that the purity of the purified and assembled antibody is above 90%.

TABLE 4 determination of the purity of bispecific antibodies

Example 4 analysis of N-sugar modification of bispecific antibodies

1. Cleavage, recovery, and labeling of N-sugar chain

Antibody sugar chain cleavage: taking 100 mu g of antibody, adding 10 mu L G7PNGaseF enzyme digestion buffer solution, 10 mu L of 10% NP40, 1 mu L of PNGaseF and supplemented ultrapure water, enabling the final volume to reach 100 mu L, uniformly mixing, wrapping the mixture by using a sealing film, and putting the mixture in a water bath kettle at 37 ℃ for warm bath overnight.

Sugar chain recovery: adding the enzyme-digested sample into 300 mu L of anhydrous ethanol pre-cooled at-20 ℃, uniformly mixing, placing in a refrigerator at-20 ℃, standing for 30min, centrifuging at 12000rpm for 5min after antibody protein precipitation, and carefully sucking the supernatant to another new 0.5mL centrifuge tube by using a pipette. And (4) putting the sugar liquid obtained in the previous step into a vacuum centrifugal concentrator, concentrating and drying at 38 ℃ until no liquid exists.

Sugar chain labeling: 3 mu L of 2-AB labeling reagent and 2 mu L of NaBH3CN 2 (prozyme) are added into each tube, the mixture is uniformly mixed by a gun and is placed into a water bath kettle at 65 ℃ for derivatization reaction for 3 hours.

Sugar chain purification: the derivatized sample was added to 100. mu.L acetonitrile, blown with a gun and mixed well, and left for 10 min. The HILIC purification tips (Thermo) of (2) were blasted in the activation solution with a gun, and the tips were emptied. Slowly blowing the mixture for 5min at constant speed in a sample containing acetonitrile to bond sugar chains to a HILIC purification gun head, and finally emptying the HILIC purification gun head. The sugar chain was purified and recovered into 30. mu.L of purified water by slowly tapping in 200. mu.L of 95% acetonitrile, emptying the tip, and slowly tapping the tip in 30. mu.L of ultrapure water for 5 min.

2. HILIC-UPLC analysis

The instrument comprises the following steps: a WATER ACQUITY UPLC; a chromatographic column: water Acquity UPLC BEH amide1.7 μm,2.1 × 100mm Column; flow rate: 0.4 mL/min; column temperature: 40 ℃; and (3) detecting fluorescence: lambda [ alpha ]_ex＝330nm；λ_em420 nm; sample introduction amount: 2 mu L of the solution; the elution gradient is shown in table 5.

TABLE 5 elution gradient

(3) The results of N-glycoform analysis of the purified and assembled antibody are shown in Table 6, and it can be seen from Table 6 that the ratio of the core fucose glycoform contained in the antibody expressed by CHO-KF cells is not higher than 4.5%.

TABLE 6 core fucose-containing ratio of bispecific antibodies

Example 5 affinity analysis of bispecific antibodies with the antigen EGFR or MET

(1) Measurement method

Surface plasmon resonance biosensors are used to measure the binding kinetics and affinity of antibodies to EGFR and MET antigens. Unless otherwise indicated, all reagents and materials were purchased from GE corporation and measurements were made at 25 ℃. Affinity analysis was performed by SPR (Biacore X100) instrument, coupling anti-human IgG Fc antibody to CM5 chip by amino coupling, flowing each antibody to be tested at a flow rate of 10 μ l/min, and capturing the antibody to be tested with the anti-human IgG Fc antibody coupled to the chip; after dilution of the analyte (EGFR or MET ECD) in a gradient (200nM, 100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM, 1.56nM, 0.78nM, 0.39nM and 0nM), the antibody to be tested was flowed in at a flow rate of 30. mu.l/min for a binding time of 200s with the analyte (EGFR or MET ECD) and a dissociation time of 1500 s; HBS-EP was used as the running buffer throughout the experiment and the chip was regenerated with a 60 second pulse of 10mM glycine HCl, pH 2.0. The data were fit to a 1:1 binding model to determine the association rate Ka, dissociation rate Kd, and equilibrium dissociation constant Kd.

