KR20150055676A - Method for preparing the plant producing bispecific antibody - Google Patents

Abstract

The present invention relates to a method for preparing plants capable of producing bispecific antibodies. More specifically, the method includes the steps of: (a) forming a single chain antibody by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of an monoclonal antibody by a linker; (b) preparing a transgenic plant by introducing the genes of the single chain antibody formed in step (a); (c) forming another single chain antibody by connecting the heavy chain variable region (VH) and the light chain variable region (VL) of another monoclonal antibody by the linker; (d) preparing another transgenic plant by introducing the genes of the single chain antibody formed in step (c); and (e) breeding the transgenic plants prepared in the step (b) and step (d). The present invention also relates to an antibody preparation method including a step of obtaining proteins from the transgenic plants prepared by the previous preparation method. The present invention can mass-produce the bispecific antibodies by using the preparation method and mass-produce the low-cost monoclonal antibodies.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing a bispecific antibody,

The present invention relates to a method for producing a plant producing a bispecific antibody, and more particularly, to a method for producing a bispecific antibody, which comprises (a) linking a heavy chain variable region (VH) of a monoclonal antibody and a light chain variable region (B) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant; (c) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody different from the monoclonal antibody of the step (a) by a linker to form a single chain antibody; ) Introducing the gene of the single chain antibody prepared in the step (c) into a plant cell to obtain a transformed plant; And (e) crossing the transformed plant individuals produced in steps (b) and (d) above, and a method for producing a transgenic plant producing the bispecific antibody, To obtain a protein from the transformed plant.

A monoclonal antibody (mAb) is called a 'guided missile' because it has two roles of recruiting specific cells in the body and killing cancer cells. These monoclonal antibodies are immunotherapeutic proteins that have been recently studied as one of chemotherapy and radiotherapy as well as chemotherapeutic agents that are used in combination with cancer patients. The production of monoclonal antibodies is mainly produced by using hybridoma cell lines or Chinese-hamster ovary (CHO) cells using animal cells. However, since the present production technology or ability does not satisfy the increasing demand of single antibody, The phenomenon is expected to happen. In 2015, there will be around $ 30 billion worldwide of therapeutic and diagnostic monoclonal antibody markets.

Antibodies are glycoproteins that can affect the biological activity, blood clearance rate, and anti-tumor activity of the antibody, because the elements, structure or location of glycosylation can affect the anticancer activity glycosylation machinery is different from human or animal glycosylation pattern. On the other hand, plants have a glycosylation machinery similar to animals and have a subcellular compartment called endoplasmic reticulum (ER) capable of folding / assembling antibody domains, making it possible to use the plant as an antibody protein production plant . Therefore, one of the research fields that have recently attracted worldwide attention is the development of inexpensive therapeutic antibody production using biotechnology.

The inventors of the present invention have been studying a method for producing a high-efficiency plant conjugate antibody, confirming that a bispecific antibody is produced in a plant produced by crossing transformed plants to produce a specific monoclonal antibody, respectively Thus completing the present invention.

Accordingly, an object of the present invention is to provide a method for preparing a monoclonal antibody, which comprises: (a) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody to a single chain antibody by linking;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant; And

and (e) crossing the transformed plant individuals produced in steps (b) and (d) above, to produce a bispecific antibody.

Another object of the present invention is to provide a method for preparing a monoclonal antibody, comprising: (a) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody to a single chain antibody by linking;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;

(e) crossing the transgenic plants produced in steps (b) and (d) above; And

(f) obtaining a protein from the crossed plant produced in the step (e).

In order to accomplish the above object, the present invention provides a method for producing a monoclonal antibody, comprising the steps of (a) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker to form a single chain antibody ;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant; And

and (e) crossing the transgenic plants produced in the steps (b) and (d). The present invention also provides a method for producing a bispecific antibody.

(A) a single chain antibody is prepared by linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker; Creating;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;

(e) crossing the transgenic plants produced in steps (b) and (d) above; And

(f) obtaining a protein from the crossed plant produced in the step (e).

Hereinafter, the present invention will be described in detail.

(A) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker to form a single chain antibody;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant; And

and (e) crossing the transgenic plants produced in the steps (b) and (d). The present invention also provides a method for producing a bispecific antibody.

The monoclonal antibody refers to a protein molecule that is directed against and specifically binds to a single antigenic site (single epitope). The monoclonal antibody can be made by a fusion method well known in the art (Kohler et al., European Journal of Immunology 6; 511-519). Generally, hybridoma cells that secrete monoclonal antibodies are made by fusing immune cells from cancer cells with immunologically appropriate host animals, such as mice injected with antigen proteins. The cell fusion of these two groups is fused using a method known in the art such as polyethylene glycol, and the antibody producing cells are proliferated by a standard culture method. After subcloning by limited dilution to obtain a uniform cell population, hybridoma cells capable of producing an antigen-specific antibody are cultured in vitro or in vivo.

Natural antibodies produced in vivo are typically about 150,000 daltons, heterotetrameric glycoproteins, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by one covalent disulfide bond, but the disulfide chain number varies between the heavy chains of the different immunoglobulin isoforms. Each heavy and light chain also has regularly spaced intra-chain disulfide bridges. Each heavy chain has a variable domain (VH) followed by a number of constant domains at one end. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; The constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that a particular amino acid residue forms an interface between the light chain variable domain and the heavy chain variable domain.

"Variable domain" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable region of the heavy chain is referred to as "VH" or "V _H ", and the variable region of the light chain is referred to as "VL" or "V _L ". These domains are generally the most variable part of the antibody and comprise an antigen binding site.

The whole antibody form of the monoclonal antibody is composed of two pairs of a light chain (LC) and a heavy chain (HC). The light and heavy chains of such antibodies are polypeptides composed of several domains. In whole antibodies, each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region comprises a heavy chain constant domain CH1, CH2 and CH3 (antibody classes IgA, IgD and IgG) and optionally a heavy chain constant domain CH4 (antibody classes IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The variable domains VH and VL can be subdivided into more conserved, hypervariable regions called complementarity determining regions (CDRs) scattered within sites called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged at the amino-terminus carboxy-terminal (Janeway, CA, Jr. et al. (2001) Immunobiology., 5th ed., Garland Publishing; and Woof, J., Burton, D., Nat Rev Immunol 4 (2004) 89-99).

In step (a), the whole antibody is in the form of two pairs of heavy chain and light chain, and the heavy chain and the light chain are connected to each other through a single chain. This step is a process of transforming into a plant-specific sequence to facilitate expression in the plant. Generally, in order to express the whole type of monoclonal antibody, it is necessary to use a binary expression vector having two promoters in order to express each of a heavy chain and a light chain. This has some limitations in the selection of expression vectors (limitations of the final clinical trial with antibiotic marker selection, number of available vectors, etc.). However, by carrying out this step, it is possible to express it in a single promoter.

The method of 'linking with one chain' is not limited as long as the method of linking the heavy chain and the light chain of the antibody by a single chain. For example, when the entire heavy chain and the light chain are linked to the VH region of the heavy chain and the CL region of the light chain Linker, or a method of linking the VL region of the entire heavy chain and the light chain to the linker at the heavy chain VH region and the light chain VL region. Preferably in the order of CH3-CH2-CH1-VH-VL in the N-terminal direction at the C-terminus by linking the VL region of the heavy chain and the light chain to the heavy chain variable region (VH) and the light chain variable region (VL) Lt; / RTI >

The inventors of the present invention named a single-chain antibody made by linking the VL region of the heavy chain and the light chain at the heavy chain VH region and the light chain VL region with a linker, as a large single chain antibody.

The linker refers to a polypeptide sequence capable of linking each domain of an antibody. The linker may be any (G4S) ₃ amino acid sequence as shown in SEQ ID NO: 1, although it is not limited as long as it does not affect the antigen recognition and binding ability of the monoclonal antibody.

In one embodiment of the present invention, a single-chain antibody was prepared by linking the heavy chain VH region of the whole antibody sequence of CO17-1A with the VL region of the light chain with a linker (amino acid sequence, (G4S) ₃ ] 1A. CO17-1A is a monoclonal antibody that recognizes the surface protein GA733-2 as an epitope, which is overexpressed on the surface of colon cancer.

(b) is a step of introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant.

In the step (b) of the present invention, the 'gene coding for a single chain antibody' may further comprise an ERK storage induction sequence (KDEL) shown in SEQ ID NO: 2. By inserting KDEL into a specific gene (a single-chain antibody gene in the present invention), the KDEL can be exposed at the amino acid end of the final product. This induces the produced protein to be present in the endoplasmic reticulum in the transformed cell without being secreted outside the plant cell. Proteins produced in the osteocyte cells into which the specific gene is introduced are post-translationally modified by the KDEL sequence, which can be stored in the endoplasmic reticulum and can be made in the plant. This plays an important role in solving the problems caused by the increased amount of intracellular antibody protein and the difference in interspecific sugar structure, which plays an important role in the interspecific immune response.

The insertion site of the ERK storage-inducing sequence (KDEL) is not limited so long as it does not affect the antigen recognition and binding ability of the short-chain monoclonal antibody, but preferably it may be a monoclonal antibody protein protein peptide sequence, Terminal region of the monoclonal antibody protein peptide sequence.

