Critical contribution of VH-VL interaction to reshaping of an antibody: The case of humanization of anti-lysozyme antibody, HyHEL-10
Critical contribution of VH-VL interaction to reshaping of an antibody: The case of humanization of anti-lysozyme antibody, HyHEL-10
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DOI：
10.1110/ps.073156708
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发表时间：
2008-02-01
期刊：
影响因子：
8
通讯作者：
Kumagai, Izumi
中科院分区：
文献类型：
--
作者：
Nakanishi, Takeshi;Tsumoto, Kouhei;Kumagai, Izumi
为了阐明鼠抗体人源化对其靶点特异性和亲和力的影响，我们检查了鸡蛋清溶菌酶 (HEL) 与其抗体 HyHEL-10 可变结构域片段 (Fv) 之间的相互作用。我们选择了同源性较高的人源抗体框架序列，将鼠HyHEL-10的6个互补决定区序列移植到该框架上，并研究了突变体Fvs与HEL之间的相互作用。等温滴定量热法表明，由于不利的熵变，人源化导致抗体与其靶标的亲和力降低 10 倍。然而，由于不利的熵变的减少，两个突变一起进入可变结构域的界面，导致抗体对靶标的亲和力和特异性完全恢复。人源化抗体复合物（包括两个突变体）与 HEL 的 X 射线晶体学表明，除了可变域的互补关联外，复合物具有几乎相同的结构，并且互补位和表位残基几乎是保守的。我们得出的结论是，可变域界面结构的调整可以有助于逆转由鼠抗体人源化引起的亲和力或特异性损失，这表明可变域的适当关联对于鼠抗体人源化而不丧失功能至关重要。
To clarify the effects of humanizing a murine antibody on its specificity and affinity for its target, we examined the interaction between hen egg white lysozyme (HEL) and its antibody, HyHEL-10 variable domain fragment (Fv). We selected a human antibody framework sequence with high homology, grafted sequences of six complementarity-determining regions of murine HyHEL-10 onto the framework, and investigated the interactions between the mutant Fvs and HEL. Isothermal titration calorimetry indicated that the humanization led to 10-fold reduced affinity of the antibody for its target, due to an unfavorable entropy change. Two mutations together into the interface of the variable domains, however, led to complete recovery of antibody affinity and specificity for the target, due to reduction of the unfavorable entropy change. X-ray crystallography of the complex of humanized antibodies, including two mutants, with HEL demonstrated that the complexes had almost identical structures and also paratope and epitope residues were almost conserved, except for complementary association of variable domains. We conclude that adjustment of the interfacial structures of variable domains can contribute to the reversal of losses of affinity or specificity caused by humanization of murine antibodies, suggesting that appropriate association of variable domains is critical for humanization of murine antibodies without loss of function.