KR20150041588A - Bicistronic expression vector for expressing antibody and method for producing antibody using the same - Google Patents

Abstract

The present invention relates to a bicistronic expression vector for expressing antibody which comprises: a first expression cassette including ′promoter-UTR-intron-antibody light chain gene-polyA′ and a second expression cassette including ′promoter-UTR-intron-antibody heavy chain gene- IRES (internal ribosome entry site)-amplification gene-polyA′, the animal cell wherein the expression vector is transformed and the manufacturing method including the stage of culturing the animal cell. Using the bicistronic expression vector for expressing antibody including the intron, an expression vector which expresses the antibody with high efficiency can be produced, and the expression vector can produce antibodies with high efficiency and stability by culturing transformed animal cells.

Description

Bicystronic Expression Vector for Expression of Antibody and Method of Producing Antibody Using the Bicistronic Expression Vector for Expressing Antibody

(I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -introne-antibody light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR-intron-antibody heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA', a second expression cassette for expression of the antibody, A vector transformed animal cell, and a method for producing an antibody comprising culturing the animal cell.

Although various human-derived proteins are expressed using genetic recombination technology, protein having normally biological activity is synthesized only when mammalian cells are used due to a series of processes such as eukaryotic cell-specific glycation and phosphorylation. The immunoglobulin G (IgG) gene, which is widely used as a therapeutic antibody, is composed of a heavy chain gene and a light chain gene. The heavy and light chain proteins from each gene are secreted out of the cell by binding to each other during folding within the cell. In order to produce the recombinant antibody protein, CHO cells (Chinese hamster ovary cells, Chinese hamster ovary cells), which is one of the mammalian cells, are frequently used (Kaufman et al., Mol. Cell. Biol. (1985) 5, 1750).

Among the various strategies used to produce recombinant expression vectors capable of inducing stronger and more stable gene expression in mammalian cells, a representative method is to select a strong promoter to increase gene expression rate. The promoter is a component of the DNA on which mRNA synthesis starts by binding of various transcription factors and TATA box, which binds with RNA polymerase, and the amount of gene expression depends on the performance of the promoter. For high antibody expression, it is preferable to always have a high expression. Typical strong promoters include SV40 (simian virus 40) promoter, CMV (Cytomegalovirus) promoter and EF1-? (Elongation factor 1?) Promoter.

In addition to enhancing the mRNA synthesis ability of the vector itself through the use of a strong promoter, there is a gene amplification method widely used to increase the productivity of the therapeutic protein. To summarize the method, an external gene is transformed into a cell, and then the cell is isolated by using a chemical agent to selectively cultivate only the cell that has been recombined with the chromosome of the foreign gene so that more proteins can be expressed , An increased amount of the chemical is added to the medium to induce amplification of the foreign gene recombined into the chromosome. Representative genes used for gene amplification include DHFR (dehydrofolate reductase) and GS (glutamine synthetase). Methotrexate (hereinafter referred to as 'MTX') is used for DHFR and Methionine sulfoximine (hereinafter referred to as 'MSX') is used for GS for the amplification of these amplified genes. Through such a gene amplification process, it becomes possible to prepare a cell line having more than 1,000 amplified foreign genes.

Generally, in order to produce a cell line that continuously expresses an antibody gene, a plasmid vector DNA containing a heavy chain and a light chain gene to be expressed and a vector DNA having an amplification gene are co-transformed into cells together, (Kaufman, Methods in Enz. (1990) 185, 487), or a method in which an amplified gene is expressed by the SV40 promoter in one vector, and a cell line in which the vector is inserted into the chromosome is isolated .

Another method for producing bi-systolic vectors by an amplification gene expression method is available. Unlike prokaryotes, eukaryotes, in principle, produce one mRNA in a single promoter and only the gene is decoded to synthesize the protein. However, some viruses have a sequence that can bind to ribosomes in the middle of mRNA, allowing the synthesis of multiple proteins in one mRNA. When the antibody gene and the amplified gene are expressed by different promoters, only the amplified gene part can be amplified and the amplification of the antibody gene may not be performed during gene amplification. If the internal ribosome entry site (IRES) is used, it is expressed from the same promoter This problem can be avoided. However, such a bi-systolic vector strategy still has a disadvantage in that it can not produce an excessive amount of desired protein, and a stronger strategy is required.

Meanwhile, bevacizumab (also called Avastin ^® ) is a humanized antibody against VEGF developed by Genentech, Inc. of the United States. It acts as an inhibitor of angiogenesis and is useful for the treatment of colorectal cancer, lung cancer, kidney cancer, And is being used as an anti-tumor agent for treatment. Tocilizumab (also known as Actemra ^® ) is a human monoclonal antibody to the IL-6 receptor developed by Roche, a Swiss pharmaceutical company. It is marketed as Actemra in the US, Europe, Japan Have been approved for rheumatoid arthritis. Denosumab (also known as Prolia ^® ) is a human monoclonal antibody to the Receptor activator of NF-kB ligand (RANKL) developed by Amgen, USA, Canada, and Europe, and has been approved as a drug to prevent bone fracture in patients who have undergone transplantation of solid tumors to bone in Europe under the product name Xgeva.

Belimumab (also known as Benlysta ^® ) is a human monoclonal antibody against a soluble B lymphocyte stimulator (BLyS) developed by GlaxoSmithKline (GSK), a multinational pharmaceutical company, , Lupus (systemic lupus erythematosus, SLE) in Canada and Europe. Golimumab (also known as Simponi ^® ) is a human monoclonal antibody against TNFα developed by Janssen Biotech, Inc. (Centocor), a subsidiary of global pharmaceutical company Johnson & Johnson. As a clone antibody, it has been approved for the indication of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis in the United States, Canada and Korea under the product name Simponi.

On the other hand, Wu Ste Thank Kinu (Ustekinumab, also known as Stelara ^®) is a multinational pharmaceutical company Janssen Pharmaceutical Companies (Janssen Pharmaceuticals, Inc.) by interleukin-12 (Interleukin-12, IL- 12) and interleukin-23, developed by the (Interleukin- 23, IL-23), approved by the US Food and Drug Administration (FDA) for psoriatic arthritis, has been approved for severe psoriasis, - are in clinical trials for moderate-to-severe plaque psoriasis, multiple sclerosis and sarcoidosis. Thank Ibis Limousin (Ipilimumab, also known as Yervoy ^®) is a US pharmaceutical company Bristol-Myers Squibb (Bristol-Myers Squibb Co., BMS ) used a cytotoxic T lymphocyte antigen -4 (Cytotoxic T-Lymphocyte Antigen, CTLA developed -4), approved by the US Food and Drug Administration (FDA) under the product name Yervoy for melanoma and skin cancer, and non-small cell lung carcinoma (NSCLC), small cell lung cancer (SCLC), bladder cancer, and metastatic hormone-refractory prostate cancer.

The development of mass production methods for such blockbuster class antibody therapeutics is required.

Under these circumstances, the present inventors have made intensive efforts to find a method capable of inducing strong and stable gene expression in a variety of mammalian cells. As a result, the present inventors have found that bi-systolic expression including UTR (untranslation region) And the light and heavy chain genes of various antibody therapeutic agents can be highly expressed simultaneously with the amplified gene. Thus, the present invention has been completed.

One object of the present invention is to provide a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-antibody light chain gene -polyA' and a 'promoter-UTR-intron-antibody heavy chain-IRES (internal ribosome entry site) Lt; / RTI > expression vector comprising a second expression cassette comprising a first expression cassette comprising the first expression cassette and a second expression cassette comprising the second expression cassette.

Another object of the present invention is to provide an animal cell in which the expression vector is transformed.

It is still another object of the present invention to provide a method for producing an antibody comprising culturing the animal cells.

In one aspect of the present invention, the present invention provides a recombinant vector comprising (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-antibody light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR-intron-antibody heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA' .

The present invention provides a bi-systolic expression vector designed to express the heavy and light chain genes of the antibody and the amplified gene as a single gene, including UTR and intron, so as to induce strong and stable gene expression in various mammalian cells There is that feature in what can be done. In particular, the present invention provides a novel expression cassette comprising a UTR and an intron.

Specifically, the present invention relates to a method for producing a recombinant vector comprising (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-bevacizumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-bevacizumab internal ribosome entry site-amplification gene -polyA ' to provide. (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-Tocilizumab light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR-intron-tosylidium heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA' to provide. (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -termine-denosumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-denosumab internal gene-IRES-amplification gene-polyAA ' to provide. (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-belimab light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR-intron-valimabeat heavy chain gene-IRES (internal ribosome entry site)-amplification gene -polyA' to provide a biSystronic expression vector for antibody expression do. (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-Golimumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-golimumab heavy chain gene-IRES (internal ribosome entry site) -enzyme gene -polyA 'to provide a biSystronic expression vector for antibody expression do. (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -introne-Ustekinumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-utestinumum heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA 'Lt; / RTI > (I) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-ipilimumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-internal ribosome entry site-IRES-amplification gene -polyA ' to provide.

The term " bicistronic " in the present invention means a system in which a ribosomal polypeptide can be synthesized in the interior of mRNA in eukaryotic cells, and various proteins can be synthesized from one mRNA. A vector was designed so that an antibody gene and an amplified gene can be expressed simultaneously.

Generally, contrary to the fact that prokaryotic cells such as Escherichia coli have a polycistronic system in which a ribosome binds to various positions of one mRNA to synthesize multiple proteins at once, eukaryotic cells decode proteins from mRNA ), The ribosome is attached to the 5 'end of the mRNA, and then the AUG, which is the initiation codon, is searched through a scanning method. As the synthesis of the protein starts from the AUG codon, one polypeptide is necessarily formed from one mRNA (Kozac sequence) plays an important role in the recognition of ATG and the start of protein synthesis. However, in the case of the EMC virus (Encephalomyocarditis virus, hereinafter referred to as "EMCV"), the RNA sequence has a special structure called an internal ribosome entry site (IRES) Several proteins can be synthesized from an mRNA in eukaryotic cells (Jang et al., J. Virol. (1989) 63, 1651). In the present invention, in order to allow simultaneous expression of an antibody gene and an amplified gene, a vector was designed so that IRES follows the heavy chain gene of the antibody, followed by an amplified gene.

The term "vector" in the present invention refers to a gene construct containing an essential regulatory element such as a promoter so that a desired gene can be expressed in a suitable host cell. For the purpose of the present invention, And may be a bi-systronic expression vector. For example, the vector of the present invention may comprise (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -introne-bevacizumab light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR (untranslation region) -introne-bevacizumab internal gene-IRES (internal ribosome entry site) -enzyme gene -polyA' Lt; / RTI > expression vector.

In yet another example, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-tocisylum light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-tosylidium-internal ribosome entry site (IRES) -amplification gene -polyA ' Lt; / RTI > In yet another embodiment, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR-intron-denosumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-denosumab internal gene-IRES-amplification gene-polyAA ' Lt; / RTI > In yet another example, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-Golimumab light chain gene -polyA'; And (ii) a second expression cassette comprising the promoter-UTR-intron-golimumab heavy chain-IRES (internal ribosome entry site) -enzyme gene -polyA '. . In yet another example, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-valimabeat light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR (untranslation region) -introne-valimabeat heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA' Expression vector. In yet another embodiment, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -intron-utestinimab light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR (untranslation region) -introne-ustecunimab heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA' Lt; / RTI > expression vector. In yet another embodiment, the vector of the present invention comprises (i) a first expression cassette comprising a 'promoter-UTR-intron-ephilimumab light chain gene -polyA'; And (ii) a second expression cassette comprising a 'promoter-UTR (untranslation region) -introne-ephilimumab gene-IRES (internal ribosome entry site) -amplification gene -polyA' Lt; / RTI > expression vector.

In the present invention, the term "promoter " means a DNA base sequence region to which transcription regulatory elements bind, and for the purpose of the present invention, a promoter capable of inducing strong and stable gene expression can be used.

The promoter may be selected from the group consisting of SV40 (simian virus 40) early promoter, CMV (cytomegalovirus) promoter and EF1-? (Elongation factor 1?) Promoter, May be hEF1-alpha (human elongation factor 1-alpha). More preferably a cytomegalovirus promoter, and most preferably a promoter of strain Cytomegalovirus strain ADAD169. In one embodiment of the present invention, a promoter in the Cytomegalovirus strain AD169 genome (genBank: X17403.1) sequence, which is one of the most powerful animal cell promoters known so far to induce high antibody expression, Respectively.

In the present invention, the term "UTR (untranslation region)" refers to the docking site of mRNA and generally refers to both ends of the coding region. In particular, the 5 'end portion is referred to as the 5' UTR and the 3 'end portion is referred to as the 3' UTR. In particular, the UTR in the present invention is not limited as long as it has an effect of increasing the expression level of the gene contained in the bi-systolic vector of the present invention. In particular, it may be a UTR derived from CMV. The present invention is characterized in that the expression level of a gene into which a biSystronic expression vector containing an expression cassette containing both UTR and intron is simultaneously increased to provide a novel antibody expression vector.

The UTR of the present invention may be, but is not limited to, the UTR of Cytomegalovirus strain AD169 of SEQ ID NO: 2. In one embodiment of the present invention, an expression vector was prepared by inserting the UTR in the Cytomegalovirus strain AD169 genome (genBank: X17403.1) sequence between promoter and antibody light chain and heavy chain gene, respectively, for more powerful gene expression.

