De Monte L, Nisticó P, Tecce R, Dellabona P, Momo M, Tarditi L, Natali P G, Mariani M, Malavasi F
Dipartimento di Genetica, Biologia e Chimica Medica, Università di Torino, Italy.
Dev Biol Stand. 1990;71:15-22.
This report deals with the use of gene transfer by retrovirus-derived shuttle vectors in a novel model aimed at the generation of hybrid hybridomas secreting bispecific monoclonal antibodies (biMAbs). Following this approach, two genes conferring dominant resistance trait to the neomycine analogue geneticin (G418) and to methotrexate (MTX) respectively, were infected in two established hybridoma lines, each producing a well characterized MAb. The vectors used here were replication-deficient, being dependent on the complementation of helper virus provided by packaging lines. The infection procedure involved co-cultivation of the hybridomas with irradiated packaging cell lines, previously transfected with the vectors and producing the recombinant retroviruses, not inclusive of helper virus in their genome. The packaging lines used were psi2 ecotropic cells made able to produce high titers of virus. Further, the vector pMV7 was carrying G418 resistance while the pSDHT render the cells able to survive MTX. Easy and fast transfer of the dominant selection markers yielded lines of hybridomas to be fused according to the conventional somatic fusions. The resulting double hybridomas were tested for the production of hybrid molecules retaining parental specificity and successively underwent extensive cloning. The purification system featuring the most efficiently between the true biMAbs and the parental immunoglobulins (or other combination products) proved to be HPLC on hydroxylapatite column. The method described above was successful in producing two biMAbs targeting simultaneously molecules expressed on cytotoxic cells (such as CD3 on T-lymphocytes and CD16 on NK cells) and the melanoma-associated antigen Ep2.
本报告涉及逆转录病毒衍生穿梭载体在一种新型模型中的基因转移应用，该模型旨在产生分泌双特异性单克隆抗体（双特异性单克隆抗体）的杂交杂交瘤。按照这种方法，分别将赋予对新霉素类似物遗传霉素（G418）和甲氨蝶呤（MTX）显性抗性特征的两个基因感染两个已建立的杂交瘤细胞系，每个细胞系产生一种特征明确的单克隆抗体。这里使用的载体是复制缺陷型的，依赖于包装细胞系提供的辅助病毒的互补作用。感染过程包括将杂交瘤与经辐照的包装细胞系共培养，这些包装细胞系先前已用载体转染并产生重组逆转录病毒，其基因组中不包括辅助病毒。所使用的包装细胞系是能够产生高滴度病毒的嗜亲性psi2细胞。此外，载体pMV7携带G418抗性，而pSDHT使细胞能够在MTX中存活。显性选择标记的轻松快速转移产生了可根据传统体细胞融合进行融合的杂交瘤细胞系。对产生的双杂交瘤进行测试，以检测保留亲本特异性的杂交分子的产生，并随后进行广泛的克隆。在真正的双特异性单克隆抗体与亲本免疫球蛋白（或其他组合产物）之间具有最高效率的纯化系统被证明是羟基磷灰石柱上的高效液相色谱法。上述方法成功地产生了两种双特异性单克隆抗体，它们同时靶向细胞毒性细胞上表达的分子（如T淋巴细胞上的CD3和NK细胞上的CD16）以及黑色素瘤相关抗原Ep2。
