Development of human monoclonal antibodies using TC(Trans Chromosomic) mice

使用TC（Trans Chromosomic）小鼠开发人单克隆抗体

• 批准号: 12470347
• 负责人: NOZAWA Shiro
• 金额: $ 8.96万
• 依托单位: Keio University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12470347/
• 关键词: TC mice; humanized monoclonal antibodies; immunohistochemical staining; endometrial carcinoma; ovarian clear cell carcinoma; western blotting; ミサイル療法

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 … More 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

目前的研究是以开发针对女性生殖系统癌症的人源化单克隆抗体的策略开始的。用新建立的表达人抗体（重链和轻链K）的TC （Trans Chromo）小鼠制备这些抗体。基础研究已经启动，对未来的临床应用有影响；得到了以下结果：以目前课组长建立的子宫内膜癌细胞系培养物（SNG-S）为抗原，研制出一种人单克隆抗体。用PEG 4000按照小鼠单克隆抗体标准方案进行细胞融合；将小鼠脾细胞与骨髓瘤细胞按1:5的比例融合，对子宫内膜癌组织切片进行免疫组化染色（ABC法）筛选杂交瘤。人单克隆抗体1-1C染色结果显示，该抗体对子宫内膜癌组织标本的染色率约为70%，而对正常子宫内膜组织标本几乎没有染色。该抗体除对子宫内膜癌呈阳性染色外，对正常子宫颈腺和宫颈腺癌也表现出较强的反应性，对其他几种与女性生殖系统无关的癌症也表现出阳性染色。对该抗体识别抗原表位的初步研究表明，胰蛋白酶处理不降低抗体染色，而定期酸处理完全消除组织标本的阳性染色；因此，一个碳水化合物片段可能是该抗体的表位。此外，对SNG-S细胞裂解液进行SDS-PAGE电泳和western blotting，发现膜部分呈阳性带，说明1-1C识别的抗原可能是细胞表面膜糖蛋白。除上述研究外，还研究了以卵巢透明细胞癌细胞系培养物（RMG-1）为抗原制备人单克隆抗体。初步研究表明建立了一种单克隆抗体，该抗体对卵巢透明细胞癌具有很强的反应性。少

项目成果

Yoshibumi KANETA., et al.: "Selective cytotoxicity of adriamycin immunoconjugate of monoclonal antibody MSN-1 to endometrial adenocarcinoma in vitro and in vivo"Oncol.Rep.. 7. 1099-1106 (2000)

Yoshibumi KANETA.等：“单克隆抗体MSN-1的阿霉素免疫缀合物对子宫内膜腺癌的体外和体内选择性细胞毒性”Oncol.Rep..7.1099-1106(2000)

Kaneyuki Kubushiro, et al 6: "Biological characteristics of human uterine endometrial cancer variant cells selected for blood group H type 1 antigen : adhesion to vascular endothelial cells."Acta Histocemica et Cytochemica. 33・3. 209-213 (2000)

Kaneyuki Kubushiro等6：“针对H型1型抗原选择的人子宫内膜癌变异细胞的生物学特性：与血管内皮细胞的粘附”。 Acta Histochemica et Cytochemica 33·3（2000）。

Eiko SAITOH., et al.: "The expression of β 1, 4-garactosyltransferase in human trophoblasts"Acta Histochem.Cytochem.. 34(1). 62(A24) (2001)

Eiko SAITOH.等：“β1,4-半乳糖基转移酶在人滋养层中的表达”Acta Histochem.Cytochem..34(A24)(2001)。

Nobuyuki SUSUMU., et al.: "Detection of chromosomal alterations in ovarian cancers using doubletargeted FISH"Acta Hictochem.Cytochem.. 34(1). 77(A39) (2001)

Nobuyuki SUSUMU., et al.：“使用双靶点 FISH 检测卵巢癌中的染色体改变”Acta Hictochem.Cytochem.. 34(1)。

Yuuki, KOMURO et al 5: "Paclitaxel and SN-38 Overcome cisplatin resistance of ovarian cancer cell lines by down-regulating the infflux system of cisplatin"Jpn. J. Cancer Res.. 92. 1241-1250 (2001)

Yuuki、KOMURO 等 5：“紫杉醇和 SN-38 通过下调顺铂流入系统克服卵巢癌细胞系的顺铂耐药性”Jpn。