JP2009136243A - TRANSFORMANT PRODUCING FUSION PROTEIN OF HUMAN Delta1 AND Fc FRACTION OF HUMAN IMMUNOGLOBULIN G - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing human Delta 1 with high purity in large quantities and conveniently in order to put a Delta1-utilizing technology into practical use, wherein a utilizing technology for Delta1 being Notch ligand, for example an immunotherapy utilizing the anticancer effect of Delta1, has been developed. <P>SOLUTION: Disclosed are: a transformant of Accession No. NITE AP-460 which produces a protein comprising an amino acid sequence represented by SEQ ID NO: 4, wherein the protein is a fusion protein of human Delta1 and the Fc fraction of human immunoglobulin G; and a method for producing the fusion protein using the transformant. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a transformant producing human Delta1, which is a Notch ligand. More specifically, a CHO cell that produces a fusion protein of human Delta1 and the Fc portion of human immunoglobulin G, comprising the amino acid sequence shown in SEQ ID NO: 4, the trait of receipt number NITE AP-460 The present invention relates to a transformant and a method for producing a fusion protein using the same.

  The term “Notch” was first used in 1917 in the description of Drosophila with a V-shaped notch at the heel end by TH Morgan et al. Was reported for the first time. A Notch molecule is a membrane protein or transmembrane receptor consisting of a signal peptide occupying its N-terminus, a transmembrane (extracellular) domain, an intracellular domain, etc., and the function of Notch that controls development and differentiation is a cell surface ligand. Activated by. Notch and its ligands are expressed in many cells, and in mammals 4 types of Notch, Notch1-Notch4, and 5 types of Notch ligands, Delta1, Delta3, Delta4, Jagged1 and Jagged2 are currently known (non-patent literature). 1).

  Regarding the action / function of such Notch and its ligand, for example, control of lymphocyte differentiation by these signal transmissions, that is, differentiation of hematopoietic stem cells into T cells, B cells, etc., T cells and B cells by Notch and its ligands Control of functionalization of the Notch (Non-patent document 2), differentiation into mature T cells by Notch and control of its activity (Non-patent document 3), signal transduction of Notch by glycosylation, a post-transcriptional modification Non-Patent Documents 1 to 3 are reviewed as described above.

  Furthermore, regarding the use of the above-mentioned mammalian Notch and its ligands and these genes, the following are known: feeders for totipotent stem cell culture (Patent Document 1), stem cell culture medium (Patent Document 2), bone marrow transplantation, Suppression of rejection and allergic reactions in organ transplantation (Patent Document 3), therapeutic agents for osteoporosis and osteoclast differentiation (Patent Document 4), vascular cell regulator (Patent Document 5), control of cell differentiation (Patent Document) 6), Proliferation suppression of prostate cells (Patent Literature 7), Amplification of progenitor cells by suppression of cell differentiation (Patent Literature 8), Delta or Jagged gene, and immunity responsible for transferring genes such as allergen, MHC antigen, cancer antigen, etc. A vaccine using cells (Patent Document 9), an inhibitor of rejection in organ transplantation using Delta and a therapeutic agent for diseases associated with bone marrow transplantation (Patent Document 11), Suppression of growth of myeloid leukemia cell line by immunotherapy agent for cancer, allergy, infectious disease, etc. (patent document 12), cytokine expression regulator (patent document 13), Delta1 and Jagged1. (Nonpatent literature 4) etc.

  In addition to the above, a regulator of IL4 expression and Th1 / Th2 balance using a fusion protein of the extracellular domain of Delta and the Fc domain of IgG is known (Patent Document 10). It is also known that a fusion protein of Delta1 or Delta4 and the Fc part of human IgG promotes the differentiation of CD8-positive T lymphocytes into CTLs and produces an antitumor effect (Patent Document 14). Although Patent Document 14 describes that a fusion protein obtained by fusing human Delta1 or mouse Delta1 with the Fc part of human IgG is produced by a transformant, mouse Delta1 is actually used in the examples. Only fusion protein. Even if this transformant is cultured, it produces only a few mg of fusion protein per liter of culture supernatant.

  As can be seen from the above, various techniques for utilizing the function of Delta1, a notch ligand, have already been developed. However, in order to put these technologies into practical use, for example, to carry out immunotherapy utilizing the antitumor effect of Delta1, methods for producing high-purity Delta1 easily in large quantities at low cost are still known. Absent.