In the present invention, the term "intron" refers to a DNA sequence that does not have any genetic information as a characteristic sequence found in eukaryotic cells and thus can not produce a protein. When mRNA is synthesized by RNA polymerase in the nucleus, But it is the part removed through the splicing process in the process of getting out of the nucleus. As a result of studies that the amount of gene expression of the plasmid containing the untranslated exon is higher than that of the intron without the intron, introduction of the heterologous intron into the recombinant expression vector results in more stable and continuous gene expression Improved vectors have been developed and are being used. However, the existing intron insertion vector has been disadvantageous in that it is used in a monocystic vector and expresses only one gene. Therefore, the inventors of the present invention have devised that, in order to overcome such disadvantages, when the external intron gene is inserted into the bi-systolic vector system after the promoter, highly efficient gene expression can be achieved. In particular, the intron of the present invention may be an intron derived from CMV.

The intron of the present invention may be an intron of Cytomegalovirus strain AD169 of SEQ ID NO: 3, though it is not limited thereto. In one embodiment of the present invention, an expression vector was prepared by inserting the intron on the Cytomegalovirus strain AD169 genome (genBank: X17403.1) sequence between the promoter and the antibody light chain and the heavy chain gene, respectively, in order to construct a stronger promoter .

In the expression vector of the present invention, the origins of the promoter, the UTR and the intron may be the same or different. For example, the promoter may be an SV40 early promoter, the UTR and the intron may be SV40; The promoter is a CMV promoter, the UTR and the intron may be CMV-derived; The promoter may be an hEF1-? Promoter, and the UTR and the intron may be derived from hEF1?.

The term "amplified gene" in the present invention means a gene amplified to induce expression with an antibody gene stably expressing in the present invention, and the amplified gene and the antibody gene are recombined with an intracellular chromosome In order to allow growth only, cells that do not contain amplified genes can be amplified using chemicals, using the principle that they do not grow in the presence of the treated chemicals. When the biSystronic expression vector of the present invention is used, the amplification gene and the antibody gene are induced to be expressed together as a single gene, and ultimately, the desired antibody gene can be highly expressed.

The amplified gene may be, but is not limited to, GS (glutamine synthetase) or DHFR (dehydrofolate reductase). In one embodiment of the present invention, pCYB204ID expression vector using GS as an amplified gene and pCYB204ID expression vector using DHFR as an amplified gene were constructed (Figs. 5 and 6), pCYBBSS001 (Fig. 8), pCYBBSS002 10), pCYBBSS004 (FIG. 11), pCYBBSS005 (FIG. 12) and pCYBBSS006 (FIG. 13) expression vectors were constructed and it was confirmed that the expression vectors were useful for highly expressing the antibody gene.

In the present invention, the term "antibody" refers to a substance produced by stimulation of an antigen in the immune system, which specifically binds to a specific antigen and migrates lymph and blood to cause an antigen-antibody reaction. For the purpose of the present invention, the antibody is intended for continuous high-level expression. The antibody gene of the present invention can be induced to express the antibody gene stably and stably using the expression vector of the present invention, and the expression vector Animal cells can be used to efficiently produce antibodies.

The antibody is a protein bound to an amino acid and an oligosaccharide, and two light chains and two heavy chains are formed as Y-shaped proteins through disulfide bonds. In one embodiment of the present invention, an expression vector capable of producing antibodies having both light and heavy chains was prepared by preparing a vector comprising a light chain expression cassette and heavy chain expression cassette of an antibody comprising UTR and intron in one vector.

The antibody of the present invention is not limited thereto, but may preferably include all therapeutic antibodies conventionally used in the art. Examples include Bevacizumab, an antibody that targets vascular endothelial growth factor-A (VEGF-A), Tocilizumab, an antibody targeting the IL-6 receptor, a receptor activator of NF-kB ligand (Golimumab), which is an antibody that targets TNFα, is an antibody that targets the RANKL (Denosumab), an antibody against B lymphocyte stimulator (BLyS), Belimumab, which is an antibody targeting RANKL, , Ustekinumab and Cytotoxic T lymphocyte Antigen-4 (Interleukin-12, IL-12), which are antibodies to interleukin-12 and interleukin-23 Lt; RTI ID = 0.0 > Ipilimumab < / RTI >

The above-mentioned bevacizumab, also called Avastin, is a humanized antibody developed by Genentech, USA, which is known as a therapeutic agent for colorectal cancer, lung cancer, kidney cancer and brain cancer. According to the embodiment of the present invention, it was confirmed that, when a typical therapeutic antibody, bevacizumab, was introduced into a bi-systolic expression vector for antibody expression of the present invention, the antibody was effectively produced.

The above-mentioned tosicilimum is a human monoclonal antibody developed by Chugai, a Japanese subsidiary of Roche, a Swiss pharmaceutical company, and is marketed as Actemra in the US, Europe and Japan as a therapeutic agent for rheumatoid arthritis I got approval. In the examples of the present invention, when the tosicilimum is introduced into the bi-systolic expression vector for antibody expression of the present invention, it is confirmed that the biosyntronic expression vector of the present invention is effectively produced by the above- It was confirmed that the system is a high-expression system of zymed antibody.

The above-mentioned denosumab was a human monoclonal antibody developed by Amgen, USA and approved for the indications of osteoporosis in USA, Canada and Europe under the product name of Prolia, and the product name Xgeva , Which has been approved as a drug to prevent bone fracture in patients with prostate cancer in Europe. According to an embodiment of the present invention, it was confirmed that when the denosumab was introduced into the biSistronic expression vector for antibody expression of the present invention, the biosystronic expression vector of the present invention effectively produced the antibody, It was confirmed that this system is a system for high antibody expression.

The belimab is a human monoclonal antibody against a soluble B lymphocyte stimulator (BLyS; or B cell activating factor, BAFF) developed by GlaxoSmithKline (GSK), a multinational pharmaceutical company. As an antibody, Lupus (systemic lupus erythematosus, SLE) was approved in the United States, Canada and Europe. In the examples of the present invention, it was confirmed that velimovat was introduced into the bi-systolic expression vector for antibody expression of the present invention and that the antibody was effectively produced, whereby the bi-systronic expression vector of the present invention was highly expressed .

Golimumab is a human monoclonal antibody developed by Janssen Biotech, Inc. (Centocor), a subsidiary of Johnson & Johnson, a global pharmaceutical company, Has been approved for treatment of rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis in the United States, Canada and Korea. In an embodiment of the present invention, when gollymum was introduced into a bi-systolic expression vector for antibody expression of the present invention, it was confirmed that the antibody was effectively produced, so that the bi-systolic expression vector of the present invention inhibited High expression system.

The above-mentioned uestekinum is a human single antibody against interleukin-12 (IL-12) and interleukin-23 (Interleukin-23, IL-23) developed by a multinational pharmaceutical company, Janssen Pharmaceuticals, As a clonal antibody, the US Food and Drug Administration (FDA) has been approved for psoriatic arthritis and has been approved for severe psoriasis, moderate-to-severe psoriasis, plaque psoriasis, multiple sclerosis, and sarcoidosis. In the examples of the present invention, it was confirmed that Ustekinumab was introduced into a bi-systolic expression vector for antibody expression of the present invention and that the antibody was effectively produced. Thus, the bi-systunic expression vector of the present invention was found to contain Ustekinumab It was confirmed that this system is a system for high antibody expression.

The humanized monoclonal antibody against the Cytotoxic T-Lymphocyte Antigen (CTLA-4) developed by Bristol-Myers Squibb Co. (BMS), an American pharmaceutical company, As an antibody, Yervoy has been approved by the US Food and Drug Administration (FDA) for melanoma and skin cancer, and has been approved for non-small cell lung carcinoma (NSCLC), small cell carcinoma lung cancer, SCLC, bladder cancer, and metastatic hormone-refractory prostate cancer. In particular, it is administered in the form of an intravenous injection of the approved example of the FDA as a melanoma therapeutic agent, and is effective by helping the human immune system to recognize and attack melanoma-forming tumor cells as targets. In an embodiment of the present invention, it has been confirmed that eicosamimetic is introduced into a bi-systolic expression vector for antibody expression of the present invention to effectively produce the antibody, whereby the bi-systolic expression vector of the present invention is capable of expressing the ephilimumim antibody High expression system.

"Antibody light chain gene" in the present invention means a gene encoding a light chain part in an antibody that is a Y-shaped protein in which two light chains and two heavy chains are combined with an amino acid and oligosaccharide. In the present invention, an antibody light chain gene includes, but is not limited to, a light chain gene for any antibody whose expression level is increased using the bi-systolic vector of the present invention. For example, therapeutic antibodies bevacizumab, A nucleotide sequence encoding a light chain portion of dehydroepiandol, deisosumab, belimomat, golimumat, ustekinumab, or eicilimumab. Specifically, SEQ ID NO: 13 for the light chain portion of bevacizumab, SEQ ID NO: 17 for the light chain portion of tosyljewax, SEQ ID NO: 21 for the light chain portion of denosumab, SEQ ID NO: 25 for the light chain portion of velimovat, A nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 29 for the light chain portion of golimumab, SEQ ID NO: 33 for the light chain portion of uestekinum and SEQ ID NO: 37 for the light chain portion of epticimumab SEQ ID NO: 11 encoding the light chain portion of bevacizumab, SEQ ID NO: 15 encoding the light chain portion of tosyloidsum, SEQ ID NO: 19 encoding the light chain portion of denosumab, light chain SEQ ID NO: SEQ ID NO: 23 coding for the light chain portion of Ustekim map, SEQ ID NO: 27 coding for the light chain portion of golimumab, SEQ ID NO: It may be one consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO: 35 encoding the light chain portion of the Philippines mumap.

"Antibody heavy chain gene" in the present invention means a gene that encodes a heavy chain portion in an antibody that is a Y-shaped protein in which two light chains and two heavy chains are combined with an amino acid and oligosaccharide. In the present invention, an antibody heavy chain gene includes, but is not limited to, a heavy chain gene for an antibody whose expression level is increased using the bissistronic vector of the present invention. For example, therapeutic antibodies bevacizumab, A nucleotide sequence that encodes the heavy chain portion of deoxysuccine, belimomat, golimumoo, uestekinum, or eicilimumab. Specifically, SEQ ID NO: 14 for the heavy chain portion of bevacizumab, SEQ ID NO: 18 for the heavy chain portion of tosyljewax, SEQ ID NO: 22 for the heavy chain portion of denosumab, SEQ ID NO: 26 for the heavy chain portion of velimovat, A nucleotide sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 30 for the heavy chain portion of golimumab, SEQ ID NO: 34 for the heavy chain portion of uestekinumab, and SEQ ID NO: 38 for the heavy chain portion of epticimumab , More specifically SEQ ID NO: 12 encoding the heavy chain portion of bevacizumab, SEQ ID NO: 16 encoding the heavy chain portion of tosyloids, SEQ ID NO: 20 encoding the heavy chain portion of denosumab, SEQ ID NO: 24 encoding the portion of SEQ ID NO: 24, SEQ ID NO: 28 encoding the heavy chain portion of golimunatum, SEQ ID NO: It may be one consisting of the nucleotide sequence selected from the group consisting of SEQ ID NO: 36 encoding the heavy chain portion of the Philippines mumap.

The term "IRES (internal ribosome entry site, internal ribosome entry point, SEQ ID NO: 4)" in the present invention refers to a specific region existing in the mRNA in which ribosomes are directly bound so that various proteins can be synthesized from one mRNA in eukaryotic cells , And serves to enable the expression vector of the present invention to become a bi-systronic expression vector.

The decipherment of mRNA in eukaryotic cells is generally cap-structure dependent on the 5 'end of the mRNA. However, in the case of this cap structure-independent translation, the ribosome directly binds to a specific region existing in the mRNA, This is called an internal start mechanism. The site on the mRNA to which the ribosome directly binds in the internal initiation mechanism is called IRES. In addition to virus mRNA such as poliovirus and EMC virus, immunoglobulin heavy chain binding protein (BiP) mRNA, cell mRNA such as Drosophila antenna gene (Antp) mRNA .

The term "polyA" in the present invention is referred to as a polyadenylic acid or polyadenylic acid fragment, and refers to a contiguous sequence of adenylic acid that is commonly present at the 3 'end of the eukaryotic mRNA. Its length is on the order of 10 to 200 nucleotides and can be variably controlled depending on the allowable size of the expression vector backbone. polyA is known to be involved in mRNA stabilization, detoxification, and transport from the nucleus to the cytoplasm.

The bSystronic expression vector for producing an antibody of the present invention is not limited thereto, but preferably includes pCYB204IG shown in Fig. 5, pCYB204ID shown in Fig. 6, pCYBBSS001 shown in Fig. 8, pCYBBSS002 shown in Fig. 9 PCYBBSS003 shown in Fig. 10, pCYBBSS004 shown in Fig. 11, pCYBBSS005 shown in Fig. 12, or pCYBBSS006 shown in Fig.

