a Biologics Discovery , Janssen Research & Development, LLC , Spring House , PA , USA.
b Department of Medicinal Chemistry , University of Washington , Seattle , WA , USA.
MAbs. 2017 Nov/Dec;9(8):1306-1316. doi: 10.1080/19420862.2017.1375639. Epub 2017 Sep 12.
The increased number of bispecific antibodies (BsAb) under therapeutic development has resulted in a need for mouse surrogate BsAbs. Here, we describe a one-step method for generating highly pure mouse BsAbs suitable for in vitro and in vivo studies. We identify two mutations in the mouse IgG2a and IgG2b Fc region: one that eliminates protein A binding and one that enhances protein A binding by 8-fold. We show that BsAbs harboring these mutations can be purified from the residual parental monoclonal antibodies in one step using protein A affinity chromatography. The structural basis for the effects of these mutations was analyzed by X-ray crystallography. While the mutation that disrupted protein A binding also inhibited FcRn interaction, a bispecific mutant in which one subunit retained the ability to bind protein A could still interact with FcRn. Pharmacokinetic analysis of the serum half-lives of the mutants showed that the mutant BsAb had a serum half-life comparable to a wild-type Ab. The results describe a rapid method for generating panels of mouse BsAbs that could be used in mouse studies.
治疗性开发的双特异性抗体（BsAb）数量增加，导致需要小鼠替代 BsAb。在这里，我们描述了一种一步法，可用于生成适合体外和体内研究的高纯度小鼠 BsAb。我们在小鼠 IgG2a 和 IgG2b Fc 区域鉴定出两个突变：一个消除了蛋白 A 结合，另一个使蛋白 A 结合增强了 8 倍。我们表明，含有这些突变的 BsAb 可以使用蛋白 A 亲和层析一步从残留的亲本单克隆抗体中纯化出来。通过 X 射线晶体学分析了这些突变的影响的结构基础。虽然破坏蛋白 A 结合的突变也抑制了 FcRn 相互作用，但一个亚基仍保留与蛋白 A 结合能力的双特异性突变体仍能与 FcRn 相互作用。对突变体的血清半衰期的药代动力学分析表明，突变体 BsAb 的血清半衰期与野生型 Ab 相当。该结果描述了一种快速生成可用于小鼠研究的小鼠 BsAb 组合的方法。
