Lindhofer H, Mocikat R, Steipe B, Thierfelder S
GSF, Immunology Institute, Munich, Germany.
J Immunol. 1995 Jul 1;155(1):219-25.
Conventional mouse/mouse or rat/rat hybrid-hybridoma supernatants contain up to 10 different IgG molecules consisting of various combinations of heavy and light chains. Hence, the yield of functional bispecific Ab is low, and purification is often complicated, hampering a general preclinical evaluation of, e.g., bispecific Ab-mediated tumor immunotherapy in animal models. In experiments to overcome this drawback we found that fusion of rat with mouse hybridomas opens the possibility of large scale production of bispecific Ab due to the increased incidence of correctly paired Ab and facilitated purification. In essence, rat/mouse quadroma-derived bispecific Ab have the following advantages: 1) enrichment of functional bispecific Ab because of preferential species-restricted heavy/light chain pairing (observed in four of four rat-mouse quadromas) in contrast to the random pairing in conventional mouse/mouse or rat/rat quadromas, and 2) a possible one-step purification of the quadroma supernatant with protein A. This simple chromatography step does not bind unwanted variants with parental rat/rat heavy chain configuration, and the desired rat/mouse bispecific Ab are retained, which can then easily be separated from parental mouse Ab by sequential pH elution.
传统的小鼠/小鼠或大鼠/大鼠杂交瘤上清液含有多达10种不同的IgG分子，这些分子由重链和轻链的各种组合构成。因此，功能性双特异性抗体的产量很低，而且纯化过程通常很复杂，这妨碍了在动物模型中对双特异性抗体介导的肿瘤免疫疗法等进行全面的临床前评估。在克服这一缺点的实验中，我们发现大鼠杂交瘤与小鼠杂交瘤融合，由于正确配对抗体的发生率增加以及纯化变得容易，从而为大规模生产双特异性抗体开辟了可能性。从本质上讲，大鼠/小鼠四瘤细胞产生的双特异性抗体具有以下优点：1）由于优先发生物种限制的重链/轻链配对（在四个大鼠-小鼠四瘤细胞中均观察到），与传统的小鼠/小鼠或大鼠/大鼠四瘤细胞中的随机配对相比，功能性双特异性抗体得到富集；2）四瘤细胞上清液可能通过蛋白A进行一步纯化。这一简单的色谱步骤不会结合具有亲本大鼠/大鼠重链构型的不需要的变体，所需的大鼠/小鼠双特异性抗体被保留下来，然后通过连续的pH洗脱可以很容易地将其与亲本小鼠抗体分离。