JP2005-34 WO02 / 016556 JP 2003-93048 A JP 2001-122798 A JP-A-10-316582 JP 2004-65243 A Special table 2004-524269 Special table 2000-511043 WO04 / 083372 WO04 / 062686 WO04 / 022730 WO03 / 087159 WO03 / 011317 JP 2006-241087 Nature Reviews Molecular Cell Biology, Volume 4, 786-797, October 2003. Nature Immunology, Volume 5 (3), 247-253, March 2004. Journal of Immunology, 173, 7109-7113, 2004. International Journal of Molecular Medicine, 14 (2), 223-226, 2004. Gene Therapy, Volume 7, 1063-1066, 2000.

  Techniques utilizing Delta1, which is a Notch ligand, have been developed, for example, immunotherapy utilizing the antitumor effect of Delta1. However, in order to put the technology using Delta1 into practical use, there has been no method for producing high-purity human Delta1 in large quantities and simply.

  In order to solve the above problems, an object of the present invention is a trait that is a cell that produces a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, which is a fusion protein of human Delta1 and the Fc part of human immunoglobulin G. It is to provide a method for producing a Delta1 as a fusion protein by providing a transformant and using the transformant.

  Since the transformant of the present invention releases a large amount of soluble Delta1 into the medium, Delta1 can be easily and rapidly isolated and purified from the culture supernatant as a fusion protein with the Fc portion of IgG. .

(Explanation of sequence listing)
SEQ ID NO: 1: Human Delta1 full-length amino acid sequence encoded by human Delta1 full-length gene DNA (NCBI accession: NM_005618).
SEQ ID NO: 2: A nucleotide sequence encoding a fusion protein consisting of the extracellular region of human Delta1 and the Fc portion of human immunoglobulin G, which was used to produce the transformant of the present invention.
SEQ ID NO: 3: Amino acid sequence of the fusion protein encoded by the base sequence of SEQ ID NO: 2.
SEQ ID NO: 4: Amino acid sequence of a soluble fusion protein consisting of the extracellular region of human Delta1 and the Fc portion of human immunoglobulin G, produced by the transformant of the present invention.
SEQ ID NO: 5: Base sequence of vector pMKITneo used for production of the transformant of the present invention.

  In light of the above-mentioned problems and circumstances, the present inventors have obtained a fusion protein of human Delta1 and the Fc part of human immunoglobulin G consisting of the amino acid sequence shown in SEQ ID NO: 4 as a result of many years of deep insight and trial and error. In the present specification, the transformant of the present invention is often referred to as “D1Fc transformant”, and the fusion protein produced by the transformant is referred to as “D1-F1”. Referred to as "Fc protein"). This transformant was deposited on November 14, 2007 to the National Institute of Technology and Evaluation, the Patent Microorganism Deposit Center (2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818). The receipt date of the deposited microorganism is November 16, 2007, and the receipt number is NITE AP-460 (note that the microorganism identification is “DIF 144”, but this cell is the “D1Fc transformation of the present invention. Body "). The D1Fc transformant of the present invention produces 200 mg or more of fusion protein per liter of culture supernatant. Therefore, by culturing the transformant of the present invention and recovering the culture supernatant, it is possible to isolate and purify highly purified Delta1 protein from the culture supernatant simply, rapidly and in large quantities.

According to the present invention, the following (1) and (2) are provided as means for solving the above-described problems.
(1) A transformant of accession number NITE AP-460, which is a cell that produces a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, which is a fusion protein of human Delta1 and the Fc portion of human immunoglobulin G.
(2) A method for producing a fusion protein of human Delta1 and the Fc part of human immunoglobulin G, comprising the amino acid sequence shown in SEQ ID NO: 4, characterized by using the transformant of item 1 above.

The D1Fc transformant of the present invention was prepared by the following method.
First, a base sequence encoding a fusion protein of human Delta1 and the Fc portion of human immunoglobulin G was prepared. The prepared base sequence is shown in SEQ ID NO: 2. This base sequence is obtained by removing 4 amino acids from the C-terminus of the amino acid sequence corresponding to the extracellular region of human Delta1 (amino acids 1 to 541 of SEQ ID NO: 1) and 2 for fusion with IgG. A base sequence encoding an amino acid (Asp and Pro) inserted and a genomic DNA corresponding to a human IgG Fc region are linked.