(2) Results

The results of the measurements of the binding rate Ka, dissociation rate Kd, and equilibrium dissociation constant Kd of the purified and assembled antibodies to EGFR or MET are shown in table 7, and it can be seen that each antibody has strong affinity to EGFR or MET; after the sequence of the anti-EGFR gene is mutated at 1 or 3 variable region sites, the affinity is not obviously changed.

TABLE 7 affinity analysis of bispecific antibodies to EGFR or MET

Example 6 bispecific antibody and Fc receptor CD16a (Fc gamma RIIIa,176V) affinity determination (1) determination method

Surface plasmon resonance biosensors were used to measure the affinity of antibodies to the Fc receptor CD16a (Fc γ RIIIa, 176V).

Affinity analysis was performed on SPR (Biacore X100) instrument by coupling anti-His antibody to CM5 chip by amino coupling, flowing His-tagged CD16a (176V) at 10 μ l/min, and capturing CD16a (176V) with anti-His antibody coupled to the chip; after the antibody to be tested is diluted in a gradient way (720nM, 240nM, 80nM, 26.7nM, 8.89nM, 2.96nM, 0.99nM and 0nM), the antibody to be tested flows in at the flow rate of 30 μ l/min, the binding time of the antibody to be tested and CD16a (176V) is 200s, and the dissociation time is 200 s; HBS-EP is used as an operation buffer solution in the whole experiment, a regeneration chip is regenerated by using 60-second pulses of 10mM glycine HCl and pH 2.0 solution, and a graph is fitted in a steady-state mode by using an affinity analysis method to obtain an affinity value.

(2) Results

The affinity KD determination results of the purified and assembled antibody with CD16a (176V) are shown in table 8, and it can be seen from table 8 that the affinity of the antibody with CD16a (176V) is significantly enhanced after reducing the core fucose or DE mutation.

TABLE 8 affinity analysis of bispecific antibodies with CD16a (176V)

Example 7 affinity assay for FcRn (1) assay method

The affinity of the antibody for the Fc receptor FcRn was measured using a surface plasmon resonance biosensor.

Affinity analysis was performed on SPR (Biacore X100) instrument by coupling anti-His antibody to CM5 chip by amino coupling, flowing His-tagged FcRn at 10 μ l/min and capturing FcRn with anti-His antibody coupled to the chip; after the antibody to be detected is diluted in a gradient way (720nM, 240nM, 80nM, 26.7nM, 8.89nM, 2.96nM, 0.99nM and 0nM), the antibody to be detected flows in at a flow rate of 30 μ l/min, the binding time of the antibody to be detected and FcRn is 200s, and the dissociation time is 200 s; HBS-EP is used as an operation buffer solution in the whole experiment, a regeneration chip is regenerated by using 60-second pulses of 10mM glycine HCl and pH 2.0 solution, and a graph is fitted in a steady-state mode by using an affinity analysis method to obtain an affinity value.

(2) Results

The affinity KD determination results of the purified and assembled antibody to FcRn are shown in table 9, and it can be seen that YTE mutations significantly enhance the affinity to FcRn.

TABLE 9 affinity assay for bispecific antibodies to FcRn

Example 8 ADCC Activity assay of bispecific antibodies

(1) The method comprises the following steps:

stomach cancer SNU-5(

CRL-5973^TM) Target cells and effector cells NK92MI-CD16a were centrifuged at 1000rpm for 5min and resuspended, then added to a 96-well plate at an effective target ratio of 10:1, and the antibodies (1X 10) were added in different concentrations by gradient dilution (1X 10)^-7～-10nM) and incubated at 37 ℃ for 8 hours, added with LDH developing solution, placed at room temperature in the dark for 20 minutes, and detected by an enzyme-linked immunosorbent assay.

(2) Results

The ADCC assay results of the purified and assembled antibody are shown in table 10 and fig. 2, and it can be seen from table 10 and fig. 2 that the ADCC activity of the antibody is significantly enhanced after reducing the core fucose or the DE mutation.

TABLE 10 ADCC Activity assay of bispecific antibodies

Example 9 therapeutic Effect of bispecific antibodies on tumors

The tumor-inhibiting experiment in nude mice was used to study the therapeutic effect of bispecific antibody on tumor.

