Generation of human chromosome-specific monoclonal antibodies using trans-chromosomic (TC) mice

拥有包含整个人免疫球蛋白重链位点和kappa轻链位点的人染色体片段（hCFs）的TC小鼠产生完整的人单克隆抗体。由于TC小鼠既不表达内源性免疫球蛋白重链，也不表达kappa轻链，因此我们从TC小鼠中制备了抗人黑色素瘤IgM单克隆抗体，用于免疫含有人类6号染色体的微细胞杂交小鼠黑色素瘤细胞B16-F10。此外，我们还从含有人3号染色体的B16-F10杂交细胞免疫的小鼠中获得了人细胞表面分子特异性的单克隆IgG2a抗体。该抗体对人类细胞系有特异性，对小鼠细胞系无特异性。通过质谱分析，我们检测到该抗体的抗原为位于3号染色体上的人转铁蛋白受体（hTfR）。此外，我们还研究了利用体外分化的TC-ES （8dTC-ES）细胞作为免疫原产生针对人细胞表面抗原的单克隆抗体。在一个测试案例中，我们试图通过化学定义培养基（CDM）培养含有人4号染色体的dTC-ES细胞，产生针对人神经祖细胞（NPC）抗原的单克隆抗体。结果，我们分离出了B6-13抗体，该抗体对该细胞系的表面有特异性识别反应。B6-13在dTC-ES细胞和人胚胎癌（EC）细胞中的表达谱与NPCs细胞内标志物nestin的表达谱相似。我们还通过质谱分析确定了B6-13抗原是4p15.32上的CD133。因此，这些结果表明，通过免疫含有人类染色体的恶性或胚胎干细胞分离特异性人类抗体的系统是药物和理解发育中调节分化的机制的宝贵资源。

TC mice that possess the human chromosome fragments (hCFs) containing the entire human immunoglobulin heavy chain locus and the kappa light chain locus generate fully human monoclonal antibody. Because the TC mice are engineered to neither express endogenous immunoglobulin heavy chain nor kappa light chain, we have generated the anti-human melanoma IgM monoclonal antibody from TC mice that immunize microcell-hybrid mouse melanoma cells, B16-F10 containing a human chromosome 6 as antigen. Furthermore, we also generated a human cell surface molecule specific monoclonal IgG2a antibody from mouse immunized by B16-F10 hybrid cells containing a human chromosome 3. This antibody has a specificity to human cell lines, not to mouse cell lines. Using mass spectrometric analyses, we detected the antigen of this antibody as human Trasferrin Receptor (hTfR), which are located in chromosome 3. In addition, we examined the generation of monoclonal antibody against human cell surface antigens utilizing in vitro differentiated TC-ES 8dTC-ES) cells as immunogens. In a test case, we attempted to generate monoclonal antibody against human neural progenitor cell (NPC) antigen(s) by using the chemically defined medium (CDM) culture for dTC-ES cells containing a human chromosome 4. As a result, we isolated B6-13 antibody that responses to specifically recognize the surface of this cell line. The staining profiles of dTC-ES cells and human embryonic carcinoma (EC) cells with B6-13 were similar to the expression profile of nestin, a well characterized intracellular marker for NPCs. We also identified that B6-13 antigen is CD133 on 4p15.32 using mass spectrometric analyses. Thus, these results suggest that this system for the isolation of a specific human antibody by immunization of malignant or ES cells containing a human chromosome is a valuable resource for drugs and understanding the mechanism that regulates the differentiation in the development.