The term " introduction " refers to an operation of inserting a gene or a gene group into a cell that artificially targets and expressing the gene group, or adding another gene (group) to the genome of the cell. The introduction of these genes can be achieved by indirect methods such as transfection by bacteriophage (bacteria), Agrobacterium spp. Which is a soil bacterium, genegun, electrophoresis, microinjection, etc. In addition, depending on the characteristics of the target cell and the insert gene, a gene transfer technique known to a person skilled in the art can be selectively used. The main methods for stably inserting the exogenous DNA into the plant genome DNA are the following two main approaches / RTI >

(i) Agrobacterium-mediated gene transfer. See, e.g., Klee, H. J. et al. (1987). Annu Rev Plant Physiol 38, 467-486; Klee, H.J. and Rogers, S.G. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 2-25, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, Cal .; and Gatenby, A. A. (1989). Regulation and Expression of Plant Genes in Microorganisms, pp. 93-112, Plant Biotechnology, S. Kung and C. J. Arntzen, eds., Butterworth Publishers, Boston, Mass.

(ii) direct DNA uptake (see, e.g., Paszkowski, J. et al. (1989). Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, pp. 52-68, J. Schell and L. K. Vasil, eds., Academic Publishers, San Diego, CA; and Toriyama, K. et al. (1988). Bio / Technol 6, 1072-1074 (methods for direct uptake of DNA into protoplasts)]. [Reference example: Zhang et al. (1988). Plant Cell Rep 7, 379-384; and Fromm, M.E. et al. (1986). Stable transformation of maize after gene transfer by electroporation. Nature 319, 791-793 (reference example: Klein et al. (1988). Bio / Technology 6, 559-563; McCabe, DE et al. Stable transformation of soybean (Glycine max) by particle acceleration. Bio / Technology 6, 923-926 and Sanford, JC (1990) Biolistic plant transformation.Physiol Plant 79, 206-209 (DNA injection into plant cells or tissues by particle Physiol Plant 79, 213-217 (use of micropipette systems (1987)), and Neuhaus, JM and Spangenberg, GC (1990) (See, for example, DeWet, JMJ et al. (1985), "Exogenous gene transfer in (Zea mays) using DNAtreated pollen, " Experimental Manipulation of Ovule Tissue, GP Chapman et al., eds., Longman, New York- London, pp. 197-209 and Ohta, fficiency Genetic Transformation of Maize by a Mixture of Pollen and exogenous DNA. Proc Natl Acad Sci USA 83, 715-719 (direct incubation of DNA with germinating pollen).

Preferably, the gene introduction method of the present invention is an Agrobacterium-mediated gene transfer method. The Agrobacterium-mediated system involves the use of a plasmid vector with a defined DNA segment inserted into the plant genomic DNA. The method of inoculation of plant tissue depends on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf-dic method, which can be performed with any tissue excision providing an excellent source for the initiation of whole-plant differentiation (Horsch, RB et al. (1988). "Leaf disc transformation." Plant Molecular Biology Manual A5, 1-9, Kluwer Academic Publishers, Dordrecht). An additional approach uses the Agrobacterium delivery system with vacuum infiltration. The Agrobacterium system is particularly useful for the generation of transgenic dicot plants.

The term "transformation" refers to a modification of the genotype of a host cell by the introduction of a foreign polynucleotide (meaning a polynucleotide encoding the short-chain monoclonal antibody produced in step (a) in the present invention) Means that the exogenous polynucleotide has been introduced into the host cell irrespective of the method used for its transformation. The exogenous polynucleotide introduced into the host cell may be maintained integrated or maintained in the genome of the host cell, but the present invention encompasses both.

Step (b) of the present invention can be carried out by a known plant cell transformation method, for example, by introducing a gene of a short-chain antibody into a plant cell to obtain a transformed plant, (In the present invention, a gene coding for a single-chain antibody produced in step (a)) is inserted into a vector to form a recombinant vector, and the recombinant vector is transformed into a strain of the genus Agrobacterium. And infecting the plant cell.

The vector is preferably an expression vector comprising at least one of a signal sequence, a replication origin, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. The expression vector is a form of a vector into which a selected polynucleotide can express. One polynucleotide sequence is "operably linked" to the regulatory sequence when the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the polynucleotide sequence . The modulatory sequence is a sequence that affects the expression (e.g., level, timing, or location of expression) of the nucleic acid to which it is operatively linked. The modulation sequence can be, for example, a nucleic acid whose effect is directly or indirectly affected by the action of one or more other molecules (e. G., Polypeptides that bind to the regulatory sequence and / . The regulatory sequence includes promoters, enhancers, and other expression control elements.

Standard recombinant DNA and molecular cloning techniques, which are known in the art, are described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory, NY, 1984, and by Ausubel, FM (1987), Cold Spring Harbor Laboratory, NY, 1989; by Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions, et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-lnterscience, 1987).

After stable transformation, the transformed plants are then propagated. This breeding means increasing the number of plants. The propagation of the plant is not limited as long as the characteristics of the regenerating plant and the expression of the parent gene transplanting plant remain the same, but may be preferably microproliferation.

The microproliferation is a method of growing a second generation plant from a single tissue sample cut from a selected parent plant or cultivar. By this method, it is possible to mass-reproduce a plant having a desired tissue and expressing the fusion protein. The newly created plant is genetically identical to the original plant and has all of the characteristics of the original plant. Microproliferation allows mass production of excellent plant material in a short period of time and enables rapid propagation of selected crops while preserving the characteristics of the original transgenic or transgenic plant. Advantages of plant cloning methods include the rapidity of plant propagation and the superiority and uniformity of the plant produced.

In one embodiment of the present invention, large single chain CO17-1AK having a nucleotide sequence encoding KDEL was inserted into the heavy chain terminal (c-terminal terminal of heavy chain CH3) nucleotide sequence of large single chain CO17-1A, which is a short chain monoclonal antibody Respectively. Also, a plant expression vector containing the gene encoding the large single chain CO17-1AK was prepared, and the plant expression vector was transformed into Agrobacterium was transformed into tumefaciens (LB4404) strains, transformed by infecting the transformed strain in the leaf segment of the tobacco plant culture was prepared in the stems and leaves, the roots are all derived transgenic tobacco plants.

(c) is a step of linking the CH1 domain of the heavy chain constant region and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) to form a single chain antibody.

The 'other monoclonal antibody' refers to a monoclonal antibody that recognizes a monoclonal antibody of step (a) as an epitope from a substance different from a substance recognized as an epitope.

The step (c) of the present invention is a process for connecting a heavy chain and a light chain of an antibody to a single chain, except that the monoclonal antibody used is different from that of the step (a) ) The purpose of the step is the same. The preparation of the single-chain antibody of this step is as described above in step (a).

In one embodiment of the present invention, a single-chain antibody is prepared by linking the heavy chain VH region of the whole antibody sequence of BR55 and the VL region of the light chain with a linker (amino acid sequence, (G4S) ₃ ] Large single chain BR55. BR55 is a monoclonal antibody that recognizes Lewis Y as an epitope, an over-expressed oligosaccharide on the surface of breast cancer.

(d) is a step of introducing a gene of the single chain antibody prepared in the step (c) into a plant cell to obtain a transformed plant.

The 'short chain antibody gene' in the step (d) may further include an ERK storage induction sequence (KDEL) shown in SEQ ID NO: 2. The endoplasmic reticulum storage induction sequence is the same as that described above in step (b).

The introduction of the gene and the production of the transgenic plant in step (d) are the same as described above in step (b).

In one embodiment of the present invention, a large single chain BR55K having a nucleotide sequence encoding KDEL was prepared at the heavy chain end (c-terminal end of heavy chain CH3) nucleotide sequence of large single chain BR55, which is a short chain monoclonal antibody. Also, a plant expression vector containing the gene coding for the large single chain BR55K was prepared, and the plant expression vector was transformed into Agrobacterium was transformed into tumefaciens (LB4404) strains, transformed by infecting the transformed strain in the leaf segment of the tobacco plant culture was prepared in the stems and leaves, the roots are all derived transgenic tobacco plants.

(e) is a step of crossing the transgenic plants produced in the steps (b) and (d).

The 'mating' refers to a method in which, for sexual reproduction, a modification is made between two individuals of a female, a male, or a different mating type, and the male and female spouses are joined to form a conjugate At this time, the genotype of the parents is the same, and the difference is not a problem. Crossing of two individuals having different genotypes is called crossing, and the 'crossing' of the present invention includes crossing.

The species of the plant in the step (a) to the step (e) of the present invention may be the same species as the plant used in step (b) and step (d) ) And the plants used in step (d) are heterogeneous and the plants in step (e) in which they are crossed are also heterogeneous (in particular, hybrid). Preferably, the plants used in steps (b) and (d) are homologous and the plants in step (e) in which they are crossed may be homologous.

The 'plant' in the steps (a) to (e) of the present invention is not limited as long as the plant is capable of introducing a foreign gene. For example, rice, wheat, barley, bamboo shoot, corn, taro, asparagus, There are onions, garlic, leeks, leeks, soya, hemp and ginger. Examples of dicotyledonous plants include, but are not limited to, Arabidopsis thaliana, eggplant, cigarette, red pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, cabbage, Watermelon, melon, cucumber, pak, strawberry, soybean, mung bean, kidney bean, buzz foot trefoil, potato, fowl, perilla, dove bean and pea. Preferably tobacco ( Nicotiana tabacum ).

The hybrid plant produced in the step (e) is characterized in that a bispecific antibody antibody is expressed in plant cells. By "bispecific" is meant that one antibody recognizes two different antigens or epitopes at the same time. Preferably means recognizing and binding the monoclonal antibody of step (a) and the respective epitope recognized by the monoclonal antibody of step (c) simultaneously.

