JP2007044035A - Preparation of antibodies against cell membrane surface antigen epitopes - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a monoclonal antibody reactive with an extracellular domain of an insoluble cell membrane antigen. <P>SOLUTION: A cell derived from an animal belonging to another order of an animal that is to be immunized and expresses a cell membrane antigen is combined with a cell in which the antigen is expressed in a host cell belonging to the same order as that of the animal to be immunized by genetic recombination and used for immune sensitization of the animal to be immunized. Thereby, an antibody clone recognizing a natural three-dimensional structure of the extracellular domain of the cell membrane antigen is obtained. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a monoclonal antibody against a cell membrane surface antigen (extracellular domain of cell membrane antigen).

  Antibodies against soluble antigens in cytokines, chemokines or serum are generally prepared by genetic recombination using Escherichia coli or yeast as a host, and of these antigen proteins containing sugar chains, CHO cells, NSO cells, SP2 / O It is produced by genetic recombination using rodent-derived animal cells such as cells as hosts. On the other hand, molecules expressed on the cell membrane such as receptors and adhesion molecules generally have only an extracellular domain prepared by genetic recombination because they have an insoluble transmembrane portion. (Non-Patent Document 1)

  However, some cell membrane antigens are insoluble not only in the transmembrane domain but also in the extracellular domain. In addition, the three-dimensional structure presented in a natural state may not always be easy for the receptor and the adhesion molecule depending on the properties and expression modes. That is, molecules expressed on the cell membrane often form a complex rather than a monomer, and often have a biological function only in cooperation with other membrane molecules. Therefore, no matter which host cell is used, the method of preparing the extracellular domain by gene recombination may not reproduce the naïve epitope of the cell membrane antigen.

For example, the extracellular domain of the CD20 molecule, which is one of the human leukocyte antigens, is insoluble, and it is considered extremely difficult to obtain an antibody having biological activity or a high-affinity antibody against it. Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). In such a case, in general, there is nothing but to use the cell itself expressing the antigen as an immunogen. Rituxan (trademark) is chimerized based on a monoclonal antibody obtained by immunizing mice with SB cells, which is a human B cell line, and it is also necessary to produce another commercially available anti-CD20 monoclonal antibody 2H7. Human B cell line 6.16c1.3 is used. However, these human cell lines do not express such antigens in sufficient amounts to induce sufficient immune stimulation and are heterogeneous to the immunized animal (mouse or rat), so that the entire cell Since it has antigenicity, it is difficult to obtain an antigen-specific immune response.
Francois Baneyx (Editor), Protein Expression Technologies: Current Status and Future Trends, BIOS Scientific Publishers, April 1, 2004 .; Barry Steven Selinsky (Editor), Membrane Protein Protocols: Expression, Purification, and Characterization., Methods in Molecular Biology, V.228, Clifton, NJ, June 1, 2003 Julie PD et al., Imminol 2002; 107: 176-82 Maria J. Polyak and Julie P. Deans, Studies of Existing CD Molecules: 93-95 MS Cragg et al., Studies of Existing CD Molecules: 95-97 Anderson KC et al., Blood 1984; 63: 1424-33, Data Sheet: FITClabeled CD20, Medical & Biological Laboratories Co., Ltd.

  An object of the present invention is to provide a method for producing a monoclonal antibody that binds to a cell membrane antigen epitope whose extracellular domain is insoluble and whose steric structure may be changed by solubilization treatment.

  In order to produce an antibody that reacts with an insoluble extracellular domain, the present inventors have repeatedly studied a method for preparing an immunizing antigen and a method for sensitizing an immunized animal. As a result, the present inventors have found a method capable of maintaining a naïve three-dimensional structure of the extracellular domain and providing a large amount of antigen. Furthermore, it was found that immunization using this induces many antibodies against various epitopes of the extracellular domain.

