'Knobs-into-holes' engineering of antibody C(H)3 domains for heavy chain heterodimerization
'Knobs-into-holes' engineering of antibody C(H)3 domains for heavy chain heterodimerization
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DOI：
10.1093/protein/9.7.617
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发表时间：
1996-07-01
期刊：
影响因子：
--
通讯作者：
Carter, P
中科院分区：
文献类型：
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作者：
Ridgway, JBB;Presta, LG;Carter, P
“打结成洞”最初是由克里克在1952年提出的，作为一种在相邻的α-螺旋之间填充氨基酸侧链的模型。这里展示了一种新颖而有效的设计策略，用于工程抗体重链同源二聚体的异二聚化。在这种方法中，首先用CD4-Ig G免疫粘附素：T366Y的C(H)3区域中的一个较大的氨基酸代替一个较小的氨基酸来获得一个“旋钮”变异体。该旋钮被设计用来插入人源化抗CD3抗体C(H)3结构域中的一个洞，该人源化抗CD3抗体是通过明智地用较小的残基替换较大的残基而产生的：Y407T，在这两个不同的重链与抗CD3轻链共同表达后，抗CD3/CD4-Ig G杂交物代表了高达92%的蛋白A纯化蛋白库，相比之下，在重链包含野生型C(H)3结构域的共表达后，仅有57%的抗CD3/CD4-Ig G杂交物被回收。因此，打结成孔工程有助于构建抗体/免疫粘附素杂交物以及可能的其他含有Fc的双功能治疗药物，包括双特异性免疫粘附素和双特异性抗体。
'Knobs-into-holes' was originally proposed by Crick in 1952 as a model for the packing of amino acid side chains between adjacent alpha-helices. 'Knobs-into-holes' is demonstrated here as a novel and effective design strategy for engineering antibody heavy chain homodimers for heterodimerization. In this approach a 'knob' variant was first obtained by replacement of a small amino acid with a larger one in the C(H)3 domain of a CD4-IgG immunoadhesin: T366Y. The knob was designed to insert into a 'hole' in the C(H)3 domain of a humanized anti-CD3 antibody created by judicious replacement of a large residue with a smaller one: Y407T, The anti-CD3/CD4-IgG hybrid represents up to 92% of the protein A purified protein pool following co-expression of these two different heavy chains together with the anti-CD3 light chain, In contrast, only up to 57% of the anti-CD3/CD4-IgG hybrid is recovered following co-expression in which heavy chains contained wild-type C(H)3 domains. Thus knobs-into-holes engineering facilitates the construction of an antibody/immunoadhesin hybrid and likely other Fc-containing bifunctional therapeutics including bispecific immunoadhesins and bispecific antibodies.