(1) The method comprises the following steps:

a tumor transplantation model using CR17/SCID mice (24-28 g) as hosts. Mice were inoculated subcutaneously with SNU-5 gastric cancer cells (5X 10) expressing human HGF⁶Cells) until the volume of the subcutaneous transplanted tumor reaches 100-300 mm³At times, the groups were randomized according to tumor volume. Blank control (PBS), C1+ C2, EXM2, EXM4, EXM4-KF, EXM4-DE, EXM4-YTE-KF were each administered intraperitoneally at a schedule of C1+ C2 (5 mg/kg each), EXM2, EXM4, EXM4-KF, EXM4-DE, EXM4-YTE-KF of 10mg/kg, respectively, 1 time per week for 4 times. Animals were sacrificed by cervical dislocation at the end of the experiment by measuring tumor volume 2 times per week, and tumors visible to the naked eye were peeled off and weighed to record data.

(2) Results

The results are shown in fig. 3, where the tumor volume increased rapidly in PBS mice; the C1+ C2 (5 mg/kg each) group showed some inhibition of tumor growth, but the tumors grew gradually with time; the EXM2 and EXM4 groups have stronger tumor inhibition capability than the C1+ C2 groups, but the tumors still partially grow; the three groups of EXM4-KF, EXM4-DE and EXM4-YTE-KF have almost no tumor growth and the best tumor inhibiting effect.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Sequence listing

<110> Beijing Kogaku (Biotechnology Co., Ltd.)

<120> bispecific antibody against EGFR protein and MET protein

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

35 40 45

Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe

50 55 60

Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala 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 Thr Tyr Arg Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

130 135 140

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ser Ser

145 150 155 160

Gln Ser Leu Leu Tyr Thr Ser Ser Gln Lys Asn Tyr Leu Ala Trp Tyr

165 170 175

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

180 185 190

Thr Arg Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Val Glu Ile Lys

245

<210> 9

<211> 107

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<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> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 10

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 11

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 12

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 12

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 13

<211> 227

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<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 Glu Glu Met 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> 14

<211> 592

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 14

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

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

325 330 335

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

340 345 350

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

355 360 365

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

370 375 380

Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Met Ile Asp Pro Ser Asn

385 390 395 400

Ser Asp Thr Arg Phe Asn Pro Asn Phe Lys Asp Arg Phe Thr Ile Ser

405 410 415

Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg

420 425 430

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Thr Tyr Arg Ser Tyr Val

435 440 445

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

450 455 460

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

465 470 475 480

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

485 490 495

Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Thr Ser

500 505 510

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

515 520 525

Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro

530 535 540

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

545 550 555 560

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr

565 570 575

Tyr Ala Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

580 585 590

<210> 15

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 15

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 16

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 16

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Asp Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Glu Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 17

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 17

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 18

<211> 330

<212> PRT

<213> Artificial Synthesis (Artificial Sequence)

<400> 18

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 Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

