A phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries

The development of a new strategy for antibody humanization is described. This strategy incorporates key recognition sequences from the parental rodent antibody into a phage display-based selection strategy. The original sequences of the third complementarity-determining regions (CDRs) of heavy and light chains, HCDR3 and LCDR3, were maintained and all other sequences were replaced by human sequences selected from phage-displayed antibody libraries. This approach was applied to the humanization of mouse mAb LM609 that is directed to human integrin α(v)β(3) and has potential applicability in cancer therapy as an antiangiogenic agent. We demonstrate this approach (i) provides a rapid route for antibody humanization constraining the content of original mouse sequences in the final antibodies to the most hypervariable of the CDRs; (ii) generates several humanized versions with different sequences at the same time; (iii) results in affinities as high as or higher than the affinity of the original antibody; and (iv) retains the antigen and epitope specificity of the original antibody. The production of multiple humanized variants may present advantages in the selection of antibodies that are more readily expressed on a large scale and could be important in therapeutic regimens that call for long-term treatment with antibodies in which antiidiotypic responses might be avoided by administration of alternative antibodies.

本研究报道了一种全新的抗体人源化策略。该策略将亲本啮齿类抗体的关键识别序列整合至基于噬菌体展示（phage display）的筛选流程中。研究保留了重链与轻链的第3互补决定区（complementarity-determining regions, CDRs）——即重链互补决定区3（HCDR3）与轻链互补决定区3（LCDR3）——的原始序列，其余所有序列均替换为从噬菌体展示抗体库中筛选得到的人源序列。该策略被应用于靶向人源整合素αvβ3的小鼠单克隆抗体（monoclonal antibody, mAb）LM609的人源化改造，该抗体作为抗血管生成剂在癌症治疗领域具有潜在应用价值。本研究证实该策略具备四大优势：其一，可实现抗体人源化的快速制备，且将最终抗体中保留的小鼠原始序列限制在互补决定区中高变程度最高的区域；其二，可同时获得多种序列各异的人源化抗体变体；其三，所得抗体的亲和力可达甚至高于原始抗体的亲和力；其四，可保留原始抗体的抗原与表位结合特异性。制备多种人源化抗体变体的优势在于，可筛选出更易于大规模表达的抗体；同时，在需要长期使用抗体的治疗方案中，通过轮换使用不同抗体可避免抗独特型应答，该策略在此类场景中具备重要应用价值。