The present invention further provides a method for producing a transgenic plant for producing a bispecific antibody comprising the steps (a) to (e) described above,

(a) A single chain antibody (heavy chain variable region (VH) and light chain variable region (VL) linked by a linker to a monoclonal antibody recognizing GA733-2 epitope) Creating;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) A single chain antibody is obtained by linking the heavy chain variable region (VH) and the light chain variable region (VL) of a monoclonal antibody different from the monoclonal antibody recognizing Lewis Y as a epitope of breast cancer cell surface glycoprotein, ;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;

(e) producing transgenic plants by crossing the transgenic plants produced in steps (b) and (d) above, to produce a transgenic plant producing bispecific antibodies .

The hybridization plant produced by the above method is characterized in that a bispecific antibody that simultaneously recognizes and binds to the colon cancer cell surface protein GA733-2 and Lewis Y, a breast cancer cell surface sugar chain, is expressed.

(A) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker to form a single chain antibody;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;

(e) crossing the transgenic plants produced in steps (b) and (d) above; And

(f) obtaining a protein from the crossed plant produced in the step (e).

The steps (a) to (e) of the above antibody production method are the same as the above-mentioned (a) to (e) in the method for producing a transgenic plant producing a bispecific antibody, ) To (e).

The step (f) is a step of obtaining a protein from a crossed plant produced through steps (a) to (e). In particular, the hybridization plant produced in step (e) contains a large amount of bispecific antibody, thus aiming to obtain the bispecific antibody.

The step of obtaining the protein from the hybrid plant in step (f) may be carried out by a method of obtaining a known plant cell-derived protein, but is not limited thereto. For example, the hybrid plant is disrupted and pulverized to prepare an extraction buffer Solution). ≪ / RTI > The extraction buffer may be, for example, but not limited to, phosphate buffered saline (PBS), or Tris-HCl pH 8, dithiothreitol (DTT), protease inhibitors aprotinin, pepstatin, leupeptine, phenyl methylsulphonyl fluoride, and [(N- (N- (L-3- trans- carboxyoxirane) 2-carbonyl) -Lucyl) -agmantine], and the like.

The step (f) may further include a protein purification process. The 'protein purification' can be purified in a conventional manner, for example, by salting out (eg, ammonium sulfate precipitation, sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, etc.) Techniques such as filtration, ion exchange, column chromatography such as reversed phase column chromatography and ultrafiltration can be applied alone or in combination (Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. , San Diego, CA (1990)).

The method for producing an antibody comprising the steps (a) to (f) is a method for producing an antibody having a high production rate because it can obtain a bispecific antibody with high yield without chimerism.

The present invention further provides a method for producing an antibody comprising the steps (a) to (f) described above,

(a) A single chain antibody (heavy chain variable region (VH) and light chain variable region (VL) linked by a linker to a monoclonal antibody recognizing GA733-2 epitope) Creating;

(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;

(c) A single chain antibody is obtained by linking the heavy chain variable region (VH) and the light chain variable region (VL) of a monoclonal antibody different from the monoclonal antibody recognizing Lewis Y as a epitope of breast cancer cell surface glycoprotein, ;

(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;

(e) preparing transgenic plants by crossing the transgenic plants produced in the steps (b) and (d); And

(f) obtaining a protein from the crossed plant produced in the step (e).

The antibody obtained by the antibody production method is characterized by simultaneously recognizing GA733-2, a surface protein of colon cancer cells, and Lewis Y, a breast cancer cell surface sugar chain, as epitopes. Therefore, the antibody produced by the above method is characterized in being able to simultaneously diagnose and treat colorectal cancer and breast cancer.

Accordingly, the present invention provides a bispecific antibody that simultaneously recognizes or binds to GA733-2 and Lewis Y produced by the above method.

Using the method of producing a plant for producing a bispecific antibody of the present invention, it is not only easy to produce a large amount of bispecific antibodies, but also is economically effective for mass production of monoclonal antibodies.

Fig. 1 shows a schematic diagram of a final plant expression vector into which a large single chain antibody gene is inserted into pBIN PLUS used as a plant expression vector.
FIGS. 2A and 2B show results obtained by isolating genomic DNA from randomly selected individuals among several transgenic tobacco plants produced, followed by PCR and electrophoresis for the insertion of a large single chain (LSC) antibody gene M: DNA marker, P: positive control (LSC Ab easy vector), NT: non-transgenic plant genomic DNA, LSC CH: plant-derived LSC CO17-1A gene, LSC CK: plant- LSC BK: plant-derived LSC BR55K gene, Numbers: plant transformant numbers).
FIG. 3 shows Western blot analysis of the expression of antibodies from transgenic tobacco plant individuals in which large single chain (LSC) antibody genes were confirmed (NT: non-transgenic plant leaf extract, LSC CH : LSC CO17-1A transgenic plant leaf extract, LSC CK: LSC CO17-1AK transgenic plant leaf extract, LSC BK: LSC BR55K transgenic plant leaf extract, Numbers: plant transformant numbers).
4 is a schematic diagram of the protein structure of a large single chain CO17-1A antibody.
FIG. 5 is a protein structure diagram of large single chain CO17-1AK and large single chain BR55K antibodies.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

≪ Example 1 >

Preparation of single chain antibody of CO17-1A

mAb CO17-1A is a monoclonal antibody (light chain; SEQ ID NOs: 15 and 17, heavy chain; SEQ ID NOs: 16 and 18) that recognizes the surface protein GA733-2 as an epitope overexpressed on the surface of colon cancer. In the nucleotide sequence of CO17-1A, the VH region of the heavy chain and the VL region of the light chain were linked by a linker (amino acid sequence, (G4S) ₃ ] so that they could be expressed in one promoter. 11 and SEQ ID NO: 23). The nucleotide sequence of KDEL (5'-aaggacgaactt-3 ', SEQ ID NO: 26), which is an endoplasmic reticulum retention peptide, was inserted at the end of the nucleotide sequence of the large single chain CO17-1A antibody, followed by a termination codon sequence And named Large single chain CO17-1AK (SEQ ID NOS: 12 and 24). The above procedure was performed by synthesizing a gene in pGEM T easy vector using the PCR method in the mAb CO17-1A gene sequence. The primer information used to construct large single chain CO17-1A and large single chain CO17-1AK is shown in [Table 1]. Specifically, the gene synthesis method for large single chain CO17-1A and large single chain CO17-1AK is as follows. Large single chain CO17-1A with linker to mAb CO17-1A and mAb CO17-1A with restriction 5 'end primer (carrying restriction enzyme, Nco I) and restriction 3' end primer ( Xba I) Large single chain CO17-1AK gene with linker and KDEL were synthesized. The genes were synthesized using an automated synthesizer that elongates the base chain (oligonucleotides) with single bases one after the other in the 3 'to 5' direction. The oligomer was synthesized by sequencing dA, dG, dT, and dC in the order of nucleotide sequence to be synthesized by combining one nucleotide for one cycle in the order of detritylation, coupling, capping, and oxidation. . When synthesis is complete, add ammonium hydroxide to separate the oligomer from the support. Oligonucleotides were synthesized by binding to the solid support to immobilize the nucleoside at the 3 'end and then reacting in the column. The synthesized oligomers were purified by PAGE prep to extract only desired oligomers.

Name of Antibody Primer order
number LSC CO17-1A Forward primer:
5'-cgctcgagggatccatgggcattaaaatgcg-3 ' 28 Reverse primer:
5'-gcggtacctctagaatcgattttacccgggc-3 ' 29 LSC CO17-1AK Forward primer:
5'-cgctcgagggatccatgggcattaaaatgcg-3 ' 30 Reverse primer:
5'-gcggtacctctagattaaagttcgtccttatcgatgc-3 ' 31 LSC BR55K Forward primer:
5'-cggtcgactctagaatgctcccggccgctcg-3 ' 32 Reverse primer:
5'-gcactagtggatcctcaaagttcatctttacccggc-3 ' 33

(LSC = large single chain)

< Example  2>

BR55 of Short chain  Preparation of antibodies

mAb BR55 is an antibody that recognizes Lewis Y, an oligosaccharide that is overexpressed on the surface of breast cancer, as an epitope (light chain; SEQ ID NO: 19 and SEQ ID NO: 21, heavy chain; SEQ ID NOs: 20 and 22). The VH region of the heavy chain of the mAb BR55 and the VL region of the light chain were linked by a linker (amino acid sequence, (G4S) ₃ ] so that they could be expressed in one promoter. Respectively. The nucleotide sequence of KDEL (5'-aaagatgaactt-3 ', SEQ ID NO: 27), which is an endoplasmic reticulum retention peptide, was inserted at the end of the large single chain BR55 antibody nucleotide sequence, followed by insertion of a termination codon sequence , Named Large single chain BR55K (SEQ ID NOS: 13 and 25). The above procedure was performed by synthesizing a gene in pGEM T easy vector using the PCR method in the mAb BR55 gene sequence. The primer information used to synthesize large single chain BR55K is shown in [Table 1]. Specifically, the gene synthesis method for LSC BR55K is as follows. Large single chain BR55K gene with linker and KDEL was synthesized in mAb BR55 using restriction 5 'end primer (carrying restriction enzyme, Xba I) and restriction 3' end primer (carrying restriction enzyme, Bam HI) The genes were synthesized using an automated synthesizer that elongates the base chain (oligonucleotides) with single bases one after the other in the 3 'to 5' direction. The oligomer was synthesized by sequencing dA, dG, dT, and dC in the order of nucleotide sequence to be synthesized by combining one nucleotide for one cycle in the order of detritylation, coupling, capping, and oxidation. . When synthesis is complete, add ammonium hydroxide to separate the oligomer from the support. Oligonucleotides were synthesized by binding to the solid support to immobilize the nucleoside at the 3 'end and then reacting in the column. The synthesized oligomers were purified by PAGE prep to extract only desired oligomers.

