Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.
Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.
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作者：
Asano R;Shimomura I;Konno S;Ito A;Masakari Y;Orimo R;Taki S;Arai K;Ogata H;Okada M;Furumoto S;Onitsuka M;Omasa T;Hayashi H;Katayose Y;Unno M;Kudo T;Umetsu M;Kumagai I
产生更有益的治疗性抗体的一种方法是开发双特异性抗体（bsAb），特别是具有四价的IgG样形式，其可以提供若干优点，例如与每种靶抗原的多价结合。虽然已经研究了构型和抗体片段类型对IgG样bsAb功能的影响，但是只有少数关于可变片段结构域顺序的影响的详细研究。在这里，我们制备了四种类型的hEx 3-scDb-Fc，IgG样bsAb，由单链hEx 3-Db（靶向表皮生长因子受体和CD 3的人源化双特异性双抗体[bsDb]）构建，以研究结构域顺序和融合方式对具有Fc融合形式的bsDb功能的影响。与VH-VL顺序相比，用具有可变轻链结构域（VL）-可变重链结构域（VH）顺序的hEx 3-scDb-Fc（hEx 3-scDb-Fc-LH）观察到更高的细胞毒性，表明Fc融合方式的差异不影响bsDb活性。此外，流式细胞术表明，hEx 3-scDb-Fc-LH的较高细胞毒性可能归因于交联的结构优势。有趣的是，用hEx 3-scDb-Fc-LH也观察到增强的降解抗性和延长的体内半衰期。hEx 3-scDb-Fc-LH及其IgG 2变体表现出强烈的体内抗肿瘤作用，表明Fc介导的效应子功能对于有效的抗肿瘤活性是有利的，这可能导致较少的副作用。我们的研究结果表明，仅仅重排IgG样bsAb的结构域顺序不仅可以增强其抗肿瘤活性，还可以增强其抗降解性和体内半衰期，并且hEx 3-scDb-Fc-LH是下一代治疗性抗体的有效候选物。
One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format. Higher cytotoxicities were observed with hEx3-scDb-Fcs with a variable light domain (VL)–variable heavy domain (VH) order (hEx3-scDb-Fc-LHs) compared with a VH–VL order, indicating that differences in the Fc fusion manner do not affect bsDb activity. In addition, flow cytometry suggested that the higher cytotoxicities of hEx3-scDb-Fc-LH may be attributable to structural superiority in cross-linking. Interestingly, enhanced degradation resistance and prolonged in vivo half-life were also observed with hEx3-scDb-Fc-LH. hEx3-scDb-Fc-LH and its IgG2 variant exhibited intense in vivo antitumor effects, suggesting that Fc-mediated effector functions are dispensable for effective anti-tumor activities, which may cause fewer side effects. Our results show that merely rearranging the domain order of IgG-like bsAbs can enhance not only their antitumor activity, but also their degradation resistance and in vivo half-life, and that hEx3-scDb-Fc-LHs are potent candidates for next-generation therapeutic antibodies.