  Next, the base sequence encoding the fusion protein was inserted and linked into the plasmid vector pMKITneo to obtain a D1-Fc fusion protein expression vector. The plasmid vector pMKITneo used in the present invention is a vector prepared by Dr. Kazuo Maruyama using the SRalpha promoter developed by Dr. Yutaka Takebe [Takebe et.al. Mol. Cell Biol., 8, 466, 1988 and See Maruyama et al., Transfection of cultured mammalian cells by mammalian expression vectors, Methods in Nucleic Acids Res., CRC Press, 283 (1991)]. The base sequence of the vector is shown in SEQ ID NO: 5.

  CHO-K1 cells were transformed with the thus constructed expression vector by a lipofection method using transfection of Bio-rad, USA. Parental strains, which were cells transformed using geneticin resistance as an index, were selected, and primary cloning by limiting dilution was performed using them. Thereafter, secondary cloning by limiting dilution was further performed to obtain a subclone in which the fusion protein productivity was increased 1.5 to 2.5 times that of the parent strain.

  In the present invention, this subclone was further acclimated to a serum-free medium to obtain the D1Fc transformant of the present invention. This transformant was deposited on November 14, 2007 to the National Institute of Technology and Evaluation, the Patent Microorganism Deposit Center (2-5-8 Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818). The receipt date of the deposited microorganism is November 16, 2007, the receipt date is November 16, 2007, and the receipt number is NITE AP-460 (note that the identification of the microorganism is “DIF 144” This cell is the same as the “D1Fc transformant” of the present invention).

  The D1Fc transformant of the present invention produces human Delta1 as a fusion protein with the Fc portion of human immunoglobulin G. Full-length human Delta1 (SEQ ID NO: 1) is a protein consisting of 723 amino acids, but Delta1 contained in the D1-Fc fusion protein (SEQ ID NO: 4) released into the medium by the transformant of the present invention is SEQ ID NO: 1 is a polypeptide corresponding to amino acids No. 22 to No. 541. This polypeptide corresponds to a polypeptide in which four amino acids have been deleted from the C-terminus of the extracellular portion of Delta1 (Nos. 22 to 545 of SEQ ID NO: 1). It has been clarified that only the extracellular part of Delta1 exerts the function of Delta1 sufficiently. Furthermore, since the sequence consisting of the extracellular portion of Delta1 is soluble, it is advantageous for producing and purifying a large amount of pure protein as described later.

  The base sequence introduced into the expression vector includes the base sequence encoding the signal sequence (No. 1 to No. 21 amino acid of SEQ ID No. 1) of Delta1, but the fusion protein is a cell. The signal sequence is cleaved when released out. Therefore, the signal sequence is not included in the D1-Fc fusion protein.

  The amino acid sequence and base sequence of the Fc part of human immunoglobulin G fused with human Delta1 are known and are used in the art for the production of various fusion proteins. Rather than producing human Delta1 or a partial sequence thereof alone, but as a fusion protein with the Fc portion of human immunoglobulin G, increasing protein yield, improving protein stability during production, There are advantages such as enhancement of biological activity in the body and improvement of in vivo stability.

  In order to obtain human Delta1 by a conventional method, for example, human cells are cultured, the cultured cells are thawed, and Delta1 is isolated and purified. Such a method has a problem that Delta1 must be isolated not only from various components contained in the medium but also from a large amount of cellular proteins. Therefore, without passing through a plurality of protein purification steps, it is not possible to obtain Delta1 having such a high purity that it can be used for immunotherapy. Also, the amount of Delta1 obtained is very limited. As a matter of course, since the cost for manufacturing Delta1 is enormous, in order to put the technology using Delta1 into practical use, a method for manufacturing Delta1 with high purity in large quantities and simply has been desired.

  Under such circumstances, the present inventors succeeded in producing a transformant that releases a large amount of human Delta1 into the medium. Since the D1Fc transformant of the present invention releases the D1-Fc fusion protein into the medium, the D1Fc transformant can be cultured, and Delta1 can be isolated and purified from the culture supernatant. In this method, since contamination of the cell protein into the medium is minimized, high-purity Delta1 can be obtained by a simple purification process.