< Example  3>

Production of a transgenic plant expressing a single-chain antibody of CO17-1A and a transgenic plant expressing a single-chain antibody of BR55

Large single chain CO17-1A, Large single chain CO17-1AK prepared in Example 1 above, or Large single chain BR55K gene prepared in Example 2 was transformed into Nicotiana tabacum ) plant cells to produce transgenic plants expressing different antibodies.

Large single chain CO17-1AK (SEQ ID NO: 12) or Large single chain BR55K (SEQ ID NO: 13) modified to a plant specific sequence to facilitate expression in plants, The gene sequences of monoclonal mAb CO17-1A or monoclonal mAb BR55 were synthesized by PCR in pGEM T easy vector, and the synthesized large single chain pGEM T easy vector was used for E.coli (DH5a) Cloning was carried out by the conversion method. In the E. coli transformation experiment, 10 μl of the synthesized gene plasmid was added to 100 μl of DH5a competenet cells, left for 15 minutes on ice, and then thermally shocked at 42 ° C. for 90 seconds. After incubation for 2 minutes on ice, the cells were spread on LB solid medium supplemented with antibiotic (+ Ampicillin, 50 μg / ml). The stained medium was incubated at 37 DEG C for 16 hours. Each large single chain antibody (large single chain CO17-1A or large single chain CO17-1AK or large single chain BR55K) was obtained by the above method.

Large single chain antibody gene was inserted into pBI 525 shuttle vector using NcoI and XbaI as restriction enzymes for the duplicated CaMV 35S promoter for the expression of large single chain antibody gene. The pBI 525 shuttle vector containing the large single chain antibody gene was inserted into the Muliple cloning site (MCS) of the pBIN PLUS vector by restriction enzymes EcoRI and HindIII. The gene expression cassette of the final plant expression vector is shown in Fig. The completed large single chain antibody plant expression vector was transformed into Agrobacterium tumefaciens (LB4404) strain. The transgenic Agrobacterium tumefaciens (LB4404) strain was infected with leaf sections of tobacco plants cultured in vitro. The leaf sections of the infected tobacco plants were cultured in Co-cultivation medium (M / S vitamin B5 0.48%, Sucrose 3%, NAA 50 ng / ml, 6-BAP 500 ng / ml) for 3 days under dark condition at 23 ° C. The cultured leaf slices were cultured in regeneration medium (M / S vitamin B5 0.48%, Sucrose 3%, NAA 100 ng / ml, 6-BAP 1 μg / ml, Kanamycin 100 μg / ml, Cefotaxime 250 μg / The transgenic tobacco plants were cultured at 23 ℃ for 8 hours under the dark condition.

<Example 4>

Identification of the presence of a single-chain antibody gene in transgenic plants

The transgenic tobacco plants expressing the large single chain CO17-1A, the transgenic tobacco plants expressing the large single chain CO17-1AK, and the transgenic tobacco plants expressing the large single chain BR55K prepared in Example 3 above Of large single chain antibody genes were introduced.

Several randomly selected transgenic tobacco plants produced at the time of Example 3 were randomly selected and the leaves of transgenic tobacco plants in which all the stem, leaf and root of the plant were cut were cut to isolate the plant genomic DNA I got it. Genomic DNA was extracted from 50 mg of leaf of transgenic plant cultured in vitro using RBC Bioscience Genomic DNA Extraction Kit (Cat. No. YGB100).

 Polymerase chain reaction (PCR) was used to confirm the presence of the large single chain antibody gene in the isolated plant genomic DNA. The primer sequences used to confirm the insertion of the large single chain antibody gene in the plant expression vector are shown in [Table 2]. To perform the PCR, iNtRON Biotechnology Maxime PCR PreMix Kit (i-Taq), Cat. PCR instrument Biometra Thermocycler (Cat. No. 25025), plant genomic DNA, LSC Ab gene forward primer (10 pmol / μl), LSC Ab gene reverse primer (10 pmol / . 050-901) was used. Specific PCR conditions were as follows; First denature 94 ℃ for 2 minutes, denature 94 ℃ for 30 seconds, anealing for 65 ℃ for 30 seconds, extention 72 ℃ for 1 minute 30 seconds. After the final extension at 72 ° C for 5 minutes, the PCR reaction was terminated at 4 ° C.

After completion of the PCR reaction, 20 μl of the PCR product on 1% agarose gel was electrophoresed in 1 × TAE buffer solution to confirm the gene product by UV light irradiation.

SEQ ID NO: Primer name order 3 lsc BR55_primer1 5'-GCTTGATCTCCAAGACTTAC-3 ' 4 lsc BR55_primer2 5'-CTGATGTTCTGGATTCCTGC-3 ' 5 lsc BR55_primer3 5'-GTAGACAGTGTAAAGGGCAG-3 ' 6 lsc BR55_primer4 5'-GACTGGATGAGTGGCAAGG-3 ' 7 lsc CO17_primer1 5'-GCTTCTGTTGATACCAGGAA-3 ' 8 lsc CO17_primer2 5'-TCTCCCAAATCTATGAGCAT-3 ' 9 lsc CO17_primer3 5'-GTACTCTGGTCACTGTATCT-3 ' 10 lsc CO17_primer4 5'-AATAGCACTCTTCGAGTGGT-3 '

As a result, as shown in FIG. 2, it was confirmed that Large single chain CO17-1A gene, Large single chain CO17-1AK gene and Large single chain BR55K gene were present in each transgenic plant cell, respectively.

< Example  5>

From transgenic plants Single-chain antibody  Confirmation of expression

In a transgenic tobacco plant expressing large single chain CO17-1A, a transgenic tobacco plant expressing large single chain CO17-1AK, and a transgenic tobacco plant expressing large single chain BR55K produced in Example 3 above Each large single chain antibody was expressed.

Several randomly selected transgenic tobacco plants produced at the time of Example 3 were screened and the leaves of transgenic tobacco plants in which all the stem, leaf and root of the plant were cut were cut into a phosphate buffered saline Western blots were used to confirm the expression of large single chain antibodies.

Specific methods of extracting antibodies in leaves and western blotting methods are as follows. 100 mg of leaf of transgenic plant cultivated in vitro was placed in a 1.5 ml tube, and 300 μl of 1 × Phosphate buffered saline was added. A homogenizer was attached to the electric drill and the plant leaf was disrupted. The crushed plant samples were mixed with 5X protein loading buffer (0.06% 1M Tris-HCl, 25% glycerol, 2% SDS, 0.05% 2-mercaptoethanol, 0.01% bromophenol blue, distilled water) + 5X protein loading buffer (24 μl) and heat-treated at 96 ° C for 5 minutes. After centrifugation at 13,000 rpm for 1 minute, the supernatant is electrophoresed on 10% SDS gel, attached to the NC membrane with antibody protein, and incubated in TBS-T solution containing 5% skimmilk for 1 hour at room temperature. After adding Goat-anti mouse IgG H + L HRP conjugate at a ratio of 1: 3000, the mixture was incubated at room temperature for 2 hours. This is followed by washing the TBS-T solution three times for 10 minutes each. Finally, the chemiluminescent substrate is treated. Antibody protein expression was confirmed by film development in the dark room.

As a result, as shown in FIG. 3, large single chain CO17-1A antibody, large single chain CO17-1AK antibody and large single chain BR55K antibody were expressed from each transgenic plant, respectively.

Each of the transgenic plants prepared above (large single chain CO17-1A antibody expressing plant, large single chain CO17-1AK antibody expressing plant and large single chain BR55K antibody expressing plant) were crossed with each other , And got new hybrid plants. Protein components contained in plant cells were extracted and examined in the new hybrid plants in the same manner as described above. As a result, it was confirmed that a bispecific antibody was produced in hybridized plant cells.

(A) a heavy chain variable region (VH) of a monoclonal antibody and a light chain variable region (VL) of a monoclonal antibody. The present invention relates to a method for producing a bispecific antibody, (B) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant; (c) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody different from the monoclonal antibody of the step (a) by a linker to form a single chain antibody; ) Introducing the gene of the single chain antibody prepared in the step (c) into a plant cell to obtain a transformed plant; And (e) crossing the transformed plant individuals produced in steps (b) and (d) above, and a method for producing a transgenic plant producing the bispecific antibody, To obtain a protein from the transformed plant.

