Antibody engineering of non-human antibodies has focused on reducing immunogenicity by humanization, being a major limitation in developing monoclonal antibodies. We analyzed four series of antibody binding fragments (Fabs) and a variable fragment (Fv) with structural information in different stages of humanization to investigate the influence of the framework, point mutations and specificity on the complementarity determining region (CDR)-H3 loop dynamics. We also studied a Fv without structural information of the anti-idiotypic antibody Ab2/3H6, because it completely lost its binding affinity upon superhumanization, as an example of a failed humanization. Enhanced sampling techniques in combination with molecular dynamics simulations allow to access micro- to milli-second timescales of the CDR-H3 loop dynamics and reveal kinetic and thermodynamic changes involved in the process of humanization. In most cases, we observe a reduced conformational diversity of the CDR-H3 loop when grafted on a human framework and find a conformational shift of the dominant CDR-H3 loop conformation in solution. A shallow side minimum of the conformational CDR-H3 loop ensemble attached to the murine framework becomes the dominant conformation in solution influenced by the human framework. Additionally, we observe in the case of the failed humanization that the potentially binding competent murine CDR-H3 loop ensemble in solution shows nearly no kinetical or structural overlap with the superhumanized variant, thus explaining the loss of binding.

中文翻译：

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抗体人源化-溶液中抗体框架对 CDR-H3 环整体的影响。


非人类抗体的抗体工程重点是通过人源化降低免疫原性，这是开发单克隆抗体的主要限制。我们分析了人源化不同阶段的四个系列的抗体结合片段（Fab）和具有结构信息的可变片段（Fv），以研究框架、点突变和特异性对互补决定区（CDR）-H3环动态的影响。我们还研究了没有抗独特型抗体 Ab2/3H6 结构信息的 Fv，因为它在超人化后完全失去了结合亲和力，作为人源化失败的一个例子。增强的采样技术与分子动力学模拟相结合，可以获取微秒到毫秒的 CDR-H3 环动力学时间尺度，并揭示人源化过程中涉及的动力学和热力学变化。在大多数情况下，我们观察到当移植到人类框架上时 CDR-H3 环的构象多样性降低，并发现溶液中主要 CDR-H3 环构象的构象变化。附着在鼠框架上的构象CDR-H3环整体的浅侧最小值成为受人类框架影响的溶液中的主要构象。此外，我们观察到在人源化失败的情况下，溶液中具有潜在结合能力的鼠CDR-H3环整体几乎没有与超人​​源化变体在动力学或结构上重叠，从而解释了结合的丧失。