Generation and application of fully human antibodies to a new tumor-associated antigen, Ep-CAM

目前有许多肿瘤相关抗原用于癌症的诊断和治疗。然而，没有一种肿瘤相关抗原可以始终是精确和完整的靶分子，因为几乎所有的肿瘤相关抗原都不是绝对的肿瘤特异性。在本研究中，我们利用含有人类免疫球蛋白基因的基因工程小鼠（KM mouse^<TM>）制备了针对泛癌抗原Ep-CAM的人单克隆抗体。用重组Ep-CAM免疫KM小鼠的脾脏细胞与P3-U1小鼠骨髓瘤细胞融合。在分析的44个抗ep - cam克隆中，2个为IgG4克隆，其余为IgM克隆。这两个IgG4克隆可能识别相同的抗原决定因子或两个位置相近的决定因子。两个IgG4克隆和一个IgM克隆在体外分别对表达ep - cam的细胞显示抗体依赖性细胞介导的细胞毒性和补体依赖性细胞毒性。我们也知道IgG4几乎不与FcγIII受体结合，而FcγIII受体参与NK细胞的ADCC。然而，在IgG4克隆M13-13存在时，观察到il -2激活的PBMC对肿瘤细胞的细胞毒性略有增加。IgG4抗体能够与单核细胞上的FcγI受体结合，从而发挥ADCC作用。用M13-13观察到的ADCC活性可能是由于活化的单核细胞。因此，我们设计了IgM克隆M11-29，利用其v区基因将其转化为IgG1型，使其能够在表达ep - cam的肿瘤细胞中同时表现出ADCC和CDC。获得的v区基因也可用于制备单链Fv抗体，以重靶向细胞毒性T细胞或肿瘤细胞的逆转录载体。综上所述，这些数据表明，这些针对Ep-CAM及其v区基因的全人源单克隆抗体可用于制备工程化抗体片段，可能对基于抗体的癌症治疗有用。

There are numerous tumor-associated antigens currently used for the diagnosis and treatment of cancer. However, no tumor-associated antigen can always be an exact and complete target molecule, because almost all of them are not absolutely tumor-specific. In the present study, we prepared human mAbs against a pan-carcinoma antigen, Ep-CAM, using a genetically engineered mouse (KM mouse^<TM>) that contains the human immunoglobulin genes. Spleen cells from KM mice immunized with recombinant Ep-CAM were fused with the P3-U1 mouse myeloma cells. Of 44 anti-Ep-CAM clones analyzed, two were of IgG4 and the others of IgM clones. The two IgG4 clones were suggested to recognize the same antigenic determinant or two closely located determinants. The two IgG4 and one of the IgM clones tested revealed antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, respectively, against Ep-CAM-expressing cells in vitro. It is also known that IgG4 hardly binds to the FcγIII receptor, which involves in ADCC by NK cells. In the presence of the IgG4 clone M13-13, however, a slight increase was observed in the cytotoxicity of IL-2-activated PBMC against tumor cells. IgG4 antibodies are able to bind to the FcγI receptor on monocytes and thereby exert ADCC. The ADCC activity observed with M13-13 might be due to activated monocytes. Therefore, we engineered our IgM clone M11-29 to convert it to the IgG1 type using its V-region genes so that it can exhibit both ADCC and CDC to Ep-CAM-expressing tumor cells. The V-region genes obtained were also useful to prepare single chain Fv antibodies to retarget cylotoxic T cells or a retrovector to tumor cells. Taken together, these data suggest that these fully human mAbs produced against Ep-CAM and their V-region genes, which are applicable for preparation of engineered antibody fragments, may be useful for antibody-based therapy of cancer.