The use of the method of producing a plant for producing a bispecific antibody of the present invention is not only easy to produce a large amount of bispecific antibodies, but also is effective for mass production of economically cheap monoclonal antibodies, and thus is highly industrially applicable.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for preparing the plant producing bispecific antibody <130> NP13-0061 <160> 33 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> Linker (amino acid sequence) <400> 1 Gly Ser Ser Ser Gly Ser Ser Ser Ser Ser Ser Ser Ser Ser   1 5 10 15 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Endoplasmic Reticulum retention peptide (amino acid sequence) <400> 2 Lys Asp Glu Leu   One <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc BR55_primer1 <400> 3 gcttgatctc caagacttac 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc BR55_primer2 <400> 4 ctgatgttct ggattcctgc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc BR55_primer3 <400> 5 gtagacagtg taaagggcag 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> lsc BR55_primer4 <400> 6 gactggatga gtggcaagg 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc CO17_primer1 <400> 7 gcttctgttg ataccaggaa 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc CO17_primer2 <400> 8 tctcccaaat ctatgagcat 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc CO17_primer3 <400> 9 gtactctggt cactgtatct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> lsc CO17_primer4 <400> 10 aatagcactc ttcgagtggt 20 <210> 11 <211> 1866 <212> DNA <213> Artificial Sequence <220> <223> Large single chain CO17-1A (nucleotide sequence) <400> 11 ctcgagggat ccatgggcat taaaatggaa tcacaaactc tggtcttcat ttccattctg 60 ctctggttat atggagctga tggaaacatt gtaatgaccc aatctcccaa atctatgagc 120 atgtctgtcg gagagagggt taccttgaca tgcaaggcaa gtgaaaatgt ggttacttat 180 gtttcctggt atcaacagaa gccagagcag tcgcctaaat tactgatata cggggcctcc 240 aatcggtata ctggcgtacc tgatcgcttt acaggcagtg gatctgcaac agattttact 300 ctgaccattt catcggtgca agctgaagac cttgcagatt atcattgcgg acaaggttat 360 tcatatccgt acacgttcgg agggggcacg aagctggaaa taaaaggtgg aggtggttct 420 gggggtggag gttcaggtgg tggtggttct atggaatgga gcagagtttt tatctttctt 480 ctatcagtaa ccgcaggagt gcattcccaa gtccagttgc agcagtctgg agctgagctg 540 gtcaggcctg gtacttcagt taaggtgtct tgcaaggctt ctggatatgc ttttactaat 600 tacttaatag agtgggtcaa gcaaaggcct ggacaaggcc ttgaatggat tggggtgatt 660 aatcctggaa gtggtggtac taactacaat gaaaagttta agggcaaggc aacacttact 720 gccgataaat cctcaagcac tgcttatatg cagctatcca gcctgacaag cgatgactct 780 gcggtctatt tctgtgctag agatggcccc tggttcgctt actggggcca aggtactctg 840 gtcactgtat ctgctgctaa aactacagct ccaagtgtgt atccactggc ccctgtgtgt 900 ggagatacta ctggctcctc tgtgactcta gggtgtctgg tcaaaggtta ttttcctgag 960 ccagtaacct tgacatggaa ctctggatct ctctccagtg gtgtgcacac cttcccagct 1020 gtcctgcaat ctgacctcta cacactctct agttcagtaa ctgtaacctc gtcaacttgg 1080 ccgagtcagt ccattacatg caatgtggcc cacccggcaa gcagtaccaa agttgataag 1140 aagattgaac ctagagggcc tacaattaag ccttgtcctc catgcaaatg tccagcacca 1200 aatcttttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgttttgatg 1260 atttcactta gtcccatagt aacatgtgta gtggtggatg tgagcgagga tgacccagat 1320 gtccagatca gctggtttgt gaataacgtg gaagtacata cagcacaaac acaaacccat 1380 agagaggatt acaatagcac tcttcgagtg gtcagtgctc tcccaatcca gcaccaagac 1440 tggatgagtg gaaaggagtt taagtgcaaa gttaataata aagacctgcc agcgcccata 1500 gagagaacaa tatcaaaacc caaagggtca gtacgtgcac ctcaagtata tgtcttgcct 1560 ccaccagaag aagaaatgac gaagaaacag gtcactctta catgtatggt gacagatttc 1620 atgccagaag acatttacgt taagtggacc aacaacggga aaacagagct aaactacaaa 1680 aacactgagc cagtactgga ctctgatgga tcttacttta tgtatagcaa actgagagtg 1740 gaaaagaaga actgggtgga aagaaattcc tattcctgtt cagtggtcca tgagggtctg 1800 cacaatcatc atacgactaa gtcgttctcc agaactccgg gtaaaatcga ttaatctaga 1860 ggtacc 1866 <210> 12 <211> 1878 <212> DNA <213> Artificial Sequence <220> Large single chain CO17-1AK (nucleotide sequence) <400> 12 ctcgagggat ccatgggcat taaaatggaa tcacaaactc tggtcttcat ttccattctg 60 ctctggttat atggagctga tggaaacatt gtaatgaccc aatctcccaa atctatgagc 120 atgtctgtcg gagagagggt taccttgaca tgcaaggcaa gtgaaaatgt ggttacttat 180 gtttcctggt atcaacagaa gccagagcag tcgcctaaat tactgatata cggggcctcc 240 aatcggtata ctggcgtacc tgatcgcttt acaggcagtg gatctgcaac agattttact 300 ctgaccattt catcggtgca agctgaagac cttgcagatt atcattgcgg acaaggttat 360 tcatatccgt acacgttcgg agggggcacg aagctggaaa taaaaggtgg aggtggttct 420 gggggtggag gttcaggtgg tggtggttct atggaatgga gcagagtttt tatctttctt 480 ctatcagtaa ccgcaggagt gcattcccaa gtccagttgc agcagtctgg agctgagctg 540 gtcaggcctg gtacttcagt taaggtgtct tgcaaggctt ctggatatgc ttttactaat 600 tacttaatag agtgggtcaa gcaaaggcct ggacaaggcc ttgaatggat tggggtgatt 660 aatcctggaa gtggtggtac taactacaat gaaaagttta agggcaaggc aacacttact 720 gccgataaat cctcaagcac tgcttatatg cagctatcca gcctgacaag cgatgactct 780 gcggtctatt tctgtgctag agatggcccc tggttcgctt actggggcca aggtactctg 840 gtcactgtat ctgctgctaa aactacagct ccaagtgtgt atccactggc ccctgtgtgt 900 ggagatacta ctggctcctc tgtgactcta gggtgtctgg tcaaaggtta ttttcctgag 960 ccagtaacct tgacatggaa ctctggatct ctctccagtg gtgtgcacac cttcccagct 1020 gtcctgcaat ctgacctcta cacactctct agttcagtaa ctgtaacctc gtcaacttgg 1080 ccgagtcagt ccattacatg caatgtggcc cacccggcaa gcagtaccaa agttgataag 1140 aagattgaac ctagagggcc tacaattaag ccttgtcctc catgcaaatg tccagcacca 1200 aatcttttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgttttgatg 1260 atttcactta gtcccatagt aacatgtgta gtggtggatg tgagcgagga tgacccagat 1320 gtccagatca gctggtttgt gaataacgtg gaagtacata cagcacaaac acaaacccat 1380 agagaggatt acaatagcac tcttcgagtg gtcagtgctc tcccaatcca gcaccaagac 1440 tggatgagtg gaaaggagtt taagtgcaaa gttaataata aagacctgcc agcgcccata 1500 gagagaacaa tatcaaaacc caaagggtca gtacgtgcac ctcaagtata tgtcttgcct 1560 ccaccagaag aagaaatgac gaagaaacag gtcactctta catgtatggt gacagatttc 1620 atgccagaag acatttacgt taagtggacc aacaacggga aaacagagct aaactacaaa 1680 aacactgagc cagtactgga ctctgatgga tcttacttta tgtatagcaa actgagagtg 1740 gaaaagaaga actgggtgga aagaaattcc tattcctgtt cagtggtcca tgagggtctg 1800 cacaatcatc atacgactaa gtcgttctcc agaactccgg gtaaaatcga taaggacgaa 1860 ctttaatcta gaggtacc 1878 <210> 13 <211> 2022 <212> DNA <213> Artificial Sequence <220> <223> Large single chain BR55K (nucleotide sequence) <400> 13 gtcgactcta gaatgctccc ggccgctatg gctgcgggat ttatgaaatt gcctgttagg 60 cttttggtgc tgatgttctg gattcctgct tcgtcttctg atgttttgat gacacaaact 120 ccattatccc tgcctgtaag tcttggagat caagcatcca tatcttgcag atcgagtcaa 180 tcaattgtac atagtaatgg aaatacctat ttagaatggt acttgcaaaa accaggccaa 240 agtccaaaac tcctgatctc caaagttagt aaccgatttt ctggggttcc agataggttc 300 agtggcagtg gatcagggac agactttaca ctaaagatta gcagggttga agctgaagat 360 ttaggagttt attactgctt tcaaggttca catgttccat ttacgttcgg atcgggcaca 420 aagttggaaa taaaaggagg aggtggttct ggtggaggtg gttctggtgg tggtggttct 480 atgcatccaa cgcgttggga actctcccat atggtcgatc ttcaagccgc cgcactggtt 540 attacaatgg atttggggtt gtcgttgatt tttcttgtcc ttgttttaaa aggtgttcaa 600 tgtgaagtga agctggtgga gagtgggggg ggtttagtgc agcctggagg gtccctgaaa 660 ctatcctgtg caacctctgg atttactttc agtgactatt acatgtattg ggttcgccag 720 actccagaga agaggctcga atgggttgca tacatttcaa atggtggtgg tagtagccat 780 tatgtagaca gtgtaaaggg cagattcaca atctccagag acaatgctaa gaacaccctg 840 tacctgcaaa tgagccgtct gaggtctgaa gatacagcca tgtatcactg tgcaagggga 900 atggattatg gagcatggtt tgcttactgg gggcaaggaa ctctggtcac tgtgtcggca 960 gctactacaa cagctccatc agtctatcct ttggtcccag gctgtagtga tacatctgga 1020 tcgagcgtga cactgggatg ccttgtaaaa ggctacttcc ctgagcctgt aactgtaaaa 1080 tggaactatg gagcattatc cagcggtgtg cgcacagtca gttctgtctt gcaatctggg 1140 ttttactccc tcagctcttt ggtgactgtt ccttcctcta cgtggcccag ccaaactgtc 1200 atctgcaatg tggctcatcc agcatcaaag actgagctaa ttaagagaat tgaacctaga 1260 ataccgaagc ctagtattcc cccaggatct tcatgtccac agcctagagg accaacaata 1320 aagccatgtc ctccatgtaa atgcccagct cctaacttgt tgggtggacc aagtgtgttt 1380 atcttccctc caaaaatcaa ggatgtactc atgatatccc tgagcccgat agtcacctgc 1440 gtggtggtgg atgtgagcga ggatgaccca gatgttcaga tcagctggtt tgtgaataac 1500 gtggaagtac atacagccca gacacaaacc catcgggagg attacaacag tactctacgt 1560 gtggtcagtg ccttgcccat ccagcatcag gactggatga gtggcaagga gttcaaatgc 1620 aaggtcaaca acaaagactt accagcgccc atcgagagaa ccatctcaaa acccaaaggg 1680 tcagtaagag ctccacaggt atatgtcttg cccccacctg aagaagagat gactaaaaaa 1740 caggtaactc tgacctgtat ggttactgac ttcatgcctg aagacattta cgtggagtgg 1800 accaacaacg gtaaaactga gctaaactat aagaacactg aaccagttct ggactctgat 1860 ggttcttact tcatgtactc taagttaagg gtggaaaaga agaattgggt ggaaagaaat 1920 agttattctt gttcagtggt ccacgaaggt ctccacaatc accacacgac taagagtttt 1980 tcccggactc cgggtaaaga tgaactttga ggatccacta gt 2022 <210> 14 <211> 2010 <212> DNA <213> Artificial Sequence <220> <223> Large single chain BR55 (nucleotide sequence) <400> 14 gtcgactcta gaatgctccc ggccgctatg gctgcgggat ttatgaaatt gcctgttagg 60 cttttggtgc tgatgttctg gattcctgct tcgtcttctg atgttttgat gacacaaact 120 ccattatccc tgcctgtaag tcttggagat caagcatcca tatcttgcag atcgagtcaa 180 tcaattgtac atagtaatgg aaatacctat ttagaatggt acttgcaaaa accaggccaa 240 agtccaaaac tcctgatctc caaagttagt aaccgatttt ctggggttcc agataggttc 300 agtggcagtg gatcagggac agactttaca ctaaagatta gcagggttga agctgaagat 360 ttaggagttt attactgctt tcaaggttca catgttccat ttacgttcgg atcgggcaca 420 aagttggaaa taaaaggagg aggtggttct ggtggaggtg gttctggtgg tggtggttct 480 atgcatccaa cgcgttggga actctcccat atggtcgatc ttcaagccgc cgcactggtt 540 attacaatgg atttggggtt gtcgttgatt tttcttgtcc ttgttttaaa aggtgttcaa 600 tgtgaagtga agctggtgga gagtgggggg ggtttagtgc agcctggagg gtccctgaaa 660 ctatcctgtg caacctctgg atttactttc agtgactatt acatgtattg ggttcgccag 720 actccagaga agaggctcga atgggttgca tacatttcaa atggtggtgg tagtagccat 780 tatgtagaca gtgtaaaggg cagattcaca atctccagag acaatgctaa gaacaccctg 840 tacctgcaaa tgagccgtct gaggtctgaa gatacagcca tgtatcactg tgcaagggga 900 atggattatg gagcatggtt tgcttactgg gggcaaggaa ctctggtcac tgtgtcggca 960 gctactacaa cagctccatc agtctatcct ttggtcccag gctgtagtga tacatctgga 1020 tcgagcgtga cactgggatg ccttgtaaaa ggctacttcc ctgagcctgt aactgtaaaa 1080 tggaactatg gagcattatc cagcggtgtg cgcacagtca gttctgtctt gcaatctggg 1140 ttttactccc tcagctcttt ggtgactgtt ccttcctcta cgtggcccag ccaaactgtc 1200 atctgcaatg tggctcatcc agcatcaaag actgagctaa ttaagagaat tgaacctaga 1260 ataccgaagc ctagtattcc cccaggatct tcatgtccac agcctagagg accaacaata 1320 aagccatgtc ctccatgtaa atgcccagct cctaacttgt tgggtggacc aagtgtgttt 1380 atcttccctc caaaaatcaa ggatgtactc atgatatccc tgagcccgat agtcacctgc 1440 gtggtggtgg atgtgagcga ggatgaccca gatgttcaga tcagctggtt tgtgaataac 1500 gtggaagtac atacagccca gacacaaacc catcgggagg attacaacag tactctacgt 1560 gtggtcagtg ccttgcccat ccagcatcag gactggatga gtggcaagga gttcaaatgc 1620 aaggtcaaca acaaagactt accagcgccc atcgagagaa ccatctcaaa acccaaaggg 1680 tcagtaagag ctccacaggt atatgtcttg cccccacctg aagaagagat gactaaaaaa 1740 caggtaactc tgacctgtat ggttactgac ttcatgcctg aagacattta cgtggagtgg 1800 accaacaacg gtaaaactga gctaaactat aagaacactg aaccagttct ggactctgat 1860 ggttcttact tcatgtactc taagttaagg gtggaaaaga agaattgggt ggaaagaaat 1920 agttattctt gttcagtggt ccacgaaggt ctccacaatc accacacgac taagagtttt 1980 tcccggactc cgggttgagg atccactagt 2010 <210> 15 <211> 717 <212> DNA <213> Artificial Sequence <220> <223> mAb CO17-1A light chain (nucleotide sequence) <400> 15 atgggcatca agatggaatc acagactctg gtcttcatat ccatactgct ctggttatat 60 ggagctgatg ggaacattgt aatgacccaa tctcccaaat ccatgtccat gtcagtagga 120 gagagggtca ccttgacctg caaggccagt gagaatgtgg ttacttatgt ttcctggtat 180 caacagaaac cagagcagtc tcctaaactg ctgatatacg gggcatccaa ccggtacact 240 ggggtccccg atcgcttcac aggcagtgga tctgcaacag atttcactct gaccatcagc 300 agtgtgcagg ctgaagacct tgcagattat cactgtggac agggttacag ctatccgtac 360 acgttcggag gggggaccaa gctggaaata aaacgggctg atgctgcacc aactgtatcc 420 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 480 