BACKGROUND Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). RESULTS The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aβ) aggregates in vivo. CONCLUSIONS This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

中文翻译：

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在 Expi293 细胞中高效且廉价地瞬时表达多特异性多价抗体。


背景技术免疫治疗是药物发现中一个非常快速扩展的领域，因此，抗体的快速且廉价的表达将是极其有价值的。然而，抗体很难表达。具有额外结合域的多功能抗体使表达进一步复杂化。只有少数方案描述了四价双特异性抗体的生产，并且所有方案的表达水平都有限。 。方法在这里，我们描述了一种可以以低成本生产约 22 mg 蛋白质/升的功能性四价双特异性抗体的方案。该表达系统基于 Expi293 细胞，该细胞已适应在比 HEK293 细胞更密集的培养物中生长，并提供更高的表达产量。新方案用 PEI（成本可以忽略不计）转染 E xpi293 细胞。结果 该方案已用于生成四价和六价双特异性抗体的多种变体，10 天内产量约为 22 mg 蛋白质/升。所有材料均可在市场上买到，并且该协议的实施既便宜又简单。我们实验室产生的双特异性抗体能够以与原始抗体相似的亲和力结合所有抗原。其中两种双特异性抗体也已在转基因小鼠中用作正电子发射断层扫描 (PET) 配体，成功检测体内淀粉样蛋白 (Aβ) 聚集体。结论 该方案是第一个描述用 PEI 转染人类 Expi293 细胞的方案。它可用于大量产生功能性多特异性抗体。生物药物，特别是多特异性抗体的使用正在迅速增加，因此改进的方案（例如这里介绍的方案）非常有价值。