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

TC小鼠具有包含完整的人类免疫球蛋白重链基因和kappa轻链基因的人类染色体片段(HCF)，可产生完全的人类单抗。由于TC小鼠被改造成既不表达内源性免疫球蛋白重链也不表达kappa轻链，我们从TC小鼠中产生了抗人黑色素瘤IgM单抗，该抗体免疫微细胞-杂交小鼠黑色素瘤细胞，B16-F10含有人6号染色体作为抗原。此外，我们还从含有人3号染色体的B16-F10杂合细胞免疫的小鼠中制备了一种人细胞表面分子特异性的单抗IgG2a，该抗体对人细胞系具有特异性，对小鼠细胞系没有特异性。通过质谱分析，我们检测到该抗体的抗原为人转铁蛋白受体(HTfR)，位于3号染色体上。此外，我们还检测了以体外分化的TC-ES细胞作为免疫原，产生抗人细胞表面抗原的单抗。在一个测试案例中，我们试图用含有人4号染色体的DTC-ES细胞的化学定义培养基来产生抗人神经前体细胞抗原(S)的单抗。结果，我们分离出了能特异性识别该细胞系表面的B6-13抗体。DTC-ES细胞和人胚胎癌细胞(EC)对B6-13的染色图谱与Nestin的表达谱相似，Nestin是一种具有很好特征的鼻咽癌细胞内标志物。我们还通过质谱分析确定了B6-13抗原是位于4p15.32上的CD133。因此，这些结果表明，这个通过免疫含有人类染色体的恶性细胞或ES细胞来分离特定人类抗体的系统是一种有价值的药物来源，并有助于了解发育过程中调节分化的机制。

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.