2001 Fiscal Year Final Research Report Summary
Development of human monoclonal antibodies using TC(Trans Chromosomic) mice
| Project/Area Number |
12470347
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Obstetrics and gynecology
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| Research Institution | Keio University |
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Principal Investigator |
NOZAWA Shiro Keio University, School of Medicine, Professor, 医学部, 教授 (90051557)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIDA Isao Kirin Brewery Co. Ltd., Planning Dept. Pharmaceutical Division, Leader, Human Antibody Section, 医薬カンパニー, プロジェクトリーダー(研究職)
KUBUSHIRO Kaneyuki Keio University, School of Medicine, Assistant Professor, 医学部, 講師 (50170022)
TSUKAZAKI Katsumi Keio University, School of Medicine, Associated Professor, 医学部, 助教授 (40118972)
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| Project Period (FY) |
2000 – 2001
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| Keywords | TC mice / humanized monoclonal antibodies / immunohistochemical staining / endometrial carcinoma / ovarian clear cell carcinoma / western blotting |
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| Research Abstract |
The current study was initiated with the strategy to develop humanized monoclonal antibodies towards cancers of the female genital system. The newly established TC (Trans Chromo) mice which express human antibodies (heavy chain and light chain K) were used to develop these antibodies. Basic investigations have been launched with implications towards future clinical applications; the following results have been obtained.
A human monoclonal antibody has been developed using as antigen an endometrial carcinoma cell line culture (SNG-S), originally established by the current research group leaders. Cell fusion was performed with PEG 4000 according to standard mouse monoclonal antibody protocols; mouse splenocytes and myeloma cells were fused at a ratio of 1:5, Hybridoma screening was carried out by immunohistochemical staining (ABC method) of endometrial carcinoma tissue sections. Staining results with the human monoclonal antibody 1-1C showed that the antibody stained approximately 70% of
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endometrial carcinoma tissue specimens while it showed almost no staining towards normal endometrial tissue specimens. In addition to positive staining of endometrial carcinoma, the antibody also showed strong reactivity with normal uterine cervical glands as well as cervical adenocarcinoma in addition to positive staining of several other cancers not related to the female genital system. Preliminary investigations to elucidate the antigenic epitope recognized by this antibody revealed that trypsin treatment of the specimens did not diminish the antibody staining while periodic acid treatment completely abolished positive staining of tissue specimens; thus a carbohydrate moiety was implicated as the possible epitope of this antibody. Furthermore, SDS-PAGE electrophoresis and western blotting of the cell lysates of the SNG-S cells revealed a positive band with the membrane fraction, denoting that the antigen recognized by 1-1C is possibly a cell surface membrane glycoprotein.
In addition to the above studies, investigations have been instigated to develop a human monoclonal antibody using as antigen an ovarian clear cell carcinoma cell line culture (RMG-1). Initial studies have revealed establishment of a monoclonal antibody which shows strong reactivity with ovarian clear cell carcinoma. Less
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