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 540 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 600 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 660 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttag 717 <210> 16 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> mAb CO17-1A heavy chain (nucleotide sequence) <400> 16 atggaatgga gcagagtctt tatctttctc ctatcagtaa ctgcaggtgt tcactcccag 60 gtccagttgc agcagtctgg agctgagctg gtaaggcctg ggacttcagt gaaggtgtcc 120 tgcaaggctt ctggatacgc cttcactaat tacttgatag agtgggtaaa gcagaggcct 180 ggacagggcc ttgagtggat tggggtgatt aatcctggaa gtggtggtac taactacaat 240 gagaagttca agggcaaggc aacactgact gcagacaaat cctccagcac tgcctacatg 300 cagctcagca gcctgacatc tgatgactct gcggtctatt tctgtgcaag agatggtccc 360 tggtttgctt actggggcca agggactctg gtcactgtct ctgcagccaa aacaacagcc 420 ccatcggtct atccactggc ccctgtgtgt ggagatacaa ctggctcctc ggtgactcta 480 ggatgcctgg tcaagggtta tttccctgag ccagtgacct tgacctggaa ctctggatcc 540 ctgtccagtg gtgtgcacac cttcccagct gtcctgcagt ctgacctcta caccctcagc 600 agctcagtga ctgtaacctc gagcacctgg cccagccagt ccatcacctg caatgtggcc 660 cacccggcaa gcagcaccaa ggtggacaag aaaattgagc ccagagggcc cacaatcaag 720 ccctgtcctc catgcaaatg cccagcacct aacctcttgg gtggaccatc cgtcttcatc 780 ttccctccaa agatcaagga tgtactcatg atctccctga gccccatagt cacatgtgtg 840 gtggtggatg tgagcgagga tgacccagat gtccagatca gctggtttgt gaacaacgtg 900 gaagtacaca cagctcagac acaaacccat agagaggatt acaacagtac tctccgggtg 960 gtcagtgccc tccccatcca gcaccaggac tggatgagtg gcaaggagtt caaatgcaag 1020 gtcaacaaca aagacctccc agcgcccatc gagagaacca tctcaaaacc caaagggtca 1080 gtaagagctc cacaggtata tgtcttgcct ccaccagaag aagagatgac taagaaacag 1140 gtcactctga cctgcatggt cacagacttc atgcctgaag acatttacgt gaagtggacc 1200 aacaacggga aaacagagct aaactacaag aacactgaac cagtcctgga ctctgatggt 1260 tcttacttca tgtacagcaa gctgagagtg gaaaagaaga actgggtgga aagaaatagc 1320 tactcctgtt cagtggtcca cgagggtctg cacaatcacc acacgactaa gagcttctcc 1380 cggactccgg gtaaaatcga ttaa 1404 <210> 17 <211> 238 <212> PRT <213> Artificial Sequence <220> <223> mAb CO17-1A light chain (amino acid sequence) <400> 17 Met Gly Ile Lys Met Glu Ser Gln Thr Leu Val Phe Ile Ser Ile Leu   1 5 10 15 Leu Trp Leu Tyr Gly Ala Asp Gly Asn Ile Val Met Thr Gln Ser Pro              20 25 30 Lys Ser Met Ser Ser Ser Val Gly Glu Arg Val Thr Leu Thr Cys Lys          35 40 45 Ala Ser Glu Asn Val Val Thr Tyr Val Ser Trp Tyr Gln Gln Lys Pro      50 55 60 Glu Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser Asn Arg Tyr Thr  65 70 75 80 Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr                  85 90 95 Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys             100 105 110 Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu         115 120 125 Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro     130 135 140 Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu 145 150 155 160 Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly                 165 170 175 Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser             180 185 190 Lys Asp Ser Thr Ser Ser Met Ser Thr Leu Thr Leu Thr Lys Asp         195 200 205 Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr     210 215 220 Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 225 230 235 <210> 18 <211> 467 <212> PRT <213> Artificial Sequence <220> <223> mAb CO17-1A heavy chain (amino acid sequence) <400> 18 Met Glu Trp Ser Arg Val Phe Ile Phe Leu Leu Ser Val Thr Ala Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg              20 25 30 Pro Gly Thr Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe          35 40 45 Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu      50 55 60 Glu Trp Ile Gly Val Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn  65 70 75 80 Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser                  85 90 95 Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val             100 105 110 Tyr Phe Cys Ala Arg Asp Gly Pro Trp Phe Ala Tyr Trp Gly Gln Gly         115 120 125 Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Ala Pro Ser Val Tyr     130 135 140 Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu 145 150 155 160 Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp                 165 170 175 Ser Ser Ser Ser Ser Val Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Val Ser             180 185 190 Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser         195 200 205 Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser     210 215 220 Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys 225 230 235 240 Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro                 245 250 255 Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser             260 265 270 Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp         275 280 285 Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr     290 295 300 Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val 305 310 315 320 Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu                 325 330 335 Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg             340 345 350 Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val         355 360 365 Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr     370 375 380 Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Lys Trp Thr 385 390 395 400 Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu                 405 410 415 Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys             420 425 430 Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu         435 440 445 Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly     450 455 460 Lys Ile Asp 465 <210> 19 <211> 771 <212> DNA <213> Artificial Sequence <220> <223> mAb BR55 light chain (nucleotide sequence) <400> 19 ctcgagggat ccatgctacc ggccgctatg gcagcgggat tcatgaagct accagttagg 60 cttcttgtgc tgatgttttg gattcccgca tcctctagtg atgttttgat gactcaaact 120 ccactttccc tccctgtatc tcttggggat caagcatcta tttcatgtcg atcaagccag 180 tctatcgttc acagtaatgg aaacacatac ttagagtggt acttgcagaa accaggtcaa 240 agtcctaaac tcctgatctc caaagtttct aatcgtttct ctggtgtacc tgataggttt 300 agtggcagtg gatcagggac agattttaca ctcaagatta gcagagtgga ggctgaggat 360 ttaggagttt attactgctt ccaaggttca catgtcccat ttacctttgg ctctggaaca 420 aagttggaga taaaaagagc tgatgctgca ccaactgtat ctatcttccc accttcttca 480 gaacagttaa catcgggtgg tgcctcagtc gtttgctttt tgaacaactt ctaccctaag 540 gacataaatg tgaaatggaa aattgatggg agtgaaagac aaaatggcgt gcttaatagt 600 tggaccgatc aggacagcaa agactccacc tattctatgt catcgacact tactttgact 660 aaggacgagt atgaacggca taatagctat acctgtgaag ctactcacaa gacgtccact 720 tcacccattg tcaagagctt taacaggaac gaatgttaac tgcagggtac c 771 <210> 20 <211> 1542 <212> DNA <213> Artificial Sequence <220> <223> mAb BR55 heavy chain (nucleotide sequence) <400> 20 gtcgactcta gaatgcatcc aacgaggtgg gagttgtcgc atatggtcga tctacaggct 60 gctgcactgg tcattacaat ggatttgggg ctctctttga tttttttagt ccttgtttta 120 aaaggtgtcc agtgtgaagt gaaactggtg gaatctggag gaggcttagt gcaaccagga 180 gggtctctga aactcagttg tgcaacctct ggtttcactt tttctgatta ttatatgtat 240 tgggttcgcc aaactccaga gaagaggctg gagtgggtcg catatataag taatggtggg 300 ggtagttctc attatgtaga ttcagtaaaa ggccgattta ccatcagcag ggataatgcc 360 aagaatacac tgtacctgca aatgtcccgt ctgaggtcag aggatacagc catgtatcac 420 tgtgcaaggg gaatggatta cggggcgtgg tttgcttact ggggccaagg gactcttgtt 480 actgttagcg cagctacaac aaccgctcct tctgtctatc ctttggtccc tggttgcagt 540 gacacctctg gatcatcggt gacactggga tgccttgtta aaggctactt tcctgagccg 600 gtaactgtaa agtggaatta tggagcactg tccagcggtg ttagaacagt ctcatctgtc 660 ctgcaatctg gattctattc cctaagcagc ttggtgactg taccctccag cacctggcca 720 tctcagactg tgatatgcaa tgtagctcac ccagccagca agaccgagtt gattaagaga 780 atcgaaccta gaatacccaa accatctatc ccacctggtt cttcatgtcc acaaccgcgt 840 ggtcccacaa ttaaaccttg tcctccatgt aaatgcccag cacctaatct attaggtgga 900 ccatccgttt tcatattccc tccaaagatc aaagatgttc tcatgatttc ccttagcccc 960 attgtaacat gtgtggtcgt ggatgtgtca gaggatgatc cagacgtcca aattagctgg 1020 tttgtgaaca acgtggaggt acatacagct cagacacaaa ctcatagaga agattacaac 1080 agtactctcc gagtcgtcag tgccctcccg atacagcacc aggactggat gagtggcaaa 1140 gaatttaagt gtaaggtcaa taataaagac cttccagctc ccattgaaag aacgatttca 1200 aaacctaaag gatcagttag agctccacaa gtatatgttt tgcctccacc agaagaagaa 1260 atgactaaga aacaagtcac tttgacatgc atggttacag acttcatgcc tgaagatatt 1320 tacgttgagt ggaccaacaa cggcaaaact gaactaaact acaagaacac tgaaccagtc 1380 ctggactctg atggttcata ttttatgtac tcgaagctga gagtggaaaa aaagaattgg 1440 gtggaaagaa atagctactc ttgttcagtt gttcatgaag gtctgcataa tcatcacacg 1500 actaagagct tctcccggac tccgggttaa ggatccacta gt 1542 <210> 21 <211> 256 <212> PRT <213> Artificial Sequence <220> MAb BR55 light chain (amino acid sequence) <400> 21 Leu Glu Gly Ser Met Leu Pro Ala Ala Met Ala Ala Gly Phe Met Lys   1 5 10 15 Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala Ser Ser              20 25 30 Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu          35 40 45 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His      50 55 60 Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln  65 70 75 80 Ser Pro Lys Leu Leu Ile Ser Lys Val Ser Asn Arg Phe Ser Gly Val                  85 90 95 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys             100 105 110 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln         115 120 125 Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile     130 135 140 Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser 145 150 155 160 Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn                 165 170 175 Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu             180 185 190 Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp         195 200 205 Ser Thr Ser Ser Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr     210 215 220 Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr 225 230 235 240 Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys Leu Gln Gly Thr                 245 250 255 <210> 22 <211> 493 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > mAb BR55 heavy chain (amino acid sequence) <400> 22 Val Asp Ser Arg Met His Pro Thr Arg Trp Glu Leu Ser His Met Val   1 5 10 15 Asp Leu Gln Ala Ala Leu Val Ile Thr Asp Leu Gly Leu Ser              20 25 30 Leu Ile Phe Leu Val Leu Val Leu Lys Gly Val Gln Cys Glu Val Lys          35 40 45 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys      50 55 60 Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr  65 70 75 80 Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile                  85 90 95 Ser Asn Gly Gly Gly Ser Ser His Tyr Val Asp Ser Val Lys Gly Arg             100 105 110 Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met         115 120 125 Ser Arg Leu Arg Ser Glu Asp Thr Ala Met Tyr His Cys Ala Arg Gly     130 135 140 Met Asp Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val 145 150 155 160 Thr Val Ser Ala Ala Thr Thr Thr Ala Pro Ser Val Tyr Pro Leu Val                 165 170 175 Pro Gly Cys Ser Asp Thr Ser Gly Ser Ser Val Thr Leu Gly Cys Leu             180 185 190 Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Lys Trp Asn Tyr Gly         195 200 205 Ala Leu Ser Ser Gly Val Arg Thr Val Ser Ser Val Leu Gln Ser Gly     210 215 220 Phe Tyr Ser Leu Ser Ser Leu Val Thr Val Ser Ser Thr Trp Pro 225 230 235 240 Ser Gln Thr Val Ile Cys Asn Val Ala His Pro Ala Ser Lys Thr Glu                 245 250 255 Leu Ile Lys Arg Ile Glu Pro Arg Ile Pro Lys Pro Ser Ile Pro Pro             260 265 270 Gly Ser Ser Cys Pro Gln Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro         275 280 285 Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe     290 295 300 Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro 305 310 315 320 Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val                 