The present invention relates to the following.
(1) At least one of the multiple immunizations uses a cell derived from an animal belonging to another eye as an immunized animal expressing a cell membrane antigen, and at least once immunized with the antigen by genetic recombination A method of immunization for obtaining an antibody against the extracellular domain of the antigen, comprising using a cell expressed in a host cell belonging to the same group as that of an immunized animal.
(2) The immunized animal is a rodent animal, and the host cell belonging to the same species as the immunized animal is a CHO cell, NSO cell, SP2 / O cell, or other rodent cell line ( The immunization method as described in 1).
(3) (1) characterized in that the extracellular domain is a protein or glycoprotein that is insoluble under physiological conditions and has a possibility that a part of the three-dimensional structure is changed by solubilization treatment. The immunization method described.
(4) A method for producing a monoclonal antibody against the extracellular domain of a cell membrane antigen using the immunization method described in (1) to (3).

  By using the immunization method of the present invention, an antibody that reacts with the extracellular domain while maintaining the original three-dimensional structure can be induced in the immunized animal. By removing spleen cells containing these antibody-producing B cells and fusing them with mouse myeloma cells, a hybridoma producing the antibody can be efficiently produced. By culturing the hybridoma obtained in this manner and cloning it repeatedly, a monoclonal antibody that specifically binds to the extracellular domain can be produced. These monoclonal antibodies include antibodies that bind to various epitopes of the extracellular domain, and further include antibodies that have biological activity against target cells having the antigen.

<1> Method for producing antibody-producing hybridoma of the present invention As an immunization method for an immunized animal, immunization is performed a plurality of times, and at least one of the immunized animals expressing a cell membrane antigen is an animal belonging to another eye. And a cell in which the antigen is expressed in a host cell belonging to the same species as the immunized animal is used at least once by genetic recombination. After sufficient immunostimulation, spleen cells are removed from the immunized animal and fused with lymphocytes contained therein and mouse myeloma cells according to a conventional method. This procedure is described in, for example, Ailsa M. Campbell (Author), Toshiaki Osawa (Translation) Biochemical Experimental Method Monoclonal Antibody, Tokyo Chemical Dojin, 1989.

  The animal to be immunized is not particularly limited, but usually a mouse or a rat is used. The immunization of the immunized animal is usually performed by performing injection of the immunization antigen a plurality of times. Setting conditions regarding the number of immunizations, the interval, the amount of antigen, and the like may be the same as in a normal method. When three or more immunizations are performed, the first immunization is generally called the first immunization, the last immunization is called the final immunization (boost), and the immunization after the first immunization is called the booster immunization. Is done.

In the present invention, during immunization a plurality of times, a cell derived from an animal belonging to another eye is used as an immunized animal that expresses a cell membrane antigen at least once, and the antigen is at least once by genetic recombination. Is used in a host cell belonging to the same order as the immunized animal. In this case, it is preferable to use different cells for the initial immunization and the final immunization. In the case of obtaining a monoclonal antibody against a human cell membrane antigen, the former is a human cell line, and the latter is a rodentia-derived cell in which the human antigen gene is expressed on the membrane surface by genetic recombination. Representative examples of rodent cells include CHO cells, NSO cells, SP2 / O cells, and the like.

  Examples of cell membrane antigens include leukocyte differentiation antigen (CD antigen), such as CD9, CD20, CD37, CD47, and the like.

  The expression density of the cell membrane antigen on the cell membrane surface of a cell line derived from an animal belonging to the same species as the immunized animal is preferably simply high. It is preferable that a fluorescence intensity of 5 times or more is detected as compared with the cells possessed.

  In order to produce a hybridoma after sufficient immunostimulation, spleen cells are extracted from the immunized animal and fused with lymphocytes contained therein and mouse myeloma cells according to a conventional method. The fusion environment includes a cell culture method and mouse ascites, which can be used.

  The reason why the effect of the present invention can be obtained is considered as follows. That is, as a sensitizing antigen, cells different from the immunized animal (cell lines derived from animals belonging to other eyes that express the antigen, and immunized animals) and homologous or closely related cells (genes) By combining the cell line derived from the animal that belongs to the same species as the immunized animal, which expresses the antigen on the surface of the cell membrane by recombination, the immunostimulation of the target molecule alone is enhanced. The proportion of hybridomas producing various monoclonal antibodies is likely to increase.