The pCYB204IG expression vector contained a first expression cassette comprising the 'CMV promoter-UTR-intron-bevacizumab light chain gene -polyA' and a 'CMV promoter-UTR-intron-bevacizumab heavy chain gene-IRES-GS-polyA' Wherein the pCYB204ID expression vector comprises a first expression cassette comprising the 'CMV promoter-UTR-intron-bevacizumab light chain gene -polyA' and a second expression cassette comprising the 'CMV promoter-UTR-intron-beba Is an expression vector comprising a second expression cassette comprising the < RTI ID = 0.0 > ssumugum < / RTI > heavy chain gene-IRES-DHFR-polyA. The pCYBBSS001 expression vector contains a first expression cassette comprising the 'CMV promoter-UTR-intron-tosylidium light chain gene -polyA' and a 'CMV promoter-UTR-intron-tosylidium heavy chain gene-IRES-DHFR-polyA'Lt; RTI ID = 0.0 > expression cassette. ≪ / RTI > The pCYBBSS002 expression vector comprises a first expression cassette comprising the 'CMV promoter-UTR-intron-denosumab light chain gene -polyA' and a 'CMV promoter-UTR-intron-denosumab heavy chain gene-IRES-DHFR-polyA'Lt; RTI ID = 0.0 > expression cassette. ≪ / RTI > The pCYBBSS003 expression vector comprises a first expression cassette comprising the 'CMV promoter-UTR-intron-valimuvat light chain gene -polyA' and a 'CMV promoter-UTR-intron-valimuvat heavy chain gene-IRES-DHFR-polyA' RTI ID = 0.0 > expression cassette. ≪ / RTI > The pCYBBSS004 expression vector comprises a first expression cassette comprising the 'CMV promoter-UTR-intron-golimuvat light chain gene -polyA' and a 'CMV promoter-UTR-intron-golimumab heavy chain gene-IRES-DHFR-polyA' RTI ID = 0.0 > expression cassette. ≪ / RTI > The pCYBBSS005 expression vector comprises a first expression cassette comprising the 'CMV promoter-UTR-intron-utestinimab light chain gene -polyA' and a 'CMV promoter-UTR-intron-eustekinumab heavy chain gene-IRES-DHFR-polyA &Quot;'.≪ / RTI > In addition, the pCYBBSS006 expression vector is composed of a first expression cassette comprising the 'CMV promoter-UTR-intron-ephilimimab light chain gene -polyA' and a 'CMV promoter-UTR-intron-ephilimumab heavy chain gene-IRES-DHFR-polyA &Quot;'.≪ / RTI >

In one embodiment of the present invention, pCYBIG was prepared using GS as an amplification gene, a light chain expression cassette and a heavy chain expression cassette, and a pCYBID expression vector containing DHFR instead of GS as an amplified gene in the pCYBIG expression vector was prepared .

As described above, the biSystronic expression vector of the present invention is designed such that the heavy and light chain genes of the antibody and the amplified gene are expressed as one gene, and can be used to stably and efficiently express the antibody.

In another embodiment, the present invention provides an animal cell in which the expression vector is transformed.

In the present invention, an expression vector transformed into an animal cell is a biSistronic expression vector for antibody expression, as described above.

The term "transfection" in the present invention refers to a method of directly introducing DNA into cultured animal cells to mutate the genetic traits of cells, and generally, introducing a gene of interest into a medium such as a plasmid do. The transformation can be carried out according to a conventional method in the art, and examples thereof include calcium phosphate coprecipitation, DEAE-dextran treatment, electroporation, lipofectamine treatment, redistribution (an artificial membrane called ribosome, A method of fusing a cell to produce a DNA complex). In one embodiment of the present invention, the light chain / heavy chain expression vector was transformed into cells using lipofectamine.

As long as the animal cell can produce an antibody, an animal cell used in the field can be used without limitation. Preferably, Chinese hamster ovary cell (CHO) can be used.

In a specific embodiment of the present invention, pCYB204IG and pCYB204GS bicistronic vectors for the expression of bevacizumab antibody were prepared by inserting the light chain and heavy chain genes of bevacizumab into a vector in which the amplified gene was different from GS and DHFR. As a result of comparing the amount of bevacizumab antibody expressed in each of the two vectors, it was found that the cell line transformed with the pCYB204ID vector was highly expressed twice more than that of the cell line transformed with the pCYB204IG vector Respectively.

Also, in a specific embodiment of the present invention, pCYBBSS001 biSystronic vector for expression of a tosylish jumph antibody was prepared by inserting a light chain and a heavy chain gene of tosyljewax into a vector containing DHFR as an amplified gene. As a result of confirming the expression level of the tosyljirimt antibody in the cell line transformed with the vector, it was confirmed that the expression was 50.8 ㎍ / ㎖ and 51.7 ㎍ / ㎖ at the gene amplification condition of 800 nM MTX concentration.

In a specific embodiment of the present invention, pCYBBSS002 bicyclist vector for denosumab antibody expression was prepared by inserting the light chain and the heavy chain gene of denosumab into a vector containing DHFR as an amplification gene. As a result of confirming the expression level of the denosumab antibody in the cell line transformed with the above vector, it was confirmed that it was highly expressed at 81.4 / / ㎖ under the gene amplification condition of 800 nM MTX concentration.

In a specific embodiment of the present invention, pCYBBSS003 bicyclic vector for the expression of bellimidab antibody was prepared by inserting the light chain and heavy chain genes of velimovat into a vector containing DHFR as an amplified gene. The expression level of belimidab antibody was confirmed in the cell line transformed with the above vector, and it was confirmed that it was highly expressed at 4 / / ml.

In a specific embodiment of the present invention, a light chain and heavy chain gene of golimumab was inserted into a vector containing DHFR as an amplified gene to prepare a pCYBBSS004 bicistronic vector for expression of a golimimab antibody. As a result of confirming the expression level of the golimimab antibody in the cell line transformed with the above vector, it was confirmed that it was highly expressed at 120.3 / / ㎖ under gene amplification conditions of 500 nM MTX concentration.

In a specific embodiment of the present invention, a light chain and heavy chain gene of Ustekinumab was inserted into a vector containing DHFR as an amplified gene to prepare a pCYBBSS005 bistronic vector for expression of Ustekinumab antibody. The expression level of Ustekinumab antibody was confirmed in the cell line transformed with the above-mentioned vector, and it was confirmed that it was highly expressed at 2.5 / / ml.

In a specific embodiment of the present invention, pCYBBSS006 biSystronic vector for expressing eicilimumab antibody was prepared by inserting the light chain and heavy chain gene of ephilimum in a vector containing DHFR as an amplified gene. As a result of confirming the expression level of the ephilimimitic antibody in the cell line transformed with the above vector, it was confirmed that it was highly expressed at 8 ㎍ / ㎖ under the condition of 500 nM methotrexate (MTX) gene amplification.

In summary, as described above, in the present invention, the biSistronic expression vector for antibody expression of the present invention was applied to various therapeutic antibodies and the expression level thereof was measured. As a result, using the expression vector of the present invention, .

In another aspect, the present invention provides a method for producing an antibody comprising culturing the animal cell.

In the present invention, the animal cell is an animal cell transformed with a bicistronic expression vector containing an intron according to the present invention, as described above.

The method for producing an antibody of the present invention can stably and efficiently produce an antibody by culturing the animal cell. The method may further comprise the step of purifying the antibody, preferably in a culture in which the animal cells have been cultured.

Such antibodies which may be produced according to the present invention are as described above and include, but are not limited to, bevacizumab, tocilizumab, denosumab, valimomat, golimumamp, May be good.

By using the bi-systolic expression vector for antibody expression comprising the UTR and intron according to the present invention, it is possible to produce an expression vector capable of expressing the desired antibody with high efficiency, and the animal cells transformed with the expression vector are cultured The antibody can be produced stably and with high efficiency.

FIG. 1 is a diagram showing the construction of the gene fragments Gln-LC and Gln-HC according to the present invention.
FIG. 2 is a view showing a manufacturing process of the GS-Gln-LC of the present invention.
FIG. 3 is a diagram illustrating a process for producing a pCYBIG vector containing a GS gene according to the present invention by inserting Gln-HC into GS-Gln-LC of the present invention.
FIG. 4 is a diagram showing a process for producing a pCYBID vector containing the DHFR gene according to the present invention from the pCYBIG vector of the present invention.
FIG. 5 is a diagram showing a process for producing bevacizumab light chain / heavy chain expression vector pCYB204IG containing the GS gene according to the present invention.
FIG. 6 is a diagram showing a process for producing bevacizumab light chain / heavy chain expression vector pCYB204ID containing the DHFR gene according to the present invention.
FIG. 7A shows the results of confirming the amount of antibody expressed in a cell line transformed with pCYB204IG at an MSX concentration of 500 uM.
FIG. 7B shows the results of confirming the amount of antibody expressed in the cell line transformed with pCYB204ID at MTX 800 nM.
Fig. 8 schematically shows a restriction map of the expression vector pCYBBSS001 used in the present invention.
9 is a schematic representation of a restriction map of the expression vector pCYBBSS002 used in the present invention.
Fig. 10 schematically shows the restriction map of the expression vector pCYBBSS003 used in the present invention.
Fig. 11 schematically shows the restriction map of the expression vector pCYBBSS004 used in the present invention.
12 schematically shows a restriction map of the expression vector pCYBBSS005 used in the present invention.
Fig. 13 schematically shows a restriction map of the expression vector pCYBBSS006 used in the present invention.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One : Light chain  Gene set for expression Gln - LC Production

The DNA sequence of the CMV promoter including the UTR part and the intron part was obtained through the nucleotide sequence of the complete genome (GenBank: X17403.1) of the human cytomegalovirus strain AD169. Since the promoter, cloning site, and poly A element are required for the expression of the target protein, the CMV promoter and the UTR, intron, cloning site, DNA of SV40 poly A are extracted using the nucleotide sequence identified in the genome of the cytomegalovirus Were purchased from Genotech (Korea).

In addition, restriction enzymes Xba I and Not I recognition sites were added between the intron and poly A to clone the light chain part of the antibody protein. The recognition site of the restriction enzyme Asc I was inserted at the 5 'end of the CMV promoter, The recognition sites of restriction enzymes BamHI and XhoI were added to the ends to be used in the subsequent cloning step.

(Gln-LC) having a constitution of [ Asc I] -CMV promoter-UTR-intron- [ Xba I / Not I] -polyA- [ BamH I / Xho I] (Fig. 1).

Example  2 : Heavy chain  Gene set for expression Gln - HC  making

Using the Gln-LC gene set prepared in Example 1 as a template, PCR was carried out using BamHI Gln-F primer (SEQ ID NO: 7) and NheI Gln-R primer (SEQ ID NO: 8) The reaction proceeded.

primer Base sequence SEQ ID NO: BamHI Gln-F GGC CGG ATC CGG CGC GCC TGC AGT GAA T 7 NheI Gln-R GGC CCT CGA GAA AGA TCT GGG CTA GCC GTG TCA AGG ACG GTG ACT GCA G 8

The restriction enzyme BamH I recognition site was added to the 3 'end of the CMV promoter for the heavy chain expression gene set Gln-HC. After cleavage of the CMV promoter, restriction enzymes Nhe I and Xho I The recognition site was added and used for subsequent cloning.

The PCR reaction was initially denatured at 94 ° C for 5 minutes and then circulated 30 times at 94 ° C for 50 seconds, 50 ° C for 30 seconds and 72 ° C for 2 minutes and 30 seconds, and finally amplified at 72 ° C for 7 minutes. The PCR reaction solution contained 25 μl of Premix Taq (Ex Taq version) (TAKARA), 2 μl each of the primers (10 pmole / μl) of SEQ ID NOs: 7 and 8, and distilled water was added so that the total volume became 50 μl.

Through the above procedure, a set (Gln-HC) for heavy chain expression having the structure of [ BamH I] -CMV promoter-UTR-intron- [ Nhe I / Xho I] - [ Nhe I] One).

The Gln-HC gene amplified by PCR was inserted into pGEM T-easy vector (Promega) according to the manufacturer's instructions.

Example  3: Light chain  A set of expression genes GS  vector Cloning  through GS - Gln - LC  Plasmid production

In order to construct an expression vector containing GS (Glutamin synthetase) gene as an amplification gene, IRES-GS vector developed by our company was used. IRES-GS gene - f1 origin - Kanamycin / neomycin resistance gene - Poly A - pUC (polymorphism) gene by treating IRES-GS vector containing IRES and GS gene and poly A with restriction enzymes Mlu I and Xho I at 37 ℃ for 4 hours. DNA vector fragments containing orogin were recovered.

On the other hand, the light chain expression gene set Gln-LC synthesized in Example 1 was Asc I Xho I was treated and cut.

The cleaved IRES-GS vector fragment and Gln-LC gene fragment fragment were subjected to 1% agarose gel electrophoresis and gel-extracted, respectively. Each recovered DNA was ligated to produce a GS-Gln-LC plasmid (Fig. 2).

Example  4 : Light chain  And Heavy chain  Expression plasmid pCYBIG Production

In order to obtain Gln-HC DNA, which is a set of light chain expression genes inserted in the T-easy vector prepared in Example 2, restriction enzymes BamHI and XhoI were treated at 37 ° C for 4 hours . The DNA treated with the restriction enzyme was subjected to 1% agaroge gel electrophoresis to recover DNA corresponding to the size of Gln-HC.

Meanwhile, the GS-Gln-LC plasmid prepared in Example 3 was digested with restriction enzymes BamHI and XhoI at 37 DEG C for 4 hours. DNA treated with restriction enzymes was subjected to 1% agaroge gel electrophoresis to recover DNA corresponding to the size of GS-Gln-LC plasmid.

