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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Expi293细胞中多特异性多价抗体的高效且廉价的瞬时表达。

背景技术免疫疗法是药物发现领域中非常快速扩展的领域，因此抗体的快速和廉价表达将非常有价值。但是，抗体很难表达。具有其他结合域的多功能抗体进一步使表达复杂化。只有很少的协议描述了四价双特异性抗体的产生以及全部具有有限的表达水平。方法在这里，我们描述了一种可以低成本生产功能性四价，双特异性抗体的方案，蛋白质价格约为22 mg / l。该表达系统基于Expi293细胞，该细胞适合于比HEK293细胞在更密集的培养物中生长，并具有更高的表达量。新协议将PEI转染Expi293细胞（成本可忽略不计）。结果该方案已用于在10天内产生四价和六价双特异性抗体的多种变体，产量约为22 mg蛋白/ l。所有材料都是可商购的，并且该协议的实现便宜且直接。在我们实验室中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。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，以在体内成功检测出淀粉样β（Aβ）聚集体。结论该方案是第一个描述用PEI转染人Expi293细胞的方案。它可以用于生成大量的功能性多特异性抗体。使用生物药物，尤其是多特异性抗体，