  In the method of the present invention, it is considered that the three-dimensional structure of the cell membrane antigen molecule can be obtained almost in its original state, and is particularly effective when the extracellular domain is insoluble. The immunosensitizing antigen is generally used by dissolving and purifying the antigen-expressing cells or mass-producing the antigen by gene recombination. In the present invention, by using a combination of two or more different immunogens sharing such an antigen or its antigenic site, the immunostimulation for the desired antigen molecule or its site was successfully enhanced.

  A method for producing a monoclonal antibody against a CD20 molecule having an insoluble membrane surface antigen will be described with reference to Examples.

<1> Preparation of monoclonal antibody (1) Preparation of mouse sensitizing immunizing antigen First, SB cells and Raji cells, which are representative B cell lines expressing CD20, were cultured in vitro. Next, from Multiple Choice cDNA human spleen, Origene Technologies, Inc. 6 Taft Court, Suite 100, Rockville, MD 20850 1) and a vector constructed by cloning using hCD20-E-Xba gctctagattaaggagagctgtcattttc (SEQ ID NO: 2) and incorporating it into pNOW (Japanese Patent No. 3582965, FIG. 1), which is a high expression vector for mammalian cells. Was transfected into CHO cells. Recombinant CHO cells (CD20 / CHO cells) that highly expressed CD20 molecules on the cell surface were established by FACS analysis. In addition, when stained with a FITC-labeled anti-CD20 monoclonal antibody, those having a fluorescence intensity of 5 times or more compared with SB cells were selected.

(2) Preparation of immunization antigen
SB cells or Raji cells were cultured using RPMI1640 medium supplemented with 10% FCS. CD20 / CHO cells were cultured using CHO-S-SFM II medium (GIBCO, Cat. No. 12052-098) supplemented with 800 μg / ml of G418. After centrifuging these cultures (1100 rpm, 5 minutes), the cells were suspended by adding Dulbecco'sPBS (-) and centrifuged again. This washing operation was repeated once more, and a suspension prepared by adding physiological saline to the cells (the number of cells per ml was 1 to 3 × 10 ⁷ ) was used for immunization.

(3) Immunity
All immunized cell preparations were intraperitoneally administered to 7-11 week old Balb / c female mice. The combination of immunogens used is the following combination (a) and (b), one of which is repeated 2-3 times at various time intervals, and then the other cell is used for the final immunization: Administered.
(B) SB cells or Raji cells (b) D20 / CHO cells The number of cells administered was 1 to 3 × 10 ⁷ cells per mouse.

(4) Cell fusion Three days after the final immunization, spleen cells were prepared from two mice, and a fusion reaction with mouse myeloma (NS-1) was performed in the presence of PEG-1500. The method followed Oi, VT and LA Herzenberg, 1980, in: Selected Methods in Cellular Immunology, eds. B. Mishell and SM Shiigi (Freeman and Co. San Francisco, CA) p. 351.

(5) Primary and secondary screening
Cell ELISA was performed using a 96-well plate to which Raji cells, CD20 / CHO cells or CHO cells (parent strain) were attached, and wells producing antibodies that specifically react with CD20 were selected. Furthermore, a 96-well plate to which the same CD20 / CHO cells were attached was used to perform a competitive reaction with a commercially available anti-CD20 monoclonal antibody Rituxan, and an antibody (well) that reacts with a similar Rituxan epitope was selected.