The Gln-HC gene set fragment recovered through the above procedure and the GS-Gln-LC plasmid fragment were ligated to produce a pCYBIG vector (FIG. 3).

Example  5: Light chain  And Heavy chain  Expression plasmid pCYBID Production

A pCYBID vector was constructed by replacing the GS amplified gene of the pCYBIG vector prepared in Example 4 with DHFR (dehydrofolate reductase) gene.

Specifically, the DHFR gene was used as a template and the PCR reaction was performed using the sal I DHFR-F primer (SEQ ID NO: 9) and the Nhe I DHFR-R primer (SEQ ID NO: 10) listed in Table 2 below.

primer Base sequence SEQ ID NO: SalI DHFR-F GGC CGT CGA CAT GGT TCG ACC GCT G 9 NheI DHFR-R GGC CGC TAG CTT AGC CTT TCT TCT CAT AGA C 10

The PCR reaction was initially denatured at 94 ° C for 5 minutes and then circulated 30 times at 94 ° C for 50 seconds, 50 ° C for 30 seconds and 72 ° C for 2 minutes and 30 seconds, and finally amplified at 72 ° C for 7 minutes. The PCR reaction solution contained 25 μl of Premix Taq (Ex Taq version) (TAKARA), 2 μl each of the primers (10 pmole / μl) of SEQ ID NOS: 9 and 10, and distilled water was added so that the total volume became 50 μl. The DHFR gene amplified by PCR as described above was inserted into pGEM T-easy vector (Promega) according to the manufacturer's instructions.

Meanwhile, the pCYBIG vector prepared in Example 4 was digested with restriction enzymes sal I and Xba I, and the DHFR gene cloned into T-easy vector was treated with sal I and Nhe I to digest the DNA.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then two DNAs were ligated to prepare a pCYBID vector substituted with DHFR gene instead of GS (FIG. 4).

Example  6: Bevacizumab  Production of expression plasmids

6-1. Bevacizumab Light chain  Insertion of genes

The bevacizumab light chain gene was inserted into the pCYBIG vector and the pCYBID vector prepared in Examples 4 and 5 to prepare a bevacizumab light chain expression plasmid.

Specifically, the pCYBIG vector and the pCYBID vector were digested with Xba I and Not I.

The light chain gene of bevacizumab replaces the amino acid sequence of bevacizumab (SEQ ID NO: 13) with the DNA base sequence (SEQ ID NO: 11). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then two DNAs were ligated to prepare a vector in which the bevacizumab light chain gene was inserted into each of pCYBIG and pCYBID vectors.

6-2. Bevacizumab Heavy chain  Insertion of genes

The light chain expression vector pCYBIG-bevacizumab light chain gene vector and the pCYBID-bevacizumab light chain gene vector prepared in Example 6-1 were digested with Nhe I and Xho I. (SEQ ID NO: 14) from the heavy chain amino acid sequence of bevacizumab (SEQ ID NO: 12). Then, as in Example 6, a Kozak sequence (GCCACC) was inserted in front of ATG, which is a start codon, and restriction enzymes NheI and XhoI recognition sites were inserted at both ends to synthesize a gene and then digested with NheI and XhoI. Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and two DNAs were ligated to prepare a vector in which the bevacizumab light chain and heavy chain genes were inserted into each pCYB vector (FIG. 5 6).

Figures 5 and 6 are schematic diagrams illustrating the method of cloning the pCYB204IG vector and pCYB204ID vector of the present invention with the bevacizumab light and heavy chains.

Example  7: pCYB204IG  Determination of Antibody Expression Rate in Vector

7-1. Transformation

To develop a cell line stably expressing an antibody protein, GS-deficient CHO-K1 cells were pre-cultured on a 6-well plate at the concentration of 4 × 10 ⁵ cells per well on the day before the transformation, and then cultured in a carbon dioxide incubator at 37 ° C . After confirming that the viability of the cells was 98% or more, the pCYB204IG vector was transformed into cells using lipofectamine (Lipofectamine, Invitrogen) in general. Transformation was carried out using Opti-MEM-1 (Invitrogen) supplemented with 20 μg of the above expression vector DNA and 10 μl of lipofectamine. From this, CHO-K1 cells transformed with pCYB204IG were selected.

7-2. Antibody gene amplification

The CHO-K1 cell line transformed with pCYB204IG in Example 7-1 was treated with 25 μM of MSX (Methionine sulfoximine) as an initial concentration, and the growth progress was observed until cell growth was restored to normal. After treatment with 25 μM of MSX, the concentration of MSX was increased to 250 μM in the passage after 7 days. In the same way, the MSX concentration was increased at the time when growth was restored to the external pressure by MSX, Respectively. The expression levels of cell line pools (pool1 and pool2) prepared at a concentration of MSX of 500 μM were measured. Measurement of the expression level was confirmed using an ELISA method using Anti-Fc (Fig. 7A).

Example  8 : pCYB204ID  Determination of Antibody Expression Rate in Vector

8-1: Transformation

In order to compare the expression level of the pCYB204ID expression vector containing the amplified gene DHFR instead of the amplified gene GS of the pCUB204IG vector, CHO-DG44 cells (Gibco, 12609- 012), pCYB204ID was transformed into cells and transformed CHO-DG44 cells were selected.

8-2: Antibody gene amplification

In Example 8-1, the CHO-DG44 cell line transformed with pCYB204ID was treated with MTX (methotrexate) to perform gene amplification. The expression level was measured by increasing the concentration of MTX when cell growth was restored in the same manner as in Example 7-2. The MTX treatment concentration was 50 nM / 200 nM / 800 nM concentration. Measurement of the expression level was confirmed using an ELISA method using Anti-Fc (Fig. 7B).

As a result of comparing the expression levels of the antibody specific for each of the vectors measured in 7-2 and 8-2, it was found that the cell line transformed with the pCYB204ID vector was highly expressed twice more than that of the cell line transformed with the pCYB204IG vector Respectively.

Example  9: Tosilicum sap  Production of expression plasmids

9-1. Tosilicum sap Light chain  Insertion of genes

The pCYBID vector prepared in Example 5 was inserted with a tosushi militant light chain gene to construct a plasmid expressing a tosushi mite light chain.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of tosicilimum was replaced with the DNA sequence encoding the light chain amino acid sequence of the tosicilimum (SEQ ID NO: 17) (SEQ ID NO: 15). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then the two DNAs were ligated to prepare a vector having the pCYBID vector inserted with the pCYBID vector.

9-2. Tosilicum sap Heavy chain  Insertion of genes

The light chain expression vector pCYBID-tosylidium light chain gene vector prepared in Example 9-1 was digested with Nhe I and Xho I. Was replaced with the DNA base sequence from the heavy chain amino acid sequence (SEQ ID NO: 18) of the tessiculimum (SEQ ID NO: 16). Then, as in Example 9-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then the two DNAs were ligated to prepare a vector in which the pCYB vector was inserted with the pseudosuccine light chain and heavy chain gene (FIG. 8).

Fig. 8 schematically shows the restriction map of the expression vector pCYBBSS001 of the present invention.

Example  10: pCYBBSS001  Determination of Antibody Expression Rate in Vector

10-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown on the plate by about 70 to 80%, the light chain / heavy chain expression vector pCYBBSS001 was transformed into cells using lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS001 were selected.

10-2. Antibody gene amplification

In Example 10-1, transgenic CHO-DG44 cell line transformed with pCYBBSS001 was treated with MTX (methotrexate) to perform gene amplification. The expression level was measured by increasing the concentration of MTX when the cell growth was normally restored. MTX treatment concentration was 50 nM / 200 nM / 800 nM concentration. As a result, the cell line transformed with the pCYBBSS001 vector showed the highest expression level at an MTX concentration of 800 nM. Protein A Dip and Read biosensors (forte BIO, Cat No. 18-5010, lot no. 120716) were equilibrated in 1 × PBS for at least 10 minutes and reacted with the culture solution. Regeneration / neutralization of the biosensor was performed in 3 cycles after each sample was analyzed. The culture was diluted with 1 × PBS to analyze within the standard range. At an MTX concentration of 800 nM, the expression level was found to be 50.8 / / ㎖ and 51.7 / / ㎖ respectively in the two pools. These results indicate that when the intron-inserted expression vector of the present invention is transformed, It can be done.

Example  11: Denosumab  Production of expression plasmids

11-1. Denosumab Light chain  Insertion of genes

A denosumab light chain gene expression plasmid was constructed by inserting the denosumab light chain gene into the pCYBID vector prepared in Example 5 above.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of denosumab replaces the amino acid sequence of denosumab (SEQ ID NO: 21) with the DNA sequence (SEQ ID NO: 19). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis and then the two DNAs were ligated to prepare a vector having a denosumab light chain gene inserted into the pCYBID vector.

11-2. Denosumab Heavy chain  Insertion of genes

The light chain expression vector pCYBID-denosumab light chain gene vector prepared in Example 11-1 was digested with Nhe I and Xho I. (SEQ ID NO: 22) from the heavy chain amino acid sequence of de nosuem (SEQ ID NO: 20). Then, as in Example 11-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then the two DNAs were ligated to prepare a vector in which the denosomeps light chain and the heavy chain gene were inserted into the pCYB vector (FIG. 9).

Fig. 9 schematically shows a restriction map of the expression vector pCYBBSS002 of the present invention.

Example  12: pCYBBSS002  Determination of Antibody Expression Rate in Vector

12-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown on the plate by about 70 to 80%, the light chain / heavy chain expression vector pCYBBSS002 was transformed into cells by using Lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS002 were selected.

12-2. Antibody gene amplification

In Example 12-1, the CHO-DG44 cell line transformed with pCYBBSS002 was treated with MTX (methotrexate) to perform gene amplification. The expression level was measured by increasing the concentration of MTX when the cell growth was normally restored. MTX treatment concentration was 50 nM / 200 nM / 800 nM concentration. As a result, the cell line transformed with the pCYBBSS002 vector showed the highest expression level at an MTX concentration of 800 nM. Protein A Dip and Read biosensors (forte BIO, Cat No. 18-5010, lot no. 120716) were equilibrated in 1 × PBS for at least 10 minutes and reacted with the culture solution. Regeneration / neutralization of the biosensor was performed in 3 cycles after each sample was analyzed. The culture was diluted with 1 × PBS to analyze within the standard range. At an MTX concentration of 800 nM, the expression level was found to be 81.4 / / ㎖. These results indicate that the expression of the intron-inserted expression vector of the present invention can result in high expression of the gene.

Example  13: Velimab  Production of expression plasmids

13-1. Velimab Light chain  Insertion of genes

The vielimov light chain gene was inserted into the pCYBID vector prepared in Example 5 to prepare a vielimab light chain expression plasmid.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of belimudat was substituted with the DNA sequence encoding the light chain amino acid sequence (SEQ ID NO: 25) of belimudat (SEQ ID NO: 23). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each DNA fragment was digested with 1% agarose gel electrophoresis, and then two DNAs were ligated to prepare a vector in which the belyimab light chain gene was inserted into the pCYBID vector.

13-2. Velimab Heavy chain  Insertion of genes

The light chain expression vector pCYBID-valimuvet light chain gene vector prepared in Example 13-1 was digested with Nhe I and Xho I. (SEQ ID NO: 24) from the heavy chain amino acid sequence (SEQ ID NO: 26) of belimudat. Then, as in Example 13-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then two DNAs were ligated to prepare a vector in which the velimovat light chain and the heavy chain gene were inserted into each pCYB vector (FIG. 10).

Fig. 10 schematically shows a restriction map of the expression vector pCYBBSS003 of the present invention.

Example  14: pCYBBSS003  Determination of Antibody Expression Rate in Vector

14-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown to about 70 to 80% on the plate, the light chain / heavy chain expression vector pCYBBSS003 was transformed into cells using lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS003 were selected.

14-2. Antibody gene Expression level  Measure

In Example 14-1, the expression level of velimovat in the CHO-DG44 cell line transformed with the vector pCYBBSS003 was measured. The expression level was determined by ELISA using Anti-Fc and PCD (Picograms / cell / day). As a result, it was confirmed that the cell line transformed with the vector pCYBBSS003 showed an expression level of 4 / / ml, and this result shows that when the expression vector containing the intron of the present invention is transformed, the gene can be continuously and highly expressed .

Example  15: Gollymab  Production of expression plasmids

15-1. Gollymab Light chain  Insertion of genes

A golliomat light chain gene was inserted into the pCYBID vector prepared in Example 5 to prepare a plasmid expressing golliumat light chain.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of golimumab was replaced with the DNA base sequence encoding the light chain amino acid sequence (SEQ ID NO: 29) of golimumab (SEQ ID NO: 27). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and two DNAs were ligated to prepare a vector in which the pCYBID vector was inserted into the pCYBID vector.

15-2. Gollymab Heavy chain  Insertion of genes

The light chain expression vector pCYBID-goliimab light chain gene vector prepared in Example 15-1 was digested with Nhe I and Xho I. (SEQ ID NO: 30) from the heavy chain amino acid sequence of golimumab (SEQ ID NO: 28). Then, as in Example 15-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and the two DNAs were ligated to prepare a vector in which the polymorphic light chain and the heavy chain gene were inserted into the pCYB vector (FIG. 11).

Fig. 11 schematically shows the restriction map of the expression vector pCYBBSS004 of the present invention.

