Xiang J, Moyana T, Liu E
Saskatoon Cancer Center, Department of Microbiology, University of Saskatchewan, Canada.
Hum Antibodies Hybridomas. 1994;5(3-4):105-15.
A mouse/human chimeric antibody cACT19 derived from the murine ACT19 antibody was constructed; it recognizes an epitope different from the sialosyl-Tn on the TAG72 antigen as defined by the B72.3 antibody. This chimeric cACT19 antibody was constructed by using two expression vectors, the heavy chain expression vector mpSV2neo-EP1-VHC gamma 1 and the light chain expression vector mpSV2gpt-EP1-VKCK. These vectors contain the following: (i) the neo or gpt gene as a selection marker, (ii) the murine immunoglobulin promoter and enhancer (EP1), (iii) the genomic DNA fragments of human immunoglobulin constant region (CK and C gamma 1) and (iv) the murine cDNA fragments of VH or VK region cloned from the murine ACT19 cDNA library. These two vector DNAs were sequentially transfected into the SP2/0Ag14 cell line. Transfectants were selected in media containing both G418 and mycophenolic acid. The chimeric cACT19 antibody was purified from the transfectant supernate by Protein A Sepharose chromatography. We confirmed that the chimeric cACT19 antibody was reactive for an epitope that differed from the sialosyl-Tn on the TAG72 antigen. This was achieved by using the TAG72-binding inhibition ELISA utilizing various monosaccharides and disialyllato-N-tetraose with the latter containing the NeuAc2-6 alpha Ga1NAc structure. The immunohistochemical double-staining technique provided further evidence of the difference between the epitope defined by the chimeric cACT19 antibody and the sialosyl-Tn epitope by illustrating complementary as well as noncomplementary expression of these two epitopes in different areas of colon carcinoma tissues. We also demonstrated that the chimeric cACT19 antibody displayed much more effective ADCC and CDC for the human OVAR3 tumor cells than the murine ACT19 antibody. Therefore, the mouse/human chimeric anti-colorectal carcinoma cACT19 antibody may prove to be useful in cancer immunotherapy in its own right, or especially when used in combination with the chimeric B72.3 antibody.
构建了一种源自鼠源ACT19抗体的鼠/人嵌合抗体cACT19；它识别的表位不同于B72.3抗体所定义的TAG72抗原上的唾液酸化-Tn。该嵌合cACT19抗体通过使用两个表达载体构建而成，即重链表达载体mpSV2neo-EP1-VHCγ1和轻链表达载体mpSV2gpt-EP1-VKCK。这些载体包含以下成分：(i)作为选择标记的neo或gpt基因；(ii)鼠免疫球蛋白启动子和增强子(EP1)；(iii)人免疫球蛋白恒定区(CK和Cγ1)的基因组DNA片段；以及(iv)从鼠ACT19 cDNA文库克隆的VH或VK区的鼠cDNA片段。将这两个载体DNA依次转染到SP2/0Ag14细胞系中。在含有G418和霉酚酸的培养基中筛选转染子。通过蛋白A琼脂糖凝胶层析从转染子上清液中纯化嵌合cACT19抗体。我们证实嵌合cACT19抗体对不同于TAG72抗原上唾液酸化-Tn的表位具有反应性。这是通过使用TAG72结合抑制ELISA实现的，该方法利用了各种单糖和含有NeuAc2-6αGalNAc结构的二唾液酸-N-四糖。免疫组织化学双重染色技术通过说明这两个表位在结肠癌组织不同区域的互补和非互补表达，进一步证明了嵌合cACT19抗体所定义的表位与唾液酸化-Tn表位之间的差异。我们还证明，与鼠源ACT19抗体相比，嵌合cACT19抗体对人OVAR3肿瘤细胞表现出更有效的ADCC和CDC。因此，鼠/人嵌合抗结直肠癌cACT19抗体本身可能被证明在癌症免疫治疗中有用，或者特别是与嵌合B72.3抗体联合使用时。