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

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 19

<211> 1428

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 19

gaattcccca ccatggagac cgacaccctg ctgctgtggg tgctgctgct gtgggtgccc 60

ggctccaccg gccaggtgca gctgaagcag tccggccccg gcctggtgca gccctcccag 120

tccctgtcca tcacctgcac cgtgtccggc ttctccctga ccaactacgg cgtgcactgg 180

gtgaggcagt cccccggcaa gggcctggag tggctgggcg tgatctggtc cggcggcaac 240

accgactaca acaccccctt cacctccagg ctgtccatca acaaggacaa ctccaagtcc 300

caggtgttct tcaagatgaa ctccctgcag tccaacgaca ccgccatcta ctactgcgcc 360

agggccctga cctactacga ctacgagttc gcctactggg gccagggcac cctggtgacc 420

gtgtccgccg cctccaccaa gggcccctcc gtgttccccc tggccccctc ctccaagtcc 480

acctccggcg gcaccgccgc cctgggctgc ctggtgaagg actacttccc cgagcccgtg 540

accgtgtcct ggaactccgg cgccctgacc tccggcgtgc acaccttccc cgccgtgctg 600

cagtcctccg gcctgtactc cctgtcctcc gtggtgaccg tgccctcctc ctccctgggc 660

acccagacct acatctgcaa cgtgaaccac aagccctcca acaccaaggt ggacaagaag 720

gtggagccca agtcctgcga caagacccac acctgccccc cctgccccgc ccccgagctg 780

ctgggcggcc cctccgtgtt cctgttcccc cccaagccca aggacaccct gatgatctcc 840

aggacccccg aggtgacctg cgtggtggtg gacgtgtccc acgaggaccc cgaggtgaag 900

ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc cagggaggag 960

cagtacaact ccacctacag ggtggtgtcc gtgctgaccg tgctgcacca ggactggctg 1020

aacggcaagg agtacaagtg caaggtgtcc aacaaggccc tgcccgcccc catcgagaag 1080

accatctcca aggccaaggg ccagcccagg gagccccagg tgtacaccct gcccccctcc 1140

agggaggaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1200

tccgacatcg ccgtggagtg ggagtccaac ggccagcccg agaacaacta caagaccacc 1260

ccccccgtgc tggactccga cggctccttc ttcctgtact ccaagctgac cgtggacaag 1320

tccaggtggc agcagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1380

cactacaccc agaagtccct gtccctgtcc cccggcaagt aaaagctt 1428

<210> 20

<211> 724

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 20

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgacatcc tgctgaccca gtcccccgtg atcctgtccg tgtcccccgg 120

cgagagggtg tccttctcct gcagggcctc ccagtccatc ggcaccaaca tccactggta 180

ccagcagagg accaacggct cccccaggct gctgatcaag tacgcctccg agtccatctc 240

cggcatcccc tccaggttct ccggctccgg ctccggcacc gacttcaccc tgtccatcaa 300

ctccgtggag tccgaggaca tcgccgacta ctactgccag cagaacaaca actggcccac 360

caccttcggc gccggcacca agctggagct gaagaggacc gtggccgccc cctccgtgtt 420

catcttcccc ccctccgacg agcagctgaa gtccggcacc gcctccgtgg tgtgcctgct 480

gaacaacttc taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc 540

cggcaactcc caggagtccg tgaccgagca ggactccaag gactccacct actccctgtc 600

ctccaccctg accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt 660

gacccaccag ggcctgtcct cccccgtgac caagtccttc aacaggggcg agtgctaaaa 720

gctt 724

<210> 21

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 21

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctggtgga gtccggcggc ggcctggtgc agcccggcgg 120

ctccctgagg ctgtcctgcg ccgcctccgg ctacaccttc acctcctact ggctgcactg 180

ggtgaggcag gcccccggca agggcctgga gtgggtgggc atgatcgacc cctccaactc 240

cgacaccagg ttcaacccca acttcaagga caggttcacc atctccgccg acacctccaa 300

gaacaccgcc tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg 360

cgccacctac aggtcctacg tgacccccct ggactactgg ggccagggca ccctggtgac 420

cgtgtcctcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 22

<211> 763

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 22

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgacaaga cccacacctg ccccccctgc cccgcccccg agctgctggg 120

cggcccctcc gtgttcctgt tcccccccaa gcccaaggac accctgatga tctccaggac 180

ccccgaggtg acctgcgtgg tggtggacgt gtcccacgag gaccccgagg tgaagttcaa 240

ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc aagcccaggg aggagcagta 300

caactccacc tacagggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg 360

caaggagtac aagtgcaagg tgtccaacaa ggccctgccc gcccccatcg agaagaccat 420

ctccaaggcc aagggccagc ccagggagcc ccaggtgtac accctgcccc cctccaggga 480

ggagatgacc aagaaccagg tgtccctgtg gtgcctggtg aagggcttct acccctccga 540

catcgccgtg gagtgggagt ccaacggcca gcccgagaac aactacaaga ccaccccccc 600

cgtgctggac tccgacggct ccttcttcct gtactccaag ctgaccgtgg acaagtccag 660

gtggcagcag ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta 720

cacccagaag tccctgtccc tgtcccccgg caagtaaaag ctt 763

<210> 23

<211> 742

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 23

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgacatcc agatgaccca gtccccctcc tccctgtccg cctccgtggg 120