325 330 335 Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr             340 345 350 Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala         355 360 365 Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys     370 375 380 Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser 385 390 395 400 Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro                 405 410 415 Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val             420 425 430 Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly         435 440 445 Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp     450 455 460 Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp 465 470 475 480 Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu                 485 490 <210> 23 <211> 621 <212> PRT <213> Artificial Sequence <220> Large single chain CO17-1A (amino acid sequence) <400> 23 Leu Glu Gly Ser Met Gly Ile Lys Met Glu Ser Gln Thr Leu Val Phe   1 5 10 15 Ile Ser Ile Leu Leu Trp Leu Tyr Gly Ala Asp Gly Asn Ile Val Met              20 25 30 Thr Gln Ser Pro Lys Ser Met Ser Ser Ser Val Gly Glu Arg Val Thr          35 40 45 Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr Val Ser Trp Tyr      50 55 60 Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser  65 70 75 80 Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala                  85 90 95 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala             100 105 110 Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly         115 120 125 Gly Thr Lys Leu Gly Ily Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Met Glu Trp Ser Arg Val Phe Ile Phe Leu 145 150 155 160 Leu Ser Val Thr Ala Gly Val His Ser Gln Val Gln Leu Gln Gln Ser                 165 170 175 Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Val Ser Cys Lys             180 185 190 Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln         195 200 205 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Val Ile Asn Pro Gly Ser     210 215 220 Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr 225 230 235 240 Ala Asp Lys Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr                 245 250 255 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Pro Trp Phe             260 265 270 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr         275 280 285 Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr     290 295 300 Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu 305 310 315 320 Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His                 325 330 335 Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser             340 345 350 Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn         355 360 365 Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro     370 375 380 Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro 385 390 395 400 Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys                 405 410 415 Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val             420 425 430 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn         435 440 445 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr     450 455 460 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 465 470 475 480 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu                 485 490 495 Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg             500 505 510 Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys         515 520 525 Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp     530 535 540 Ile Tyr Val Lys Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys 545 550 555 560 Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser                 565 570 575 Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser             580 585 590 Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser         595 600 605 Phe Ser Arg Thr Pro Gly Lys Ile Asp Ser Arg Gly Thr     610 615 620 <210> 24 <211> 625 <212> PRT <213> Artificial Sequence <220> Large single chain CO17-1AK (amino acid sequence) <400> 24 Leu Glu Gly Ser Met Gly Ile Lys Met Glu Ser Gln Thr Leu Val Phe   1 5 10 15 Ile Ser Ile Leu Leu Trp Leu Tyr Gly Ala Asp Gly Asn Ile Val Met              20 25 30 Thr Gln Ser Pro Lys Ser Met Ser Ser Ser Val Gly Glu Arg Val Thr          35 40 45 Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr Val Ser Trp Tyr      50 55 60 Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile Tyr Gly Ala Ser  65 70 75 80 Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala                  85 90 95 Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala             100 105 110 Asp Tyr His Cys Gly Gln Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly         115 120 125 Gly Thr Lys Leu Gly Ily Lys Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly     130 135 140 Ser Gly Gly Gly Gly Ser Met Glu Trp Ser Arg Val Phe Ile Phe Leu 145 150 155 160 Leu Ser Val Thr Ala Gly Val His Ser Gln Val Gln Leu Gln Gln Ser                 165 170 175 Gly Ala Glu Leu Val Arg Pro Gly Thr Ser Val Lys Val Ser Cys Lys             180 185 190 Ala Ser Gly Tyr Ala Phe Thr Asn Tyr Leu Ile Glu Trp Val Lys Gln         195 200 205 Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Val Ile Asn Pro Gly Ser     210 215 220 Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr 225 230 235 240 Ala Asp Lys Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr                 245 250 255 Ser Asp Ser Ala Val Tyr Phe Cys Ala Arg Asp Gly Pro Trp Phe             260 265 270 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr         275 280 285 Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr     290 295 300 Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu 305 310 315 320 Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His                 325 330 335 Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser             340 345 350 Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn         355 360 365 Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro     370 375 380 Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro 385 390 395 400 Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys                 405 410 415 Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val             420 425 430 Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn         435 440 445 Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr     450 455 460 Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp 465 470 475 480 Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu                 485 490 495 Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg             500 505 510 Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys         515 520 525 Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp     530 535 540 Ile Tyr Val Lys Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys 545 550 555 560 Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser                 565 570 575 Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser             580 585 590 Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser         595 600 605 Phe Ser Arg Thr Pro Gly Lys Ile Asp Lys Asp Glu Leu Ser Arg Gly     610 615 620 Thr 625 <210> 25 <211> 673 <212> PRT <213> Artificial Sequence <220> Large single chain BR55K (amino acid sequence) <400> 25 Val Asp Ser Arg Met Leu Pro Ala Ala Met Ala Ala Gly Phe Met Lys   1 5 10 15 Leu Pro Val Arg Leu Leu Val Leu Met Phe Trp Ile Pro Ala Ser Ser              20 25 30 Ser Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu          35 40 45 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His      50 55 60 Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln  65 70 75 80 Ser Pro Lys Leu Leu Ile Ser Lys Val Ser Asn Arg Phe Ser Gly Val                  85 90 95 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys             100 105 110 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln         115 120 125 Gly Ser His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile     130 135 140 Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 145 150 155 160 Met His Pro Thr Arg Trp Glu Leu Ser His Met Val Asp Leu Gln Ala                 165 170 175 Ala Ala Leu Val Ile Thr Met Asp Leu Gly Leu Ser Leu Ile Phe Leu             180 185 190 Val Leu Val Leu Lys Gly Val Gln Cys Glu Val Lys Leu Val Glu Ser         195 200 205 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala     210 215 220 Thr Ser Gly Phe Thr Phe Ser Asp Tyr Tyr Met Tyr Trp Val Arg Gln 225 230 235 240 Thr Pro Glu Lys Arg Leu Glu Trp Val Ala Tyr Ile Ser Asn Gly Gly                 245 250 255 Gly Ser Ser His Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser             260 265 270 Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Arg Leu Arg         275 280 285 Ser Glu Asp Thr Ala Met Tyr His Cys Ala Arg Gly Met Asp Tyr Gly     290 295 300 Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 305 310 315 320 Ala Thr Thr Ala Pro Ser Val Tyr Pro Leu Val Pro Gly Cys Ser                 325 330 335 Asp Thr Ser Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr             340 345 350 Phe Pro Glu Pro Val Thr Val Lys Trp Asn Tyr Gly Ala Leu Ser Ser         355 360 365 Gly Val Arg Thr Val Ser Ser Val Leu Gln Ser Gly Phe Tyr Ser Leu     370 375 380 Ser Ser Leu Val Thr Val Ser Ser Thr Trp Ser Ser Gln Thr Val 385 390 395 400 Ile Cys Asn Val Ala His Pro Ala Ser Lys Thr Glu Leu Ile Lys Arg                 405 410 415 Ile Glu Pro Arg Ile Pro Lys Pro Ser Ile Pro Pro Gly Ser Ser Cys             420 425 430 Pro Gln Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys         435 440 445 Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro     450 455 460 Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys 465 470 475 480 Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp                 485 490 495 Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg             500 505 510 Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln         515 520 525 His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn     530 535 540 Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly 545 550 555 560 Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu                 565 570 575 Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met             580 585 590 Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu         595 600 605 Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe     610 615 620 Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn 625 630 635 640 Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr                 645 650 655 Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys Asp Glu Leu Gly Ser Thr             660 665 670 Ser     <210> 26 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding KDEL in Large single chain CO17-1AK <400> 26 aaggacgaac tt 12 <210> 27 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding KDEL in Large single chain BR55K <400> 27 aaagatgaac tt 12 <210> 28 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Large Single Chain CO17-1A <400> 28 cgctcgaggg atccatgggc attaaaatgc g 31 <210> 29 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Large Single Chain CO17-1A <400> 29 gcggtacctc tagaatcgat tttacccggg c 31 <210> 30 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Large Single Chain CO17-1AK <400> 30 cgctcgaggg atccatgggc attaaaatgc g 31 <210> 31 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Large Single Chain CO17-1AK <400> 31 gcggtacctc tagattaaag ttcgtcctta tcgatgc 37 <210> 32 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for Large Single Chain BR55K <400> 32 cggtcgactc tagaatgctc ccggccgctc g 31 <210> 33 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for Large Single Chain BR55K <400> 33 gcactagtgg atcctcaaag ttcatcttta cccggc 36