Next, a method for producing a fusion protein using the transformant of the present invention will be described.
First, the D1Fc transformant is cultured, and the culture supernatant is collected. As long as the D1Fc transformant which is CHO-K1 cells grows and the D1-Fc fusion protein is released into the medium, the medium to be used is not particularly limited, and a commercially available medium used for culturing CHO-K1 cells can be used. Can be used. Since the D1Fc transformant of the present invention is conditioned to a serum-free medium, it is preferably cultured in a serum-free medium in order to prevent contamination of the fusion protein with serum. Various amino acids, vitamins, yeast extract, soybean hydrolyzate, saccharides and the like may be added to the medium as necessary. The culture method for the D1Fc transformant is not limited, and any of batch culture, fed-batch culture, and continuous culture may be used. The culture conditions are not particularly limited as long as the D1Fc transformant grows and mutations are not induced. For example, the pH is about 6.6 to 7.8, preferably about 6.8 to under an air flow. The agitation culture can be performed for 2 days to 2 weeks, preferably 3 days to 12 days under the conditions of 7.5 and a culture temperature of about 30 to 38 ° C, preferably 35 to 37 ° C.

  The amount of D1-Fc fusion protein accumulated in the medium in one culture of the D1Fc transformant varies depending on the composition of the medium and the culture conditions, but is usually 200 mg / liter (L) or more (FIG. 2). See). When a D1Fc transformant is cultured for the purpose of producing a D1-Fc fusion protein, the collection time of the culture supernatant can be determined using the number of cells as an index. In addition, a part of the culture supernatant is collected during the culture, and the accumulated amount of the fusion protein is measured by a known method such as ELISA, and the culture supernatant is recovered when a sufficient amount of the fusion protein is detected. You can also

  There is no particular limitation on the method of recovering the culture supernatant from the culture solution of the D1Fc transformant, and a method used for separating cells from the culture solution in the art may be carried out. For example, the cells can be separated from the culture solution by centrifugation or filtration, and the culture supernatant can be collected.

  The method for isolating and purifying the D1-Fc fusion protein from the culture supernatant is not particularly limited, and a known protein purification method may be performed. Specifically, if the D1-Fc fusion protein is isolated from the collected culture supernatant by, for example, salting out, precipitation with an organic solvent, batch method using adsorption / desorption to a commercially available gel or resin, column purification, etc. Good. Since the D1-Fc fusion protein is a fusion protein with the Fc part of human immunoglobulin G, a known purification method using protein A or protein G known to bind to the Fc part of human IgG, for example, a protein A affinity column And high-purity can be easily achieved by affinity chromatography using a protein G affinity column.

  Furthermore, in the present invention, not only the culture supernatant but also the cultured transformant can be thawed and the D1-Fc fusion protein can be extracted from the cell lysate. In this case, the protein obtained from the cell is a fusion protein containing a signal sequence, that is, the amino acid sequence of SEQ ID NO: 3.

  The D1-Fc fusion protein isolated and purified from the transformant of the present invention is dispensed into ampoules, vials, etc., and stored and provided in a sealed state in the form of a liquid or a dried product (lyophilized product). can do.

  The D1-Fc fusion protein obtained by the method as described above can be used in various techniques. For example, the D1-Fc fusion protein promotes the differentiation of CD8 positive T lymphocytes into CTLs and produces an antitumor effect. The antitumor effect is obtained by administering a D1-Fc fusion protein to mice injected or transplanted with tumor cells subcutaneously and measuring the tumor diameter (diameter of a circular tumor cell region) that expands due to the growth of the tumor cells. Can be evaluated.

  Hereinafter, the configuration and effects of the present invention will be specifically described with reference to examples and reference examples. However, the present invention is not limited to these examples and reference examples.