cgacagggtg accatcacct gcaagtcctc ccagtccctg ctgtacacct cctcccagaa 180

gaactacctg gcctggtacc agcagaagcc cggcaaggcc cccaagctgc tgatctactg 240

ggcctccacc agggagtccg gcgtgccctc caggttctcc ggctccggct ccggcaccga 300

cttcaccctg accatctcct ccctgcagcc cgaggacttc gccacctact actgccagca 360

gtactacgcc tacccctgga ccttcggcca gggcaccaag gtggagatca agaggaccgt 420

ggccgccccc tccgtgttca tcttcccccc ctccgacgag cagctgaagt ccggcaccgc 480

ctccgtggtg tgcctgctga acaacttcta ccccagggag gccaaggtgc agtggaaggt 540

ggacaacgcc ctgcagtccg gcaactccca ggagtccgtg accgagcagg actccaagga 600

ctccacctac tccctgtcct ccaccctgac cctgtccaag gccgactacg agaagcacaa 660

ggtgtacgcc tgcgaggtga cccaccaggg cctgtcctcc cccgtgacca agtccttcaa 720

caggggcgag tgctaaaagc tt 742

<210> 24

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 24

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggccaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtccaacgac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 25

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 25

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggccaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtccaacgac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 26

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 26

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctggtgga gtccggcggc ggcctggtgc agcccggcgg 120

ctccctgagg ctgtcctgcg ccgcctccgg ctacaccttc acctcctact ggctgcactg 180

ggtgaggcag gcccccggca agggcctgga gtgggtgggc atgatcgacc cctccaactc 240

cgacaccagg ttcaacccca acttcaagga caggttcacc atctccgccg acacctccaa 300

gaacaccgcc tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg 360

cgccacctac aggtcctacg tgacccccct ggactactgg ggccagggca ccctggtgac 420

cgtgtcctcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 27

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 27

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggccaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtcccaggac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 28

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 28

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtcccaggac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 29

<211> 724

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 29

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgacatcc tgctgaccca gtcccccgtg atcctgtccg tgtcccccgg 120

cgagagggtg tccttctcct gcagggcctc ccagtccatc ggcaccaaca tccactggta 180

ccagcagagg acccagggct cccccaggct gctgatcaag tacgcctccg agtccatctc 240

cggcatcccc tccaggttct ccggctccgg ctccggcacc gacttcaccc tgtccatcaa 300

ctccgtggag tccgaggaca tcgccgacta ctactgccag cagaacaaca actggcccac 360

caccttcggc gccggcacca agctggagct gaagaggacc gtggccgccc cctccgtgtt 420

catcttcccc ccctccgacg agcagctgaa gtccggcacc gcctccgtgg tgtgcctgct 480

gaacaacttc taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagtc 540

cggcaactcc caggagtccg tgaccgagca ggactccaag gactccacct actccctgtc 600

ctccaccctg accctgtcca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt 660

gacccaccag ggcctgtcct cccccgtgac caagtccttc aacaggggcg agtgctaaaa 720

gctt 724

<210> 30

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 30

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtcccaggac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc cccgacgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccgaggagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 31

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 31

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctggtgga gtccggcggc ggcctggtgc agcccggcgg 120

ctccctgagg ctgtcctgcg ccgcctccgg ctacaccttc acctcctact ggctgcactg 180

ggtgaggcag gcccccggca agggcctgga gtgggtgggc atgatcgacc cctccaactc 240

cgacaccagg ttcaacccca acttcaagga caggttcacc atctccgccg acacctccaa 300

gaacaccgcc tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg 360

cgccacctac aggtcctacg tgacccccct ggactactgg ggccagggca ccctggtgac 420

cgtgtcctcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc cccgacgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccgaggagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 32

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 32

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtcccaggac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgtacatcac 840

cagggagccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtcctgc gccgtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctggtg tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 33

<211> 1429

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 33

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctggtgga gtccggcggc ggcctggtgc agcccggcgg 120

ctccctgagg ctgtcctgcg ccgcctccgg ctacaccttc acctcctact ggctgcactg 180

ggtgaggcag gcccccggca agggcctgga gtgggtgggc atgatcgacc cctccaactc 240

cgacaccagg ttcaacccca acttcaagga caggttcacc atctccgccg acacctccaa 300

gaacaccgcc tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg 360

cgccacctac aggtcctacg tgacccccct ggactactgg ggccagggca ccctggtgac 420

cgtgtcctcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgtacatcac 840

cagggagccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag taaaagctt 1429

<210> 34

<211> 1504

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 34

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctggtgga gtccggcggc ggcctggtgc agcccggcgg 120

