Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells.
Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells.
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DOI：
10.1186/s12575-017-0060-7
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发表时间：
2017
影响因子：
6.4
通讯作者：
Hultqvist G
中科院分区：
文献类型：
--
作者：
Fang XT;Sehlin D;Lannfelt L;Syvänen S;Hultqvist G
免疫疗法是药物发现中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。具有额外结合结构域的多功能抗体进一步使表达复杂化。只有少数方案描述了四价双特异性抗体的生产，并且所有方案都具有有限的表达水平。该表达系统基于Expi 293细胞，Expi 293细胞已适应于在比HEK 293细胞更密集的培养物中生长，并产生更高的表达产量。新的方案用PEI（其具有可忽略的成本）抑制E xpi 293细胞。该方案已用于产生四价和六价双特异性抗体的多种变体，在10天内产量约为22 mg蛋白质/l。所有的材料都是商业上可获得的，并且该协议的实施是廉价和简单的。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，以成功检测体内淀粉样蛋白-β（Aβ）聚集体。该方案首次描述了用PEI转染人Expi 293细胞。它可用于产生大量的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，如这里提出的方案，是非常有价值的。
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. 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). 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. 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.
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DOI：
10.1007/978-1-61779-352-3_2
发表时间：
2012-01-01
期刊：
PROTEIN EXPRESSION IN MAMMALIAN CELLS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Baldi, Lucia;Hacker, David L.;Wurm, Florian M.
通讯作者：
Wurm, Florian M.
影响因子：
3.8
作者：
Backliwal, Gaurav;Hildinger, Markus;Wurm, Florian M.
通讯作者：
Wurm, Florian M.
影响因子：
14.9
作者：
Durocher, Y;Perret, S;Kamen, A
通讯作者：
Kamen, A
影响因子：
3.8
作者：
Backliwal, Gaurav;Hildinger, Markus;Wurm, Florian M.
通讯作者：
Wurm, Florian M.
影响因子：
2.2
作者：
Eberhardy, Scott R.;Radzniak, Laurie;Liu, Zhong
通讯作者：
Liu, Zhong