Example  16: pCYBBSS004  Determination of Antibody Expression Rate in Vector

16-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown to 70 to 80% on the plate, the light chain / heavy chain expression vector pCYBBSS004 was transformed into cells using Lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS004 were selected.

16-2. Antibody gene amplification

In Example 16-1, MTX (methotrexate) was treated with CHO-DG44 cell line transformed with pCYBBSS004 to perform gene amplification. The expression level was measured by increasing the concentration of MTX when the cell growth was normally restored. MTX treatment concentration was 50 nM / 200 nM / 500 nM / 800 nM concentration. As a result, the cell line transformed with the pCYBBSS004 vector showed the highest expression level at an MTX concentration of 800 nM. Protein A Dip and Read biosensors (forte BIO, Cat No. 18-5010, lot no. 120716) were equilibrated in 1 × PBS for at least 10 minutes and reacted with the culture solution. Regeneration / neutralization of the biosensor was performed in 3 cycles after each sample was analyzed. The culture was diluted with 1 × PBS to analyze within the standard range. At an MTX concentration of 500 nM, the expression level was found to be 120.3 / / ㎖. These results indicate that the gene of the present invention can be continuously expressed when the genomic mutation of the present invention is transformed.

Example  17: Ustekinumab  Production of expression plasmids

17-1. Ustekinumab Light chain  Insertion of genes

Ustekinumab light chain gene was inserted into the pCYBID vector prepared in Example 5 to prepare a plasmid expressing Ustekinumab light chain.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of Ustekinumab was substituted with the DNA base sequence encoding the light chain amino acid sequence of Ustekinumab (SEQ ID NO: 33) (SEQ ID NO: 31). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and the two DNAs were ligated to prepare a vector containing the rightstekinemac light chain gene in the pCYBID vector.

17-2. Ustekinumab Heavy chain  Insertion of genes

The light chain expression vector pCYBID-ustestinumab light chain gene vector prepared in Example 17-1 was digested with Nhe I and Xho I. (SEQ ID NO: 34) from the heavy chain amino acid sequence of Ustekinumab (SEQ ID NO: 32). Then, as in Example 17-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and then two DNAs were ligated to prepare a vector having Ustekinemach light chain and heavy chain gene inserted into each pCYB vector (FIG. 12) .

Fig. 12 schematically shows a restriction map of the expression vector pCYBBSS005 of the present invention.

Example  18: pCYBBSS005  Determination of Antibody Expression Rate in Vector

18-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown on the plate by about 70 to 80%, the light chain / heavy chain expression vector pCYBBSS005 was transformed into cells by using Lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS005 were selected.

18-2. Antibody gene Expression level  Measure

The expression level of Ustekinumab in the CHO-DG44 cell line transformed with the vector pCYBBSS005 in Example 18-1 was measured. The expression level was determined by ELISA using Anti-Fc and PCD (Picograms / cell / day). As a result, it was confirmed that the cell line transformed with the pCYBBSS005 vector showed an expression level of 2.5 μg / ml. These results indicate that when the expression vector containing the intron of the present invention is transformed, the gene can be continuously and highly expressed .

Example  19: Emilimumum  Production of expression plasmids

19-1. Emilimumum Light chain  Insertion of genes

The ephilimimab light chain gene was inserted into the pCYBID vector prepared in Example 5 to prepare an eicilimumab light chain expression plasmid.

Specifically, the pCYBID vector was digested with Xba I and Not I.

The light chain gene of eicilimumab was substituted with the DNA base sequence encoding the light chain amino acid sequence of eicilimumab (SEQ ID NO: 37) (SEQ ID NO: 35). In DNA sequence substitution, the DNA sequence was optimized for CHO cells expressing the antibody, and restriction enzymes Nhe I and Not I recognition sites were inserted at both 5'- and 3'- ends. Kozak sequence (GCCACC) was inserted in front of the ATG, which is the start codon, to induce the expression level to be increased, and genes were synthesized by adding MGWSCIILFLVATATGVHS as a signal sequence. The synthesized genes were digested with Nhe I and Not I.

Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and the two DNAs were ligated to prepare a vector having the pCYBID vector inserted with the ephilimimab light chain gene.

19-2. Emilimumum Heavy chain  Insertion of genes

The light chain expression vector pCYBID-eicilimumab light chain gene vector prepared in Example 19-1 was digested with Nhe I and Xho I. (SEQ ID NO: 38) with the DNA base sequence (SEQ ID NO: 36). Then, as in Example 19-1, a Kozak sequence (GCCACC) was inserted in front of ATG as a start codon, and restriction enzymes Nhe I and Xho I recognition sites were inserted at both ends to synthesize genes. Then, Nhe I and Xho I Lt; / RTI > Each of the digested DNAs was recovered by 1% agarose gel electrophoresis, and the two DNAs were ligated to prepare a vector in which an ephilimumimab light chain and a heavy chain gene were inserted into each pCYB vector (FIG. 13).

Fig. 13 schematically shows a restriction map of the expression vector pCYBBSS006 of the present invention.

Example  20: pCYBBSS006  Determination of Antibody Expression Rate in Vector

20-1. Transformation

To develop a cell line stably expressing the antibody protein, to the first DHFR-deficient CHO-DG44 cells per well concentration of 8 × 10 ⁵ cells (Dr.Chasin, Univ. Columbia. USA ) transfected in 6-well plates the day before the experiment conversion The cells were cultured in a carbon dioxide incubator at 37 ° C. After confirming that the cells were grown to about 70 to 80% on the plate, the light chain / heavy chain expression vector pCYBBSS006 was transformed into cells using lipofectamine LTX and PLUS (Lipofectamine LTX and PLUS reagent, Invitrogen). At this time, for the transformation, Opti-MEM-1 (Invitrogen) was supplemented with 2.5 [micro] g of the above expression vector DNA and 10 [micro] l of lipofectamine LTX. From this, CHO-DG44 cells transformed with the light chain / heavy chain expression vector pCYBBSS006 were selected.