ctccctgagg ctgtcctgcg ccgcctccgg ctacaccttc acctcctact ggctgcactg 180

ggtgaggcag gcccccggca agggcctgga gtgggtgggc atgatcgacc cctccaactc 240

cgacaccagg ttcaacccca acttcaagga caggttcacc atctccgccg acacctccaa 300

gaacaccgcc tacctgcaga tgaactccct gagggccgag gacaccgccg tgtactactg 360

cgccacctac aggtcctacg tgacccccct ggactactgg ggccagggca ccctggtgac 420

cgtgtcctcc ggcggcggcg gctccggcgg cggcggctcc ggcggcggcg gctccgacat 480

ccagatgacc cagtccccct cctccctgtc cgcctccgtg ggcgacaggg tgaccatcac 540

ctgcaagtcc tcccagtccc tgctgtacac ctcctcccag aagaactacc tggcctggta 600

ccagcagaag cccggcaagg cccccaagct gctgatctac tgggcctcca ccagggagtc 660

cggcgtgccc tccaggttct ccggctccgg ctccggcacc gacttcaccc tgaccatctc 720

ctccctgcag cccgaggact tcgccaccta ctactgccag cagtactacg cctacccctg 780

gaccttcggc cagggcacca aggtggagat caaggacaag acccacacct gccccccctg 840

ccccgccccc gagctgctgg gcggcccctc cgtgttcctg ttccccccca agcccaagga 900

caccctgatg atctccagga cccccgaggt gacctgcgtg gtggtggacg tgtcccacga 960

ggaccccgag gtgaagttca actggtacgt ggacggcgtg gaggtgcaca acgccaagac 1020

caagcccagg gaggagcagt acaactccac ctacagggtg gtgtccgtgc tgaccgtgct 1080

gcaccaggac tggctgaacg gcaaggagta caagtgcaag gtgtccaaca aggccctgcc 1140

cgcccccatc gagaagacca tctccaaggc caagggccag cccagggagc cccaggtgta 1200

caccctgccc ccctccaggg aggagatgac caagaaccag gtgtccctgt ggtgcctggt 1260

gaagggcttc tacccctccg acatcgccgt ggagtgggag tccaacggcc agcccgagaa 1320

caactacaag accacccccc ccgtgctgga ctccgacggc tccttcttcc tgtactccaa 1380

gctgaccgtg gacaagtcca ggtggcagca gggcaacgtg ttctcctgct ccgtgatgca 1440

cgaggccctg cacaaccact acacccagaa gtccctgtcc ctgtcccccg gcaagtaaaa 1500

gctt 1504

<210> 35

<211> 2215

<212> DNA

<213> Artificial Synthesis (Artificial Sequence)