(6) Cell ELISA
Raji cells, CD20 / CHO cells, or CHO cells (parent strain) attached to Poly-L-Lysine-coated 96-well plates (Asahi Techno Glass, Cat. No. 11-023-018) were used for Cell ELISA. In each well, 150 μl of blocking solution (0.2% -Gelatine, 0.5% BSA in PBS) was added and allowed to stand at 37 ° C. for 1 hour. After washing the plate 5 times with 150 mM NaCl, 0.05% -Tween20 aqueous solution, 100 μl of the sample (diluted culture supernatant) was placed in each well, and the primary reaction was carried out at 37 ° C. for 1 hour. After washing, a diluted solution of labeled antibody [HRP-labeled anti-mouse IgG (H + L) rabbit antibody (Jackson Lab. Code No. 315-035-003) or HRP-labeled anti-mouse IgG (Fcγ) rabbit antibody (Jackson Lab. Code No. 315-035-008)]]] was added to each well, and the secondary reaction was carried out at 37 ° C. for 1 hour. The same reaction solution as the blocking solution was used for preparing the primary and secondary reaction solutions. After washing, 100 μl of color developing solution (OPD) was added to each well, 30 minutes later, 50 μl of 4N—H ₂ SO ₄ was added to stop the reaction, and A492 was measured.

(7) Competitive reaction in Cell ELISA A mixed solution of a sample (diluted culture supernatant) and a chimeric antibody (10-40 ng / ml) was prepared.
After a blocking reaction as in the above Cell ELISA, 100 μl of this mixed solution was added to each well, and the primary reaction was performed at 37 ° C. for 1 hour. After washing, add 100 μl of labeled antibody dilution solution [HRP-labeled anti-human IgG (H + L) rabbit antibody (Jackson Lab. Code No. 309-035-082)] to each well and perform the secondary reaction at 37 ° C. Done for hours. After washing, 100 μl of color developing solution (OPD) was added to each well, 30 minutes later, 50 μl of 4N—H ₂ SO ₄ was added to stop the reaction, and A492 was measured.

  Since the labeled antibody reacts only with the chimeric antibody, if the antibody in the sample added in the primary reaction competes with the chimeric antibody, a decrease in the measured value was observed.

(8) Cloning The limiting dilution method was used. After culturing the cells in a 96-well plate, Cell ELISA was performed on the culture supernatant of one colony well, and a clone producing the specific antibody was selected.

(9) Preparation of purified antibody Clone specific antibody producing clones in RPMI1640 medium supplemented with 10% FCS, and when the cell density reaches around 5 × 10 ⁵ / ml, add the medium to serum-free medium ASF-104N (Ajinomoto). The culture was performed after replacement. Two to four days later, the culture solution was centrifuged to collect the culture supernatant, and then purified using a protein G column, and the eluted monoclonal antibody solution was dialyzed against 150 mM NaCl. The solution was sterilized by filtration with a 0.2 μm filter to obtain a test antibody (anti-human CD20 mouse monoclonal antibody).

<Result>
Table 1 shows the immunization method, the results of screening hybridomas obtained by fusing the obtained spleen cells and mouse myeloma cells, and the results of the competitive reactivity test with Rituxan.

  The obtained hybridoma clones were further subcultured and selected to obtain clones having high reactivity with CD20 positive B cell lines. FIG. 2 shows the binding results of these antibody clones in Cell EISA with Raji cells attached and Cell ELISA with SB cells attached. Cell ELISA as a negative control was prepared by attaching CHO cells as host cells. A mouse antibody having a Rituxan variable region was used as a positive control antibody, and a mouse monoclonal antibody (BD PharMingen) against human CD3 antigen was used as a negative control antibody.

The structure of the vector pNOW for protein expression is shown.

Claims (4)

The immunized animal that expresses cell membrane antigen at least once in a plurality of immunizations uses cells derived from animals belonging to other eyes, and at least once, the antigen is immunized with the immunized animal by genetic recombination. A method of immunization for obtaining an antibody against an extracellular domain of the antigen, which comprises using a cell expressed in a host cell belonging to the same order. The immunized animal is a rodent animal, and the host cell belonging to the same species as the immunized animal is a CHO cell, NSO cell, SP2 / O cell, or other rodent cell line. Immunization method. 2. The immunization method according to claim 1, wherein the extracellular domain is a protein or glycoprotein which is insoluble under physiological conditions and can be partially altered by solubilization treatment. . A method for producing a monoclonal antibody against an extracellular domain of a cell membrane antigen using the immunization method according to claim 1.

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