20-2. Antibody gene amplification

In Example 20-1, the CHO-DG44 cell line transformed with pCYBBSS006 was treated with MTX (methotrexate) to perform gene amplification. The expression level was measured by increasing the concentration of MTX when the cell growth was normally restored. MTX treatment concentration was 50 nM / 200 nM / 500 nM / 800 nM concentration. As a result, the cell line transformed with the vector pCYBBSS006 showed the highest expression level at an MTX concentration of 500 nM. Protein A Dip and Read biosensors (forte BIO, Cat No. 18-5010, lot no. 120716) were equilibrated in 1 × PBS for at least 10 minutes and reacted with the culture solution. Regeneration / neutralization of the biosensor was performed in 3 cycles after each sample was analyzed. The culture was diluted with 1 × PBS to analyze within the standard range. At an MTX concentration of 500 nM, the expression level was found to be 8 쨉 g / ml. These results indicate that the expression of the intron-inserted expression vector of the present invention can result in high expression of the gene.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> HANWHA CHEMICAL CORPORATION <120> Bicistronic expression vector for expressing antibody and method          for producing antibody using the same <130> PA130872KR-P7 <150> KR10-2013-0119511 <151> 2013-10-07 <160> 38 <170> Kopatentin 2.0 <210> 1 <211> 1143 <212> DNA <213> Cytomegalovirus stain AD169 promoter <400> 1 ctgcagtgaa taataaaatg tgtgtttgtc cgaaatacgc gttttgagat ttctgtcgcc 60 gactaaattc atgtcgcgcg atagtggtgt ttatcgccga tagagatggc gatattggaa 120 aaatcgatat ttgaaaatat ggcatattga aaatgtcgcc gatgtgagtt tctgtgtaac 180 tgatatcgcc atttttccaa aagtgatttt tgggcatacg cgatatctgg cgatagcgct 240 tatatcgttt acgggggatg gcgatagacg actttggtga cttgggcgat tctgtgtgtc 300 gcaaatatcg cagtttcgat ataggtgaca gacgatatga ggctatatcg ccgatagagg 360 cgacatcaag ctggcacatg gccaatgcat atcgatctat acattgaatc aatattggcc 420 attagccata ttattcattg gttatatagc ataaatcaat attggctatt ggccattgca 480 tacgttgtat ccatatcata atatgtacat ttatattggc tcatgtccaa cattaccgcc 540 atgttgacat tgattattga ctagttatta atagtaatca attacggggt cattagttca 600 tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 660 gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 720 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 780 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 840 cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 900 cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 960 atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 1020 gtttggcac caaaatcaac gggactttcc aaaatgtcgt aacaactccg ccccattgac 1080 gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc gtttagtgaa 1140 ccg 1143 <210> 2 <211> 121 <212> DNA <213> Cytomegalovirus stain AD169 UTR (5'UTR) <400> 2 tcagatcgcc tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg 60 atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg ccaagagtga 120 c 121 <210> 3 <211> 827 <212> DNA <213> Cytomegalovirus stain AD169 intron <400> 3 gtaagtaccg cctatagagt ctataggccc acccccttgg cttcttatgc atgctatact 60 gtttttggct tggggtctat acacccccgc ttcctcatgt tataggtgat ggtatagctt 120 agcctatagg tgtgggttat tgaccattat tgaccactcc cctattggtg acgatacttt 180 ccattactaa tccataacat ggctctttgc cacaactctc tttattggct atatgccaat 240 acactgtcct tcagagactg acacggactc tgtattttta caggatgggg tctcatttat 300 tatttacaaa ttcacatata caacaccacc gtccccagtg cccgcagttt ttattaaaca 360 taacgtggga tctccacgcg aatctcgggt acgtgttccg gacatgggct cttctccggt 420 agcggcggag cttctacatc cgagccctgc tcccatgcct ccagcgactc atggtcgctc 480 ggcagctcct tgctcctaac agtggaggcc agacttaggc acagcacgat gcccaccacc 540 accagtgtgc cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga gctcggggag 600 cgggcttgca ccgctgacgc atttggaaga cttaaggcag cggcagaaga agatgcaggc 660 agctgagttg ttgtgttctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg 720 gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac cagacataat 780 agctgacaga ctaacagact gttcctttcc atgggtcttt tctgcag 827 <210> 4 <211> 576 <212> DNA <213> IRES (internal ribosome entry site) <400> 4 cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 60 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 120 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 180 ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 240 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 300 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 360 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 420 aaggggctgga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 480 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 540 ggggacgtgg ttttcctttg aaaaacacga tgataa 576 <210> 5 <211> 2399 <212> DNA <213> Artificial Sequence <220> <223> Gln-LC <400> 5 cctaggcgcg cctgcagtga ataataaaat gtgtgtttgt ccgaaatacg cgttttgaga 60 tttctgtcgc cgactaaatt catgtcgcgc gatagtggtg tttatcgccg atagagatgg 120 cgatattgga aaaatcgata tttgaaaata tggcatattg aaaatgtcgc cgatgtgagt 180 ttctgtgtaa ctgatatcgc catttttcca aaagtgattt ttgggcatac gcgatatctg 240 gcgatagcgc ttatatcgtt tacgggggat ggcgatagac gactttggtg acttgggcga 300 ttctgtgtgt cgcaaatatc gcagtttcga tataggtgac agacgatatg aggctatatc 360 gccgatagag gcgacatcaa gctggcacat ggccaatgca tatcgatcta tacattgaat 420 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat 480 tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca 540 acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 600 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 660 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 720 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 780 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 840 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 900 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 960 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 1020 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 1080 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 1140 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 1200 agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc 1260 cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttct 1320 tatgcatgct atactgtttt tggcttgggg tctatacacc cccgcttcct catgttatag 1380 gtgatggtat agcttagcct ataggtgtgg gttattgacc attattgacc actcccctat 1440 tggtgacgat actttccatt actaatccat aacatggctc tttgccacaa ctctctttat 1500 tggctatatg ccaatacact gtccttcaga gactgacacg gactctgtat ttttakhga 1560 tggggtctca tttattattt acaaattcac atatacaaca ccaccgtccc cagtgcccgc 1620 agtttttatt aaacataacg tgggatctcc acgcgaatct cgggtacgtg ttccggacat 1680 gggctcttct ccggtagcgg cggagcttct acatccgagc cctgctccca tgcctccagc 1740 gactcatggt cgctcggcag ctccttgctc ctaacagtgg aggccagact taggcacagc 1800 acgatgccca ccaccaccag tgtgccgcac aaggccgtgg cggtagggta tgtgtctgaa 1860 aatgagctcg gggagcgggc ttgcaccgct gacgcatttg gaagacttaa ggcagcggca 1920 gaagaagatg caggcagctg agttgttgtg ttctgataag agtcagaggt aactcccgtt 1980 gcggtgctgt taacggtgga gggcagtgta gtctgagcag tactcgttgc tgccgcgcgc 2040 gccaccagac ataatagctg acagactaac agactgttcc tttccatggg tcttttctgc 2100 agtcaccgtc cttgacacgt ctagaccaga tctttgcggc cgcattgatc ataatcagcc 2160 ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc 2220 tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt 2280 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 2340 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc tagctcgag 2399 <210> 6 <211> 2125 <212> DNA <213> Artificial Sequence <220> <223> Gln-HC <400> 6 cctaggcgcg cctgcagtga ataataaaat gtgtgtttgt ccgaaatacg cgttttgaga 60 tttctgtcgc cgactaaatt catgtcgcgc gatagtggtg tttatcgccg atagagatgg 120 cgatattgga aaaatcgata tttgaaaata tggcatattg aaaatgtcgc cgatgtgagt 180 ttctgtgtaa ctgatatcgc catttttcca aaagtgattt ttgggcatac gcgatatctg 240 gcgatagcgc ttatatcgtt tacgggggat ggcgatagac gactttggtg acttgggcga 300 ttctgtgtgt cgcaaatatc gcagtttcga tataggtgac agacgatatg aggctatatc 360 gccgatagag gcgacatcaa gctggcacat ggccaatgca tatcgatcta tacattgaat 420 caatattggc cattagccat attattcatt ggttatatag cataaatcaa tattggctat 480 tggccattgc atacgttgta tccatatcat aatatgtaca tttatattgg ctcatgtcca 540 acattaccgc catgttgaca ttgattattg actagttatt aatagtaatc aattacgggg 600 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 660 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 720 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 780 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 840 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 900 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 960 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 1020 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 1080 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 1140 cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 1200 agacaccggg accgatccag cctccgcggc cgggaacggt gcattggaac gcggattccc 1260 cgtgccaaga gtgacgtaag taccgcctat agagtctata ggcccacccc cttggcttct 1320 tatgcatgct atactgtttt tggcttgggg tctatacacc cccgcttcct catgttatag 1380 gtgatggtat agcttagcct ataggtgtgg gttattgacc attattgacc actcccctat 1440 tggtgacgat actttccatt actaatccat aacatggctc tttgccacaa ctctctttat 1500 tggctatatg ccaatacact gtccttcaga gactgacacg gactctgtat ttttakhga 1560 tggggtctca tttattattt acaaattcac atatacaaca ccaccgtccc cagtgcccgc 1620 agtttttatt aaacataacg tgggatctcc acgcgaatct cgggtacgtg ttccggacat 1680 gggctcttct ccggtagcgg cggagcttct acatccgagc cctgctccca tgcctccagc 1740 gactcatggt cgctcggcag ctccttgctc ctaacagtgg aggccagact taggcacagc 1800 acgatgccca ccaccaccag tgtgccgcac aaggccgtgg cggtagggta tgtgtctgaa 1860 aatgagctcg gggagcgggc ttgcaccgct gacgcatttg gaagacttaa ggcagcggca 1920 gaagaagatg caggcagctg agttgttgtg ttctgataag agtcagaggt aactcccgtt 1980 gcggtgctgt taacggtgga gggcagtgta gtctgagcag tactcgttgc tgccgcgcgc 2040 gccaccagac ataatagctg acagactaac agactgttcc tttccatggg tcttttctgc 2100 agtcaccgtc cttgacacgt ctaga 2125 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > BamHI Gln-F primer <400> 7 ggccggatcc ggcgcgcctg cagtgaat 28 <210> 8 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Nhe1 Gln-R primer <400> 8 ggccctcgag aaagatctgg cctagccgtg tcaaggacgg tgactgcag 49 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SalI DHFR-F primer <400> 9 ggccgtcgac atggttcgac cgctg 25 <210> 10 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> NheI DHFR-R primer <400> 10 ggccgctagc ttagcctttc ttctcataga c 31 <210> 11 <211> 728 <212> DNA <213> bevacizumab light chain <400> 11 gctagcgcca ccatgggctg gtcctgcatc atcctgttcc tggtggccac cgccaccggc 60 gtgcactccg acatccagat gacccagtcc ccctcctccc tgtccgcctc cgtgggcgac 120 cgggtgacca tcacctgctc cgcctcccag gacatctcca actacctgaa ctggtaccag 180 cagaagcccg gcaaggcccc caaggtgctg atctacttca cctcctccct gcactccggc 240 gtgccctccc ggttctccgg ctccggctcc ggcaccgact tcaccctgac catctcctcc 300 ctgcagcccg aggacttcgc cacctactac tgccagcagt actccaccgt gccctggacc 360 ttcggccagg gcaccaaggt ggagatcaag cggaccgtgg ccgccccctc cgtgttcatc 420 ttccccccct ccgacgagca gctgaagtcc ggcaccgcct ccgtggtgtg cctgctgaac 480 aacttctacc cccgggaggc caaggtgcag tggaaggtgg acaacgccct gcagtccggc 540 aactcccagg agtccgtgac cgagcaggac tccaaggact ccacctactc cctgtcctcc 600 accctgaccc tgtccaaggc cgactacgag aagcacaagg tgtacgcctg cgaggtgacc 660 caccagggcc tgtcctcccc cgtgaccaag tccttcaacc ggggcgagtg ctgagcggcc 720 gcctcgag 728 <210> 12 <211> 1445 <212> DNA <213> bevacizumab heavy chain <400> 12 gctagcgcca ccatgggctg gtcctgcatc atcctgttcc tggtggccac cgccaccggc 60 gtgcactccg aggtgcagct ggtggagtcc ggcggcggcc tggtgcagcc cggcggctcc 120 ctgcggctgt cctgcgccgc ctccggctac accttcacca actacggcat gaactgggtg 180 cggcaggccc ccggcaaggg cctggagtgg gtgggctgga tcaacaccta caccggcgag 240 cccacctacg ccgccgactt caagcggcgg ttcaccttct ccctggacac ctccaagtcc 300 accgcctacc tgcagatgaa ctccctgcgg gccgaggaca ccgccgtgta ctactgcgcc 360 aagtaccccc actactacgg ctcctcccac tggtacttcg acgtgtgggg ccagggcacc 420 ctggtgaccg tgtcctccgc ctccaccaag ggcccctccg tgttccccct ggccccctcc 480 tccaagtcca cctccggcgg caccgccgcc ctgggctgcc tggtgaagga ctacttcccc 540 gagcccgtga ccgtgtcctg gaactccggc gccctgacct ccggcgtgca caccttcccc 600 gccgtgctgc agtcctccgg cctgtactcc ctgtcctccg tggtgaccgt gccctcctcc 660 tccctgggca cccagaccta catctgcaac gtgaaccaca agccctccaa caccaaggtg 720 gacaagaagg tggagcccaa gtcctgcgac aagacccaca cctgcccccc ctgccccgcc 780 cccgagctgc tgggcggccc ctccgtgttc ctgttccccc ccaagcccaa ggacaccctg 840 atgatctccc ggacccccga ggtgacctgc gtggtggtgg acgtgtccca cgaggacccc 900 gaggtgaagt tcaactggta cgtggacggc gtggaggtgc acaacgccaa gaccaagccc 960 cgggaggagc agtacaactc cacctaccgg gtggtgtccg tgctgaccgt gctgcaccag 1020 gactggctga acggcaagga gtacaagtgc aaggtgtcca acaaggccct gcccgccccc 1080 atcgagaaga ccatctccaa ggccaagggc cagccccggg agccccaggt gtacaccctg 1140 cccccctccc gggaggagat gaccaagaac caggtgtccc tgacctgcct ggtgaagggc 1200 ttctacccct ccgacatcgc cgtggagtgg gagtccaacg gccagcccga gaacaactac 1260 aagaccaccc cccccgtgct ggactccgac ggctccttct tcctgtactc caagctgacc 1320 gtggacaagt cccggtggca gcagggcaac gtgttctcct gctccgtgat gcacgaggcc 1380 ctgcacaacc actacaccca gaagtccctg tccctgtccc ccggcaagtg agcggccgcc 1440 tcgag 1445 <210> 13 <211> 214 <212> PRT <213> bevacizumab light chain <400> 13 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile          35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr Val Pro Trp                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala             100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly         115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala     130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser                 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr             180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser         195 200 205 Phe Asn Arg Gly Glu Cys     210 <210> 14 <211> 453 <212> PRT <213> bevacizumab heavy chain <400> 14 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe      50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser 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 Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val             100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly         115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly     130 135 140 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe                 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val             180 185 190 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val         195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys     210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr                 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val             260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val         275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser     290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala                 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro             340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln         355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala     370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu                 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser             420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser         435 440 445 Leu Ser Pro Gly Lys     450 <210> 15 <211> 739 <212> DNA <213> tocilizumab light chain <400> 15 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgacat ccagatgacc cagtccccct cctccctgtc cgcctccgtg 120 ggcgaccggg tgaccatcac ctgccgggcc tcccaggaca tctcctccta cctgaactgg 180 taccagcaga agcccggcaa ggcccccaag ctgctgatct actacacctc ccggctgcac 240 tccggcgtgc cctcccggtt ctccggctcc ggctccggca ccgacttcac cttcaccatc 300 tcctccctgc agcccgagga catcgccacc tactactgcc agcagggcaa caccctgccc 360 tacaccttcg gccagggcac caaggtggag atcaagcgga ccgtggccgc cccctccgtg 420 ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 480 ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 540 tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 600 tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 660 gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaaccgggg cgagtgctga 720 gaattcgcgg ccgctcgag 739 <210> 16 <211> 1444 <212> DNA <213> tocilizumab heavy chain <400> 16 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actcccaggt gcagctgcag gagtccggcc ccggcctggt gcggccctcc 120 cagaccctgt ccctgacctg caccgtgtcc ggctactcca tcacctccga ccacgcctgg 180 tcctgggtgc ggcagccccc cggccggggc ctggagtgga tcggctacat ctcctactcc 240 ggcatcacca cctacaaccc ctccctgaag tcccgggtga ctatgctgcg ggacacctcc 300 aagaaccagt tctccctgcg gctgtcctcc gtgaccgccg ccgacaccgc cgtgtactac 360 tgcgcccggt ccctggcccg gaccaccgct atggactact ggggccaggg ctccctggtg 420 accgtgtcct ccgcctccac caagggcccc tccgtgttcc ccctggcccc ctcctccaag 480 tccacctccg gcggcaccgc cgccctgggc tgcctggtga aggactactt ccccgagccc 540 gtgaccgtgt cctggaactc cggcgccctg acctccggcg tgcacacctt ccccgccgtg 600 ctgcagtcct ccggcctgta ctccctgtcc tccgtggtga ctgtgccctc ctcctccctg 660 ggcacccaga cctacatctg caacgtgaac cacaagccct ccaacaccaa ggtggacaag 720 aaggtggagc ccaagtcctg cgacaagacc cacacctgcc ccccctgccc cgcccccgag 780 ctgctgggcg gcccctccgt gttcctgttc ccccccaagc ccaaggacac cctgatgatc 840 tcccggaccc ccgaggtgac ttgcgtggtg gtggacgtgt cccacgagga ccccgaggtg 900 aagttcaact ggtacgtgga cggcgtggag gtgcacaacg ccaagaccaa gccccgggag 960 gagcagtaca actccaccta ccgggtggtg tccgtgctga ccgtgctgca ccaggactgg 1020 ctgaacggca aggagtacaa gtgcaaggtg tccaacaagg ccctgcccgc ccccatcgag 1080 aagaccatct ccaaggccaa gggccagccc cgggagcccc aggtgtacac cctgcccccc 1140 tcccgggacg agctgaccaa gaaccaggtg tccctgacct gcctggtgaa gggcttctac 1200 ccctccgaca tcgccgtgga gtgggagtcc aacggccagc ccgagaacaa ctacaagacc 1260 accccccccg tgctggactc cgacggctcc ttcttcctgt actccaagct gaccgtggac 1320 aagtcccggt ggcagcaggg caacgtgttc tcctgctccg tgatgcacga ggccctgcac 1380 aaccactaca cccagaagtc cctgtccctg tcccccggca agtgagaatt cgcggccgct 1440 cgag 1444 <210> 17 <211> 233 <212> PRT <213> tocilizumab light chain <400> 17 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Leu Ser Ala              20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile          35 40 45 Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys      50 55 60 Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Ser Ser Arg  65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser                  85 90 95 Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr             100 105 110 Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr         115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu     130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly                 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr             180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His         195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val     210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 18 <211> 468 <212> PRT <213> tocilizumab heavy chain <400> 18 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg              20 25 30 Pro Ser Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile          35 40 45 Thr Ser Asp His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Arg Gly      50 55 60 Leu Glu Trp Ile Gly Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn  65 70 75 80 Pro Ser Leu Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn                  85 90 95 Gln Phe Ser Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp         115 120 125 Gly Gln Gly Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro     130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr                 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro             180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr         195 200 205 Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn     210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu             260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser         275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu     290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn                 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro             340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln         355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val     370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro                 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr             420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val         435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu     450 455 460 Ser Pro Gly Lys 465 <210> 19 <211> 742 <212> DNA <213> denosumab light chain <400> 19 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgagat cgtgctgacc cagtcccccg gcaccctgtc cctgtccccc 120 ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgcggggccg gtacctggcc 180 cgcctgcgc gccaccggca tccccgaccg gttctccggc tccggctccg gcaccgactt caccctgacc 300 atctcccggc tggagcccga ggacttcgcc gtgttctact gccagcagta cggctcctcc 360 ccccggacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc cgccccctcc 420 gtgttcatct tccccccctc cgacgagcag ctgaagtccg gcaccgcctc cgtggtgtgc 480 ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg 540 cagtccggca actcccagga gtccgtgacc gagcaggact ccaaggactc cacctactcc 600 ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 660 gaggtgaccc accagggcct gtcctccccc gtgaccaagt ccttcaaccg gggcgagtgc 720 tgagaattcg cggccgctcg ag 742 <210> 20 <211> 1441 <212> DNA <213> denosumab heavy chain <400> 20 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgaggt gcagctgctg gagtccggcg gcggcctggt gcagcccggc 120 ggctccctgc ggctgtcctg cgccgcctcc ggcttcacct tctcctccta cgccatgtcc 180 tgggtgcggc aggcccccgg caagggcctg gagtgggtgt ccggcatcac cggctccggc 240 ggctccacct actacgccga ctccgtgaag ggccggttca ccatctcccg ggacaactcc 300 aagaacaccc tgtacctgca gatgaactcc ctgcgggccg aggacaccgc cgtgtactac 360 tgcgccaagg accccggcac caccgtgatt atgtcctggt tcgacccctg gggccagggc 420 accctggtga ccgtgtcctc cgcctccacc aagggcccct ccgtgttccc cctggccccc 480 tgctcccggt ccacctccga gtccaccgcc gccctgggct gcctggtgaa ggactacttc 540 cccgagcccg tgaccgtgtc ctggaactcc ggcgccctga cctccggcgt gcacaccttc 600 cccgccgtgc tgcagtcctc cggcctgtac tccctgtcct ccgtggtgac tgtgccctcc 660 tccaacttcg gcacccagac ctacacctgc aacgtggacc acaagccctc caacaccaag 720 gtggacaaga ccgtggagcg gaagtgctgc gtggagtgcc ccccctgccc cgcccccccc 780 gtggccggcc cctccgtgtt cctgttcccc cccaagccca aggacaccct gatgatctcc 840 cggacccccg aggtgacttg cgtggtggtg gacgtgtccc acgaggaccc cgaggtgcag 900 ttcaactggt acgtggacgg cgtggaggtg cacaacgcca agaccaagcc ccgggaggag 960 cagttcaact ccaccttccg ggtggtgtcc gtgctgaccg tggtgcacca ggactggctg 1020 aacggcaagg agtacaagtg caaggtgtcc aacaagggcc tgcccgcccc catcgagaag 1080 accatctcca agaccaaggg ccagccccgg gagccccagg tgtacaccct gcccccctcc 1140 cgggaggaga tgaccaagaa ccaggtgtcc ctgacctgcc tggtgaaggg cttctacccc 1200 tccgacatcg ccgtggagtg ggagtccaac ggccagcccg agaacaacta caagaccacc 1260 ccccccatgc tggactccga cggctccttc ttcctgtact ccaagctgac cgtggacaag 1320 tcccggtggc agcagggcaa cgtgttctcc tgctccgtga tgcacgaggc cctgcacaac 1380 cactacaccc agaagtccct gtccctgtcc cccggcaagt gagaattcgc ggccgctcga 1440 g 1441 <210> 21 <211> 234 <212> PRT <213> denosumab light chain <400> 21 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu              20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val          35 40 45 Arg Gly Arg Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro      50 55 60 Arg Leu Leu Ile Tyr Gly Ala Ser Ser Ala Thr Gly Ile Pro Asp  65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                  85 90 95 Arg Leu Glu Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln Gln Tyr Gly             100 105 110 Ser Ser Pro Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 22 <211> 467 <212> PRT <213> denosumab heavy chain <400> 22 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln              20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe          35 40 45 Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu      50 55 60 Glu Trp Val Ser Gly Ile Thr Gly Ser Gly Gly Ser Thr Tyr Tyr Ala  65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Lys Asp Pro Gly Thr Thr Val Ile Met Ser Trp Phe         115 120 125 Asp Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr     130 135 140 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 145 150 155 160 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu                 165 170 175 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His             180 185 190 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser         195 200 205 Val Val Thr Val Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys     210 215 220 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu 225 230 235 240 Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala                 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275 280 285 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly                 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile             340 345 350 Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Pro Gly Lys 465 <210> 23 <211> 739 <212> DNA <213> Belimumab light chain <400> 23 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 cggctgcg cagaccgtgc gggtgacctg ccagggcgac tccctgcggt cctactacgc ctcctggtac 180 cagcagaagc ccggccaggc ccccgtgctg gtgatctacg gcaagaacaa ccggccctcc 240 ggcatccccg accggttctc cggctcctcc tccggcaaca ccgcctccct gaccatcact 300 ggcgcccagg ccgaggacga ggccgactac tactgctcct cccgggactc ctccggcaac 360 cactgggtgt tcggcggcgg caccgagctg accgtgctgg gccagcccaa ggccgccccc 420 tccgtgaccc tgttcccccc ctcctccgag gagctgcagg ccaacaaggc caccctggtg 480 tgcctgatct ccgacttcta ccccggcgcc gtgaccgtgg cctggaaggc cgactcctcc 540 cccgtgaagg ccggcgtgga gaccaccacc ccctccaagc agtccaacaa caagtacgcc 600 gcctcctcct acctgtccct gacccccgag cagtggaagt cccaccggtc ctactcctgc 660 caggtgaccc acgagggctc caccgtggag aagaccgtgg cccccaccga gtgctcctga 720 gaattcgcgg ccgctcgag 739 <210> 24 <211> 1456 <212> DNA <213> Belimumab heavy chain <400> 24 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actcccaggt gcagctgcag cagtccggcg ccgaggtgaa gaagcccggc 120 tcctccgtgc gggtgtcctg caaggcctcc ggcggcacct tcaacaacaa cgccatcaac 180 tgggtgcggc aggcccccgg ccagggcctg gagtggatgg gcggcatcat ccccatgttc 240 ggcaccgcca agtactccca gaacttccag ggccgggtgg ccatcaccgc cgacgagtcc 300 accggcaccg cctccatgga gctgtcctcc ctgcggtccg aggacaccgc cgtgtactac 360 tgcgcccggt cccgggacct gctgctgttc ccccaccacg ccctgtcccc ctggggccgg 420 ggcaccatgg tgaccgtgtc ctccgcctcc accaagggcc cctccgtgtt ccccctggcc 480 ccctcctcca agtccacctc cggcggcacc gccgccctgg gctgcctggt gaaggactac 540 ttccccgagc ccgtgaccgt gtcctggaac tccggcgccc tgacctccgg cgtgcacacc 600 ttccccgccg tgctgcagtc ctccggcctg tactccctgt cctccgtggt gactgtgccc 660 tcctcctccc tgggcaccca gacctacatc tgcaacgtga accacaagcc ctccaacacc 720 aaggtggaca agaaggtgga gcccaagtcc tgcgacaaga cccacacctg ccccccctgc 780 cccgcccccg agctgctggg cggcccctcc gtgttcctgt tccccccaa gcccaaggac 840 accctgatga tctcccggac ccccgaggtg acttgcgtgg tggtggacgt gtcccacgag 900 gaccccgagg tgaagttcaa ctggtacgtg gacggcgtgg aggtgcacaa cgccaagacc 960 aagccccggg aggagcagta caactccacc taccgggtgg tgtccgtgct gaccgtgctg 1020 caccaggact ggctgaacgg caaggagtac aagtgcaagg tgtccaacaa ggccctgccc 1080 gcccccatcg agaagaccat ctccaaggcc aagggccagc cccgggagcc ccaggtgtac 1140 accctgcccc cctcccggga cgagctgacc aagaaccagg tgtccctgac ctgcctggtg 1200 aagggcttct acccctccga catcgccgtg gagtgggagt ccaacggcca gcccgagaac 1260 aactacaaga ccaccccccc cgtgctggac tccgacggct ccttcttcct gtactccaag 1320 ctgaccgtgg acaagtcccg gtggcagcag ggcaacgtgt tctcctgctc cgtgatgcac 1380 gaggccctgc acaaccacta cacccagaag tccctgtccc tgtcccccgg caagtgagaa 1440 ttcgcggccg ctcgag 1456 <210> 25 <211> 233 <212> PRT <213> Belimumab light chain <400> 25 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala              20 25 30 Leu Gly Gln Thr Val Arg Val Thr Cys Gln Gly Asp Ser Leu Arg Ser          35 40 45 Tyr Tyr Ala Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu      50 55 60 Val Ile Tyr Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe  65 70 75 80 Ser Gly Ser Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala                  85 90 95 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Arg Asp Ser Ser             100 105 110 Gly Asn His Trp Val Phe Gly Gly Gly Thr Glu Leu Thr Val Leu Gly         115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu     130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val                 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys             180 185 190 Tyr Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser         195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu     210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser 225 230 <210> 26 <211> 472 <212> PRT <213> Belimumab heavy chain <400> 26 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys              20 25 30 Pro Gly Ser Ser Val Val Arg Ser Ser Cys Lys Ala Ser Gly Gly Thr Phe          35 40 45 Asn Asn Asn Ale Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu      50 55 60 Glu Trp Met Gly Gly Ile Ile Pro Met Phe Gly Thr Ala Lys Tyr Ser  65 70 75 80 Gln Asn Phe Gln Gly Arg Val Ala Ile Thr Ala Asp Glu Ser Thr Gly                  85 90 95 Thr Ala Ser Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ser Arg Asp Leu Leu Leu Phe Pro His His Ala         115 120 125 Leu Ser Pro Trp Gly Arg Gly Thr Met Val Thr Val Ser Ser Ala Ser     130 135 140 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr 145 150 155 160 Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro                 165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val             180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser         195 200 205 Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile     210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 240 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala                 245 250 255 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro             260 265 270 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val         275 280 285 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val     290 295 300 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 305 310 315 320 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln                 325 330 335 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala             340 345 350 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro         355 360 365 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr     370 375 380 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 385 390 395 400 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr                 405 410 415 Lys Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr             420 425 430 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe         435 440 445 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys     450 455 460 Ser Leu Ser Leu Ser Pro Gly Lys 465 470 <210> 27 <211> 742 <212> DNA <213> golimumab light chain <400> 27 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgagat cgtgctgacc cagtcccccg ccaccctgtc cctgtccccc 120 ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgtactccta cctggcctgg 180 taccagcaga agcccggcca ggccccccgg ctgctgatct acgacgcctc caaccgggcc 240 accggcatcc ccgcccggtt ctccggctcc ggctccggca ccgacttcac cctgaccatc 300 tcctccctgg agcccgagga cttcgccgtg tactactgcc agcagcggtc caactggccc 360 cccttcacct tcggccccgg caccaaggtg gacatcaagc ggaccgtggc cgccccctcc 420 gtgttcatct tccccccctc cgacgagcag ctgaagtccg gcaccgcctc cgtggtgtgc 480 ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg 540 cagtccggca actcccagga gtccgtgacc gagcaggact ccaaggactc cacctactcc 600 ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 660 gaggtgaccc accagggcct gtcctccccc gtgaccaagt ccttcaaccg gggcgagtgc 720 tgagaattcg cggccgctcg ag 742 <210> 28 <211> 1465 <212> DNA <213> golimumab heavy chain <400> 28 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actcccaggt gcagctggtg gagtccggcg gcggcgtggt gcagcccggc 120 cggtccctgc ggctgtcctg cgccgcctcc ggcttcatct tctcctccta cgccatgcac 180 tgggtgcggc aggcccccgg caacggcctg gagtgggtgg ccttcatgtc ctacgacggc 240 tccaacaaga agtacgccga ctccgtgaag ggccggttca ccatctcccg ggacaactcc 300 aagaacaccc tgtacctgca gatgaactcc ctgcgggccg aggacaccgc cgtgtactac 360 tgcgcccggg accggggcat cgccgctggc ggcaactact actactacgg catggacgtg 420 tggggccagg gcaccaccgt gaccgtgtcc tccgcctcca ccaagggccc ctccgtgttc 480 cccctggccc cctcctccaa gtccacctcc ggcggcaccg ccgccctggg ctgcctggtg 540 aaggactact tccccgagcc cgtgaccgtg tcctggaact ccggcgccct gacctccggc 600 gtgcacacct tccccgccgt gctgcagtcc tccggcctgt actccctgtc ctccgtggtg 660 actgtgccct cctcctccct gggcacccag acctacatct gcaacgtgaa ccacaagccc 720 tccaacacca aggtggacaa gaaggtggag cccaagtcct gcgacaagac ccacacctgc 780 cccccctgcc ccgcccccga gctgctgggc ggcccctccg tgttcctgtt cccccccaag 840 cccaaggaca ccctgatgat ctcccggacc cccgaggtga cttgcgtggt ggtggacgtg 900 tcccacgagg accccgaggt gaagttcaac tggtacgtgg acggcgtgga ggtgcacaac 960 gccaagacca agccccggga ggagcagtac aactccacct accgggtggt gtccgtgctg 1020 accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gtccaacaag 1080 gccctgcccg cccccatcga gaagaccatc tccaaggcca agggccagcc ccgggagccc 1140 caggtgtaca ccctgccccc ctcccgggac gagctgacca agaaccaggt gtccctgacc 1200 tgcctggtga agggcttcta cccctccgac atcgccgtgg agtgggagtc caacggccag 1260 cccgagaaca actacaagac cacccccccc gtgctggact ccgacggctc cttcttcctg 1320 tactccaagc tgaccgtgga caagtcccgg tggcagcagg gcaacgtgtt ctcctgctcc 1380 gtgatgcacg aggccctgca caaccactac acccagaagt ccctgtccct gtcccccggc 1440 aagtgagaat tcgcggccgc tcgag 1465 <210> 29 <211> 234 <212> PRT <213> golimumab light chain <400> 29 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu              20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val          35 40 45 Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg      50 55 60 Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg  65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser                  85 90 95 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn             100 105 110 Trp Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 30 <211> 475 <212> PRT <213> golimumab heavy chain <400> 30 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln              20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Ile Phe          35 40 45 Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Asn Gly Leu      50 55 60 Glu Trp Val Ala Phe Met Ser Tyr Asp Gly Ser Asn Lys Lys Tyr Ala  65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Asp Arg Gly Ile Ala Ala Gly Gly Asn Tyr Tyr         115 120 125 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser     130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp                 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr             180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr         195 200 205 Ser Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln     210 215 220 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 225 230 235 240 Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro                 245 250 255 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro             260 265 270 Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr         275 280 285 Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn     290 295 300 Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 305 310 315 320 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val                 325 330 335 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser             340 345 350 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys         355 360 365 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp     370 375 380 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 385 390 395 400 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu                 405 410 415 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe             420 425 430 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly         435 440 445 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr     450 455 460 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 465 470 475 <210> 31 <211> 739 <212> DNA <213> Ustekinumab light chain <400> 31 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgacat ccagatgacc cagtccccct cctccctgtc cgcctccgtg 120 ggcgaccggg tgaccatcac ctgccgggcc tcccagggca tctcctcctg gctggcctgg 180 taccagcaga agcccgagaa ggcccccaag tccctgatct acgccgcctc ctccctgcag 240 tccggcgtgc cctcccggtt ctccggctcc ggctccggca ccgacttcac cctgaccatc 300 tcctccctgc agcccgagga cttcgccacc tactactgcc agcagtacaa catctacccc 360 tacaccttcg gccagggcac caagctggag atcaagcgga ccgtggccgc cccctccgtg 420 ttcatcttcc ccccctccga cgagcagctg aagtccggca ccgcctccgt ggtgtgcctg 480 ctgaacaact tctacccccg ggaggccaag gtgcagtgga aggtggacaa cgccctgcag 540 tccggcaact cccaggagtc cgtgaccgag caggactcca aggactccac ctactccctg 600 tcctccaccc tgaccctgtc caaggccgac tacgagaagc acaaggtgta cgcctgcgag 660 gtgacccacc agggcctgtc ctcccccgtg accaagtcct tcaaccgggg cgagtgctga 720 gaattcgcgg ccgctcgag 739 <210> 32 <211> 1426 <212> DNA <213> Ustekinumab heavy chain <400> 32 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactccgag 60 gtgcagctgg tgcagtccgg cgccgaggtg aagaagcccg gcgagtccct gaagatctcc 120 tgcaagggct ccggctactc cttcaccacc tactggctgg gctgggtgcg gcagatgccc 180 ggcaagggcc tggactggat cggcatcatg tcccccgtgg actccgacat ccggtactcc 240 ccctccttcc agggccaggt gactatgtcc gtggacaagt ccatcaccac cgcctacctg 300 cagtggaact ccctgaaggc ctccgacacc gccatgtact actgcgcccg gcggcggccc 360 ggccagggct acttcgactt ctggggccag ggcaccctgg tgaccgtgtc ctcctcctcc 420 accaagggcc cctccgtgtt ccccctggcc ccctcctcca agtccacctc cggcggcacc 480 gccgccctgg gctgcctggt gaaggactac ttccccgagc ccgtgaccgt gtcctggaac 540 tccggcgccc tgacctccgg cgtgcacacc ttccccgccg tgctgcagtc ctccggcctg 600 tactccctgt cctccgtggt gactgtgccc tcctcctccc tgggcaccca gacctacatc 660 tgcaacgtga accacaagcc ctccaacacc aaggtggaca agcgggtgga gcccaagtcc 720 tgcgacaaga cccacacctg ccccccctgc cccgcccccg agctgctggg cggcccctcc 780 gtgttcctgt tcccccccaa gcccaaggac accctgatga tctcccggac ccccgaggtg 840 acttgcgtgg tggtggacgt gtcccacgag gaccccgagg tgaagttcaa ctggtacgtg 900 gacggcgtgg aggtgcacaa cgccaagacc aagccccggg aggagcagta caactccacc 960 taccgggtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggagtac 1020 aagtgcaagg tgtccaacaa ggccctgccc gcccccatcg agaagaccat ctccaaggcc 1080 aagggccagc cccgggagcc ccaggtgtac accctgcccc cctcccggga cgagctgacc 1140 aagaaccagg tgtccctgac ctgcctggtg aagggcttct acccctccga catcgccgtg 1200 gagtgggagt ccaacggcca gcccgagaac aactacaaga ccaccccccc cgtgctggac 1260 tccgacggct ccttcttcct gtactccaag ctgaccgtgg acaagtcccg gtggcagcag 1320 ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacccagaag 1380 tccctgtccc tgtcccccgg caagtgagaa ttcgcggccg ctcgag 1426 <210> 33 <211> 233 <212> PRT <213> Ustekinumab light chain <400> 33 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Leu Ser Ala              20 25 30 Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile          35 40 45 Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys      50 55 60 Ser Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Ser Ser Arg  65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser                  85 90 95 Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ile             100 105 110 Tyr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr         115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu     130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly                 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr             180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His         195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val     210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 34 <211> 468 <212> PRT <213> Ustekinumab heavy chain <400> 34 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys              20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe          35 40 45 Thr Thr Trp Leu Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu      50 55 60 Asp Trp Ile Gly Ile Met Ser Pro Val Asp Ser Asp Ile Arg Tyr Ser  65 70 75 80 Pro Ser Phe Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr                  85 90 95 Thr Ala Tyr Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met             100 105 110 Tyr Tyr Cys Ala Arg Arg Arg Pro Gly Gln Gly Tyr Phe Asp Phe Trp         115 120 125 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ser Thr Lys Gly Pro     130 135 140 Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr                 165 170 175 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro             180 185 190 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr         195 200 205 Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn     210 215 220 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser 225 230 235 240 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu                 245 250 255 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu             260 265 270 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser         275 280 285 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu     290 295 300 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 305 310 315 320 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn                 325 330 335 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro             340 345 350 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln         355 360 365 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val     370 375 380 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 385 390 395 400 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro                 405 410 415 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr             420 425 430 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val         435 440 445 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu     450 455 460 Ser Pro Gly Lys 465 <210> 35 <211> 742 <212> DNA <213> ipilimumab light chain <400> 35 gctagcaagc ttgccaccat gggctggtcc tgcatcatcc tgttcctggt ggccaccgcc 60 accggcgtgc actccgagat cgtgctgacc cagtcccccg gcaccctgtc cctgtccccc 120 ggcgagcggg ccaccctgtc ctgccgggcc tcccagtccg tgggctcctc ctacctggcc 180 tggtaccagc agaagcccgg ccaggccccc cggctgctga tctacggcgc cttctcccgg 240 gccaccggca tccccgaccg gttctccggc tccggctccg gcaccgactt caccctgacc 300 atctcccggc tggagcccga ggacttcgcc gtgtactact gccagcagta cggctcctcc 360 ccctggacct tcggccaggg caccaaggtg gagatcaagc ggaccgtggc cgccccctcc 420 gtgttcatct tccccccctc cgacgagcag ctgaagtccg gcaccgcctc cgtggtgtgc 480 ctgctgaaca acttctaccc ccgggaggcc aaggtgcagt ggaaggtgga caacgccctg 540 cagtccggca actcccagga gtccgtgacc gagcaggact ccaaggactc cacctactcc 600 ctgtcctcca ccctgaccct gtccaaggcc gactacgaga agcacaaggt gtacgcctgc 660 gaggtgaccc accagggcct gtcctccccc gtgaccaagt ccttcaaccg gggcgagtgc 720 tgagaattcg cggccgctcg ag 742 <210> 36 <211> 1423 <212> DNA <213> ipilimumab heavy chain <400> 36 atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcgt gcactcccag 60 gtgcagctgg tggagtccgg cggcggcgtg gtgcagcccg gccggtccct gcggctgtcc 120 tgcgccgcct ccggcttcac cttctcctcc tacaccatgc actgggtgcg gcaggccccc 180 ggcaagggcc tggagtgggt gactttcatc tcctacgacg gcaacaacaa gtactacgcc 240 gactccgtga agggccggtt caccatctcc cgggacaact ccaagaacac cctgtacctg 300 cagatgaact ccctgcgggc cgaggacacc gccatctact actgcgcccg gaccggctgg 360 ctgggcccct tcgactactg gggccagggc accctggtga ccgtgtcctc cgcctccacc 420 aagggcccct ccgtgttccc cctggccccc tcctccaagt ccacctccgg cggcaccgcc 480 gccctgggct gcctggtgaa ggactacttc cccgagcccg tgaccgtgtc ctggaactcc 540 ggcgccctga cctccggcgt gcacaccttc cccgccgtgc tgcagtcctc cggcctgtac 600 tccctgtcct ccgtggtgac tgtgccctcc tcctccctgg gcacccagac ctacatctgc 660 aacgtgaacc acaagccctc caacaccaag gtggacaagc gggtggagcc caagtcctgc 720 gacaagaccc acacctgccc cccctgcccc gcccccgagc tgctgggcgg cccctccgtg 780 ttcctgttcc cccccaagcc caaggacacc ctgatgatct cccggacccc cgaggtgact 840 tgcgtggtgg tggacgtgtc ccacgaggac cccgaggtga agttcaactg gtacgtggac 900 ggcgtggagg tgcacaacgc caagaccaag ccccgggagg agcagtacaa ctccacctac 960 cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 1020 tgcaaggtgt ccaacaaggc cctgcccgcc cccatcgaga agaccatctc caaggccaag 1080 ggccagcccc gggagcccca ggtgtacacc ctgcccccct cccgggacga gctgaccaag 1140 aaccaggtgt ccctgacctg cctggtgaag ggcttctacc cctccgacat cgccgtggag 1200 tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggactcc 1260 gcggctcct tcttcctgta ctccaagctg accgtggaca agtcccggtg gcagcagggc 1320 aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1380 ctgtccctgt cccccggcaa gtgagaattc gcggccgctc gag 1423 <210> 37 <211> 234 <212> PRT <213> ipilimumab light chain <400> 37 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu              20 25 30 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val          35 40 45 Gly Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro      50 55 60 Arg Leu Leu Ile Tyr Gly Ala Phe Ser Arg Ala Thr Gly Ile Pro Asp  65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                  85 90 95 Arg Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly             100 105 110 Ser Ser Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg         115 120 125 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln     130 135 140 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 145 150 155 160 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser                 165 170 175 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr             180 185 190 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys         195 200 205 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro     210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 38 <211> 467 <212> PRT <213> ipilimumab heavy chain <400> 38 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly   1 5 10 15 Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln              20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe          35 40 45 Ser Ser Tyr Thr Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu      50 55 60 Glu Trp Val Thr Phe Ile Ser Tyr Asp Gly Asn Asn Lys Tyr Tyr Ala  65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                  85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile             100 105 110 Tyr Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser     130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala             180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val         195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His     210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Pro Gly Lys 465