<400> 35

gaattcccca ccaatggaga ccgacaccct gctgctgtgg gtgctgctgc tgtgggtgcc 60

cggctccacc ggcgaggtgc agctgaagca gtccggcccc ggcctggtgc agccctccca 120

gtccctgtcc atcacctgca ccgtgtccgg cttctccctg accaactacg gcgtgcactg 180

ggtgaggcag tcccccggca agggcctgga gtggctgggc gtgatctggt ccggcggcaa 240

caccgactac aacaccccct tcacctccag gctgtccatc aacaaggaca actccaagtc 300

ccaggtgttc ttcaagatga actccctgca gtcccaggac accgccatct actactgcgc 360

cagggccctg acctactacg actacgagtt cgcctactgg ggccagggca ccctggtgac 420

cgtgtccgcc gcctccacca agggcccctc cgtgttcccc ctggccccct cctccaagtc 480

cacctccggc ggcaccgccg ccctgggctg cctggtgaag gactacttcc ccgagcccgt 540

gaccgtgtcc tggaactccg gcgccctgac ctccggcgtg cacaccttcc ccgccgtgct 600

gcagtcctcc ggcctgtact ccctgtcctc cgtggtgacc gtgccctcct cctccctggg 660

cacccagacc tacatctgca acgtgaacca caagccctcc aacaccaagg tggacaagaa 720

ggtggagccc aagtcctgcg acaagaccca cacctgcccc ccctgccccg cccccgagct 780

gctgggcggc ccctccgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatctc 840

caggaccccc gaggtgacct gcgtggtggt ggacgtgtcc cacgaggacc ccgaggtgaa 900

gttcaactgg tacgtggacg gcgtggaggt gcacaacgcc aagaccaagc ccagggagga 960

gcagtacaac tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct 1020

gaacggcaag gagtacaagt gcaaggtgtc caacaaggcc ctgcccgccc ccatcgagaa 1080

gaccatctcc aaggccaagg gccagcccag ggagccccag gtgtacaccc tgcccccctc 1140

cagggaggag atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc 1200

ctccgacatc gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac 1260

cccccccgtg ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa 1320

gtccaggtgg cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa 1380

ccactacacc cagaagtccc tgtccctgtc ccccggcaag ggcggcggcg gctccggcgg 1440

cggcggctcc ggcggcggcg gctccgaggt gcagctggtg gagtccggcg gcggcctggt 1500

gcagcccggc ggctccctga ggctgtcctg cgccgcctcc ggctacacct tcacctccta 1560

ctggctgcac tgggtgaggc aggcccccgg caagggcctg gagtgggtgg gcatgatcga 1620

cccctccaac tccgacacca ggttcaaccc caacttcaag gacaggttca ccatctccgc 1680

cgacacctcc aagaacaccg cctacctgca gatgaactcc ctgagggccg aggacaccgc 1740

cgtgtactac tgcgccacct acaggtccta cgtgaccccc ctggactact ggggccaggg 1800

caccctggtg accgtgtcct ccggcggcgg cggctccggc ggcggcggct ccggcggcgg 1860

cggctccgac atccagatga cccagtcccc ctcctccctg tccgcctccg tgggcgacag 1920

ggtgaccatc acctgcaagt cctcccagtc cctgctgtac acctcctccc agaagaacta 1980

cctggcctgg taccagcaga agcccggcaa ggcccccaag ctgctgatct actgggcctc 2040

caccagggag tccggcgtgc cctccaggtt ctccggctcc ggctccggca ccgacttcac 2100

cctgaccatc tcctccctgc agcccgagga cttcgccacc tactactgcc agcagtacta 2160

cgcctacccc tggaccttcg gccagggcac caaggtggag atcaagtaaa agctt 2215

<210> 36

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gggaccttat tgttttatat 20

<210> 37

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

gttttatata ggtggtcatt 20

<210> 38

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gtgtaccatg tattcctcaa 20

Claims (9)

1. A bispecific antibody comprising a light-heavy chain pair that specifically binds EGFR, and a light-heavy chain pair that specifically binds MET; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 3; the amino acid sequence of the light chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID NO.7, and the amino acid sequence of the heavy chain variable region in the light-heavy chain pair specifically binding to MET is shown as SEQ ID NO. 6;

the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to EGFR is selected from the amino acid sequences shown in SEQ ID NO.12, SEQ ID NO.15 and SEQ ID NO. 17; the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to EGFR is shown as SEQ ID NO. 9;

the amino acid sequence of the heavy chain constant region in the light-heavy chain pair specifically binding to MET is selected from the amino acid sequences shown in SEQ ID NO.11, SEQ ID NO.16 and SEQ ID NO. 18; the amino acid sequence of the light chain constant region in the light-heavy chain pair specifically binding to MET is shown in SEQ ID NO. 9.

2. The bispecific antibody of claim 1, wherein the bispecific antibody comprises a core fucose type ratio of not more than 4.5%.

3. A nucleic acid molecule encoding the bispecific antibody of claim 1, the nucleic acid molecule having the sequence shown in SEQ ID No.23, SEQ ID No.26, SEQ ID No.28, SEQ ID No.29, SEQ ID No.30, SEQ ID No.31, SEQ ID No. 32.

4. A recombinant vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the recombinant vector of claim 4.

6. A method of making a bispecific antibody of claim 1, comprising the steps of:

(1) synthesizing DNA sequences shown in SEQ ID NO.23, SEQ ID NO.26, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31 and SEQ ID NO.32 and connecting the DNA sequences to a plasmid vector to construct a recombinant vector;

(2) introducing the recombinant vector obtained in the step (1) into a host cell for expression;

(3) and (5) purifying and assembling.

7. The method of claim 6, wherein the host cell is a host cell having a frame-shift mutation in the reading frame of the Fut8 gene.

8. A pharmaceutical composition comprising the bispecific antibody of claim 1.

9. Use of a bispecific antibody of claim 1 for the preparation of a medicament for the treatment of a tumor.

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