Application of Bispecific F(ab′)2μ Fragments Prepared from IgMs against Carcinoembryonic Antigen and Alkaline Phosphatase

[摘要] Bispecific monoclonal antibodies (BsMAbs) have two different binding regions that simultaneously recognize two different antigens (1)(2). BsMAbs have been studied for use in immunodiagnosis (3)(4) and immunotherapy (5)(6).Most BsMAbs are derived from the IgG class, whereas most hybridomas raised against carbohydrate antigens, including the tumor-associated antigens, are IgM MAb producers. IgM antibody-enzyme conjugates are of limited use in enzyme immunoassays because of steric effects, reduction of their activities, and high nonspecific binding. For these reasons, BsMAbs prepared from IgM MAbs have been especially used for cancer immunodiagnosis. Their production by hybrid-hybridoma fusion is one method (7), but it is not an effective one. F(ab′)2μ fragments have been successfully prepared from their IgMs, and their performance has been evaluated (8)(9)(10)(11). In this report, homogeneous bispecific F(ab′)2μ fragments (BsFμ fragments) were efficiently prepared from IgMs.Mouse IgM monoclonal antibodies 9CA10 and APM05 recognize carcinoembryonic antigen (CEA) and calf intestine alkaline phosphatase (AP), respectively. Their F(ab′)2μ fragments were prepared by pepsin digestion and purified by hydrophobic interaction HPLC (8)(10)(11). The purities of the fragments from the IgMs were >96% by size exclusion HPLC using a TSKgel G3000SWXL column (7.8 mm i.d × 30 cm; Tosoh), and the yields were 54% (APM05) and 53% (9CA10), respectively. The F(ab′)2μ of 9CA10 was reduced to Fab′μ-SH with 50 mmol/L 2-mercaptoethylamine (Sigma) in phosphate-buffered saline (PBS) containing 10 mmol/L EDTA at 37 °C for 1 h, and …

[学科分类] 过敏症与临床免疫学