(1) Construction of D1-Fc fusion protein expression vector A base sequence encoding a fusion protein of human Delta1 and the Fc portion of human immunoglobulin G was prepared. Specifically, the amino acid sequence corresponding to the extracellular part of human Delta1 except for 4 amino acids from the C-terminus (ie, amino acids 1 to 541 of SEQ ID NO: 1) for fusion with IgG The base sequence encoding the amino acid sequence in which two amino acids (Asp and Pro) were inserted into and the genomic DNA corresponding to the human IgG Fc region were linked to construct the base sequence of SEQ ID NO: 2 encoding the fusion protein. .
Next, the base sequence encoding the fusion protein is inserted between the EcoRI site (gaattc; 2,994 to 2,999 bases) and NotI site (gcggccgc; 3,387 to 3,394 bases) of plasmid vector pMKITneo (SEQ ID NO: 5). Together, a D1-Fc fusion protein expression vector was obtained.

(2) Establishment of D1-Fc fusion protein-producing cells CHO-K1 cells were transformed with the expression vector constructed in the above section (1) by the lipofection method using transfection of Bio-rad, USA. Specifically, CHO-K1 cells are seeded in a 6-well multi-plate dish and 1 in a CO ² incubator (culture temperature: 36.5 ± 0.5 ° C., carbon dioxide concentration: 5.0 ± 0.2%). Cultured for days. For the culture, 500 ml of Ham's F-12 medium (manufactured by Sigma, USA) plus 55.6 ml of non-immobilized fetal bovine serum was used. 400 μl of the transferctin-plasmid DNA complex was dropped into a dish having a seeded cell density of 4 × 10 ⁵ cells / ml, which reached 70 to 90% confluence, and cultured for 20 to 24 hours. Introduced.

  Since transformed cells acquire resistance to geneticin, which is a selection marker, geneticin resistant strains were selected from the penicillin cup method or 96-well multiplate dishes. Geneticin (G8168 manufactured by Sigma, USA) was used for selection, and its concentration was 800 mg / L. As a result, a total of 30 geneticin-resistant strains were isolated, and 19 strains with high D1-Fc fusion protein production rate and high growth were selected from them (D1-Fc fusion protein in the medium was ELISA described later). Quantified by the method). These 19 strains were further cultured in a flask, and a strain having a high accumulation amount of D1-Fc fusion protein in the medium was selected as a strain subjected to primary cloning.

Primary cloning was performed by limiting dilution. Specifically, a cell suspension of 10 cells / ml was prepared, seeded at 0.1 ml / well in a 96-well multiplate dish, and cultured for 6 days. 0.1 ml of selective medium (5 ml of non-immobilized fetal bovine serum added to 500 ml of Ham's F-12 medium and further 800 mg / L of geneticin) was added to each well, and further cultured for 8 days. did. The culture supernatant was collected from the well in which a single colony was formed, and the accumulated amount of D1-Fc fusion protein was measured. A strain having a high production amount was selected, from which a strain having a particularly high growth rate and a high production rate of the D1-Fc fusion protein was selected, and secondary cloning was performed. Secondary cloning was performed by the same limiting dilution method as described above, and transformants with high productivity and high productivity of the D1-Fc fusion protein were selected.

(3) Acclimation of D1-Fc fusion protein-producing cells to serum-free medium D1-Fc fusion protein-producing cells that can be cultured in serum-free medium by subjecting the secondary cloning isolate to conditioned culture under the following conditions Established.

For the conditioned culture, EX-CELL 302 (SAFC, 14324-1000M, USA), which is the above-described selective medium and serum-free medium, is used, and the two media are mixed as follows and used for cell passage. : 2 passages with selective medium (50%) + serum free medium (50%), 3 passages with selective medium (10%) + serum free medium (90%), selective medium (5%) + serum free medium 3 passages (95%) and 10 passages in serum-free medium.
The culture container for conditioned culture is a T-25 flask (3103-025, manufactured by IWAKI, Japan), the culture volume is 5 ml, the target seeded cell density is 1 to 5 × 10 ⁵ cells / ml, and the culture temperature is 36.5. Under the conditions of ± 0.5 ° C. and carbon dioxide gas concentration of 5.0 ± 0.2%, the number of days of culturing was set to 2 to 5 days. Passage was performed when the survival rate determined from the cell count was 90% or more, the culture supernatant was collected, and the accumulated amount of D1-Fc fusion protein was measured by the ELISA method described later. In addition, when cells floated during subculture, they were cultured in a 125 ml Erlenmeyer flask without performing trypsin treatment. In the case where adherent-extended cells and floating cells coexist, only floating cells were collected, and the viable cell density was measured and passaged.

