MAbs. 2014 Jan-Feb;6(1):236-45. doi: 10.4161/mabs.27261.
In vitro selection technologies are an important means of affinity maturing antibodies to generate the optimal therapeutic profile for a particular disease target. Here, we describe the isolation of a parent antibody, KENB061 using phage display and solution phase selections with soluble biotinylated human IL-1R1. KENB061 was affinity matured using phage display and targeted mutagenesis of VH and VL CDR3 using NNS randomization. Affinity matured VHCDR3 and VLCDR3 library blocks were recombined and selected using phage and ribosome display protocol. A direct comparison of the phage and ribosome display antibodies generated was made to determine their functional characteristics.In our analyses, we observed distinct differences in the pattern of beneficial mutations in antibodies derived from phage and ribosome display selections, and discovered the lead antibody Jedi067 had a ~3700-fold improvement in KD over the parent KENB061. We constructed a homology model of the Fv region of Jedi067 to map the specific positions where mutations occurred in the CDR3 loops. For VL CDR3, positions 94 to 97 carry greater diversity in the ribosome display variants compared with the phage display. The positions 95a, 95b and 96 of VLCDR3 form part of the interface with VH in this model. The model shows that positions 96, 98, 100e, 100f, 100 g, 100h, 100i and 101 of the VHCDR3 include residues at the VH and VL interface. Importantly, Leu96 and Tyr98 are conserved at the interface positions in both phage and ribosome display indicating their importance in maintaining the VH-VL interface. For antibodies derived from ribosome display, there is significant diversity at residues 100a to 100f of the VH CDR3 compared with phage display. A unique deletion of isoleucine at position 102 of the lead candidate, Jedi067, also occurs in the VHCDR3.As anticipated, recombining the mutations via ribosome display led to a greater structural diversity, particularly in the heavy chain CDR3, which in turn led to antibodies with improved potencies. For this particular analysis, we also found that VH-VL interface positions provided a source of structural diversity for those derived from the ribosome display selections. This greater diversity is a likely consequence of the presence of a larger pool of recombinants in the ribosome display system, or the evolutionary capacity of ribosome display, but may also reflect differential selection of antibodies in the two systems.
体外选择技术是亲和成熟抗体的重要手段，可针对特定疾病靶标产生最佳治疗特征。在这里，我们描述了使用噬菌体展示和带有可溶性生物素化人 IL-1R1 的溶液相选择从噬菌体展示中分离出亲本抗体 KENB061。使用噬菌体展示和 VH 和 VL CDR3 的 NNS 随机化靶向突变，使 KENB061 亲和力成熟。使用噬菌体和核糖体展示方案对亲和力成熟的 VHCDR3 和 VLCDR3 文库块进行重组和选择。对噬菌体和核糖体展示产生的抗体进行了直接比较，以确定它们的功能特征。在我们的分析中，我们观察到源自噬菌体和核糖体展示选择的抗体中有益突变模式的明显差异，并发现先导抗体 Jedi067 的 KD 相对于亲本 KENB061 提高了约 3700 倍。我们构建了 Jedi067 的 Fv 区域同源模型，以映射 CDR3 环中发生突变的特定位置。对于 VL CDR3，与噬菌体展示相比，核糖体展示变体在位置 94 到 97 处具有更大的多样性。在该模型中，VLCDR3 的位置 95a、95b 和 96 构成与 VH 界面的一部分。该模型表明，VHCDR3 的位置 96、98、100e、100f、100g、100h、100i 和 101 包括 VH 和 VL 界面处的残基。重要的是，亮氨酸 96 和酪氨酸 98 在噬菌体和核糖体显示的界面位置保守，表明它们在维持 VH-VL 界面方面的重要性。对于源自核糖体显示的抗体，与噬菌体显示相比，VH CDR3 的位置 100a 到 100f 处存在显着的多样性。在先导候选物 Jedi067 的位置 102 处还发生了独特的缺失异亮氨酸。正如预期的那样，通过核糖体显示重组突变导致结构多样性增加，特别是在重链 CDR3 中，这反过来又导致效力提高的抗体。对于这项特定分析，我们还发现 VH-VL 界面位置为源自核糖体显示选择的抗体提供了结构多样性的来源。这种更大的多样性可能是核糖体显示系统中重组体池较大或核糖体显示的进化能力的结果，但也可能反映出两种系统中抗体的差异选择。
