探究用單株抗體來標的表現膜型免疫球蛋白A的B細胞的治療方策

• Main Authors: Hung, Fu-Hsin, 洪福信
• Other Authors: Pan, Rong-Long
• Format: Others
• Language: en_US
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/59185892641407127980

博士 === 國立清華大學 === 生物資訊與結構生物研究所 === 100 === In humans, secretory IgA exists as two subclasses, IgA1 and IgA2, which contain distinct ?? and ?? heavy chains, respectively. Both subclasses also have membrane-bound forms (mIgA1 and mIgA2) containing the corresponding m?? and m?? heavy chains, which differ from ?? and ?? by an additional “membrane-anchor” peptide segment extending from the CH3 domain of ?? and ??. The membrane-anchor segment has three parts: an extracellular, a transmembrane, and an intracellular segment. The heavy chain m?? exists in short and long isoforms, referred to as m??S and m??L, with the latter containing extra 6 amino acid residues, GSCSVA, at the N-terminus of the extracellular segment (residues 453-458). By studying the genomic and mRNA sequences of m?? and m?? from 30 individuals residing in Taiwan, we have found that, in addition to the known m?? allele, referred to as m??(456S), m?? also has a previously unknown allele, referred to as m??(456C) (GenBank accession no. EU431191). This newly identified allele is present in the donor population at a similar proportion to m??(456S), and appears to exist only as the long isoform, i.e. m??L, rather than the short isoform, m??S. We also confirmed that m?? exists only as the short isoform. Because the membrane-anchoring peptide segment which is only present on mIgA is unique in amino acid sequences we proposed that the extracellular segment, referred to as the mIg isotype-specific (migis-?? segment of m? could be a specific antigenic site suitable for isotype-specific targeting of mIgA-expressing B cells by antibodies. We rationalized that the migis-??specific antibodies could bind to, and in turn, regulate IgA-expressing B cells, by mechanisms such as inducing apoptosis or decreasing the production of IgA. In the study, we developed several anti-migis-? monoclonal antibodies (mAbs), such as mAb 29C11, specific to a segment towards the N-terminus of the 26 amino acid long migis-?? The mAbs bound strongly to synthetic peptides of migis-? and to various recombinant proteins containing migis-? as revealed by ELISA. On B cells, however, flow cytometric analysis suggested that these mAbs did not bind strongly to mIgA. After lipid rafts of B cells were disrupted by cholesterol extraction, the mAbs were able to bind strongly to the treated B cells. Moreover, immunoprecipitation analysis of these mAbs indicated that mIgA could only be pulled down by the mAbs when mIgA-expressing B cells were solubilized by strong detergents, such as sodium dodecyl sulfate (SDS), or when lipid rafts were disrupted. Together, these results suggest that the migis-? region of mIgA in the BCR is associated with lipid rafts, which hinder binding of migis-??specific antibodies to mIgA on the cell surface. Further studies are in progress to evaluate the suitability of 29C11 or its affinity-improved variants for targeting mIgA-expressing B cells.