The transformant thus obtained was named a D1Fc transformant, and as of November 14, 2007, the National Institute of Technology and Evaluation, the Patent Microorganism Depositary Center (Kazusa Kamashichi, Kisarazu City, Chiba Prefecture 292-0818, Japan) -5-8). The receipt date of the deposited microorganism is November 16, 2007, and the receipt number is NITE AP-460 (note that the microorganism identification is “DIF 144”, but this cell is the “D1Fc transformation of the present invention. Body ").

(4) Culture of D1Fc transformant The D1Fc transformant prepared in (3) above was cultured using a 5 L culture tank (BCP-07, Biot, Japan). EX-CELL 302 (SAFC, 14324-1000M, USA) as a serum-free medium as a medium, final concentration 4 mM L-glutamine-200 mM solution (USA, 59202-100M, SAFC), 1/100 volume of 100 × HT solution , And a final concentration of 800 mg / L of geneticin was used and fed batch culture. The culture volume is 4 L at the start of culture, the culture temperature is 36.7 ± 0.1 ° C., the stirring rate is 49 rpm, the pH target value is 7.3 ± 0.1, the dissolved oxygen concentration target value is 20% ± 10%, and the seeded cells. The density was cultured at 2 × 10 ⁵ cells / ml for 12 days, and the following fed-batch medium was added on days 3, 5, 7, 9 and 11 of the culture.
Feed medium added on days 3, 5 and 7 of culture: 0.5 ml amino acid concentrate (SAFC 60990-1000M, USA), 0.5 ml vitamins (SAFC 60989-1000M, USA), 0. 2 ml soybean hydrolysate (58903-100M, SAFC, USA), 1 ml 200 mM L-glutamine solution and 1 ml 200 g / L glucose solution; and fed-batch medium added on days 9 and 11 of culture: 0.5 ml Amino acid concentrate, 0.5 ml vitamins, 0.2 ml soybean hydrolysate, 1 ml 200 mM L-glutamine solution and 0.5 ml 200 g / L glucose solution.

The dissolved oxygen concentration was controlled by supplying oxygen to the upper surface or the culture solution. The pH was controlled by supplying carbon dioxide gas to the upper surface when rising and adding 1.5 mol / L sodium carbonate when falling. The cell density and viability were measured by analyzing the cell suspension collected from the culture tank using a cell autoanalyzer. From the third day of culture, the accumulated amount of D1-Fc fusion protein in the supernatant was measured by ELISA described in detail below.

(5) Quantification of D1-Fc fusion protein by ELISA method As a primary antibody solution, rabbit anti-human IgG (Dako Cytomation, Denmark, Polyclonal Rabbit Anti-human IgG, Code No. A0423) was used 2400 times with PBS (-). The diluted one was used. 100 μl of the primary antibody solution is added to each well of a 96-well microtiter plate (manufactured by Nalgene Nunk International Co., Ltd., NUNC-Immunoplate F96 CERT. MAXISORP, product number 439454), and allowed to stand at 2-8 ° C. overnight. The primary antibody was coated on the well. After removing the liquid in the well, the plate was struck against clean paper to drain the liquid. To wash the wells, add 300 μl of wash solution (0.5 g Tween20 plus 1000 ml PBS (−)) to all wells of the plate, remove the solution in the wells, and then place the plate on clean paper The washing operation of tapping and draining the liquid was repeated a total of 3 times. Next, 200 μl of blocking solution [1.0 g of bovine serum albumin dissolved with 100 ml of PBS (−)] was added to each well of the plate, and the plate was allowed to stand at room temperature for 60 minutes. The same washing operation as described above was repeated a total of 3 times to remove excess blocking solution.

  Human Delta1-Ig standard stock solution (lot number 050121, concentration 1.08 mg / ml, received from Tokyo University Hospital), which is an isolated and purified fusion protein of human Delta1 and IgG, was used as a standard substance, and it was used as a sample diluent [ 0.2 g BSA (fraction V) and 0.1 g Tween 20 plus 200 ml PBS (−)] were used to prepare a standard solution for preparing a calibration curve. The Delta1-Ig concentration of the standard solution was 400, 200, 100, 50, 25, 12.5, 6.25, and 0 ng / ml. A sample solution (the culture supernatant of the transformant diluted with a sample diluent) and a standard solution are added to each of two wells, and the plate is allowed to stand at room temperature for 60 minutes. Repeated 3 times.