Claims (20)

(i) a first expression cassette comprising a 'promoter-UTR (untranslation region) -introne-antibody light chain gene -polyA'; And
(ii) a second expression cassette comprising a 'promoter-UTR-intron-antibody heavy chain gene-IRES (internal ribosome entry site) -amplification gene -polyA'
BiSystronic expression vector for antibody expression.
2. The expression vector according to claim 1, wherein the promoter is selected from the group consisting of SV40 (simian virus 40) early promoter, CMV (cytomegalovirus) promoter and hEF1-alpha (human elongation factor 1-alpha) promoter.
2. The expression vector of claim 1, wherein the UTR is CMV-derived.
2. The expression vector according to claim 1, wherein the intron is derived from CMV.
2. The expression vector of claim 1, wherein the promoter is an SV40 early promoter, and the UTR and the intron are derived from SV40.
2. The expression vector according to claim 1, wherein the promoter is a CMV promoter, and the UTR and the intron are derived from CMV.
The expression vector according to claim 1, wherein the promoter is an hEF1-a promoter, and the UTR and the intron are derived from hEF1-a.
3. The expression vector according to claim 2, wherein the promoter is a CMV promoter consisting of the nucleotide sequence of SEQ ID NO: 1.
4. The expression vector according to claim 3, wherein the UTR is a CMV-derived UTR consisting of the nucleotide sequence of SEQ ID NO: 2.
6. The expression vector according to claim 5, wherein the intron comprises the nucleotide sequence of SEQ ID NO: 3.
The expression vector according to claim 1, wherein the amplified gene is GS (glutamine synthetase) or DHFR (dehydrofolate reductase).
2. The expression vector of claim 1, wherein the antibody is bevacizumab.
2. The expression vector according to claim 1, wherein the expression vector has cleavage map of pCYB204IG described in Fig. 5 or cleavage map of pCYB204ID described in Fig.
The expression vector according to claim 1, wherein the antibody light chain gene comprises the nucleotide sequence of SEQ ID NO: 11.
The expression vector according to claim 1, wherein the antibody heavy chain gene comprises the nucleotide sequence of SEQ ID NO: 12.
15. An animal cell transformed with an expression vector according to any one of claims 1 to 15.
17. The animal cell of claim 16, wherein the animal cell is a Chinese hamster ovary cell (CHO).
16. A method of producing an antibody comprising culturing the animal cell of claim 16.
19. The method of claim 18,
Further comprising the step of purifying the antibody in a culture in which the animal cell has been cultured.
20. The method of claim 18 or 19, wherein the antibody is bevacizumab.

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