Claims (6)

(a) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker to form a single chain antibody;
(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;
(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;
(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant; And
(e) crossing the transgenic plant individuals produced in steps (b) and (d) above to produce a bispecific antibody.
The method according to claim 1,
(a) A single chain antibody (heavy chain variable region (VH) and light chain variable region (VL) linked by a linker to a monoclonal antibody recognizing GA733-2 epitope) Creating;
(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;
(c) A single chain antibody is obtained by linking the heavy chain variable region (VH) and the light chain variable region (VL) of a monoclonal antibody different from the monoclonal antibody recognizing Lewis Y as a epitope of breast cancer cell surface glycoprotein, ;
(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;
(e) crossing the transgenic plants produced in steps (b) and (d) to produce a hybrid plant.
(a) linking a heavy chain variable region (VH) and a light chain variable region (VL) of a monoclonal antibody with a linker to form a single chain antibody;
(b) introducing the gene of the single chain antibody prepared in the step (a) into a plant cell to obtain a transformed plant;
(c) linking the heavy chain variable region (VH) and the light chain variable region (VL) of the monoclonal antibody different from the monoclonal antibody of the step (a) with a linker to form a single chain antibody;
(d) introducing the gene of the single chain antibody produced in the step (c) into a plant cell to obtain a transformed plant;
(e) crossing the transgenic plants produced in steps (b) and (d) above; And
(f) obtaining a protein from the crossed plant produced in step (e).
4. The method according to any one of claims 1 to 3, wherein the linker is a polypeptide represented by SEQ ID NO: 1.
4. The method according to any one of claims 1 to 3, wherein the gene coding for the short-chain antibody of steps (b) and (d) is further characterized in that it further comprises an endoplasmic reticulum residual peptide represented by SEQ ID NO: 2 Way.
6. The method according to any one of claims 1 to 5, wherein the plant is a tobacco ( Nicotiana tabacum ). &lt; / RTI &gt;

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