  As a secondary antibody, a peroxidase-labeled anti-human IgG (γ chain) rabbit polyclonal antibody (Dako Cytomation, Denmark, Code No. A0406, 1.1 mg / ml) was used, and the sample diluted solution was 4000 as described above. A solution diluted twice was used as a secondary antibody solution. After adding 100 μl of the secondary antibody solution to each well and allowing to stand at room temperature for 60 minutes, the same washing operation as described above was repeated three times. Next, 100 μl of substrate solution was added to each well of the plate. The substrate solution consists of 1 OPD tablet (10 mg o-phenylenediamine dihydrochloride / tablet, manufactured by Wako Pure Chemical Industries, Japan) in 25 ml OPD solution (10.5 g citric acid monohydrate and phosphoric acid). Dissolved by adding 35.8 g of disodium hydrogen dodecahydrate in water and making it 500 ml), dissolved it by adding 5 μl of hydrogen peroxide (30%) immediately before use. The plate was covered with a plate seal, shielded from light with aluminum foil, and allowed to stand at room temperature for 20 minutes to carry out the enzyme reaction. The enzyme reaction was stopped by adding 100 μl of a 2 mol / L sulfuric acid solution to each well of the plate and stirring with a microplate mixer (MPX96 manufactured by IWAKI, Japan).

Next, absorbance at wavelengths of 490 nm (A ₁ ) and 650 nm (A ₂ ) was measured using a microplate reader (Emax, manufactured by Molecular Devices, USA). A calibration curve based on the 4-parameter method is created by taking the concentration of Delta1-Ig on the horizontal axis and the absorbance (A ₁ -A ₂ ) on the vertical axis and plotting the concentration and absorbance of the standard solution on the graph. did. Using this calibration curve, the concentration of the D1-Fc fusion protein was determined from the absorbance of each sample solution.

The culture results of the D1Fc transformant of the present invention are shown in FIGS. FIG. 1 shows changes over time in cell proliferation and survival rate, and FIG. 2 shows changes over time in the accumulated amount of D1-Fc fusion protein in the culture supernatant. The reached cell density on day 7 of culture was 37 × 10 ⁵ cells / ml, and the accumulated amount of D1-Fc fusion protein on day 12 of culture was very high at 214 mg / L.

Reference example 1

Anti-tumor effect by Delta1-Fc fusion protein Eight week old female BALB / c mice were immunized by intraperitoneal inoculation of 3,000 rad-irradiated renal cancer cells 3T1 at 1 × 10 ⁷ cells / dose. Ten days after immunization, 3T1 was implanted subcutaneously on the back of each mouse. Subsequently, 50 μg / dose of D1-Fc fusion protein was administered intraperitoneally to each mouse at intervals of 3 days from the day of the first immunization. Thereafter, the tumor diameter of the mice was measured over time. As a result, the tumor diameter was clearly smaller in the group administered with the D1-Fc fusion protein compared to the control group not administered with the fusion protein.

  By using the transformant of the present invention, high-purity human Delta1 can be easily produced in large quantities. By providing high-purity human Delta1 at low cost, various technologies that utilize Delta1 (for example, immunotherapy that utilizes the antitumor effect of Delta1) can be put into practical use.

The time-dependent change of the cell growth and survival rate of a D1Fc transformant is shown. The time-dependent change of the accumulation amount of D1-Fc fusion protein in the culture supernatant of a D1Fc transformant is shown.

SEQ ID NO: 2: DNA encoding D1-Fc fusion protein
SEQ ID NO: 3: Synthetic construct SEQ ID NO: 4: Soluble D1-Fc fusion protein SEQ ID NO: 5: Plasmid vector pMKITNeo

Claims (2)

  A transformant of accession number NITE AP-460, which is a cell that produces a protein consisting of the amino acid sequence shown in SEQ ID NO: 4, which is a fusion protein of human Delta1 and the Fc part of human immunoglobulin G.   A method for producing a fusion protein of human Delta1 and the Fc portion of human immunoglobulin G, comprising the amino acid sequence shown in SEQ ID NO: 4, wherein the transformant of claim 1 is used.

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