Immunobiology Department, Center of Molecular Immunology, Havana, Cuba.
Immunobiology. 2011 Dec;216(12):1239-47. doi: 10.1016/j.imbio.2011.07.004. Epub 2011 Jul 7.
Gangliosides containing the N-glycolyl (NGc) form of sialic acid are tumor-associated antigens and promising candidates for cancer therapy. We previously generated the murine 14F7 monoclonal antibody (mAb), specific for the N-glycolyl-GM3 ganglioside (NGcGM3), which induced an oncosis-like type of cell death on malignant cell lines expressing this antigen and recognized breast carcinoma by immunoscintigraphy in cancer patients. As humanization is expected to enhance its use for human cancer therapy, herein we describe the design and generation of two humanized versions of the 14F7 mAb by disrupting potential human T cell epitopes on its variable region. No differences in antigen reactivity or cytotoxic properties were detected among the variants tested and with respect to the chimeric counterpart. Humanized 14F7 genes were transfected into the NGcGM3-expressing NS0 cell line. Therefore, in the industrial scaling-up of the transfectoma in serum-free medium, cell viability was lost due to the cytotoxic effect of the secreted antibody. This shortcoming was solved by knocking down the CMP-N-acetylneuraminic acid hydroxylase enzyme, thus impairing the synthesis of NGc-glycoconjugates. Humanized 14F7 mAb is of potential value for the therapy of NGcGM3-expressing tumors.
含有 N-羟乙酰神经氨酸（NGc）形式唾液酸的神经节苷脂是肿瘤相关抗原，是癌症治疗的有前途的候选物。我们之前生成了针对 N-羟乙酰神经节苷脂（NGcGM3）的鼠 14F7 单克隆抗体（mAb），该 mAb 特异性识别表达该抗原的恶性细胞系上的 N-羟乙酰-GM3 神经节苷脂（NGcGM3），并通过免疫闪烁成像在癌症患者中识别乳腺癌。由于人源化有望增强其在人类癌症治疗中的应用，因此本文描述了通过破坏其可变区上的潜在人 T 细胞表位来设计和生成两种 14F7 mAb 的人源化版本。在测试的变体之间以及与嵌合对应物相比，在抗原反应性或细胞毒性特性方面没有差异。人源化的 14F7 基因被转染到表达 NGcGM3 的 NS0 细胞系中。因此，在无血清培养基中进行转染瘤的工业放大时，由于分泌的抗体的细胞毒性作用，细胞活力丧失。通过敲低 CMP-N-乙酰神经氨酸羟化酶来解决这个缺点，从而破坏 NGc-糖缀合物的合成。人源化的 14F7 mAb 对治疗表达 NGcGM3 的肿瘤具有潜在价值。
