Early Solutions UCB Biopharma UK, Slough, UK.
Department of Biology and Biochemistry, University of Bath, Bath, UK.
MAbs. 2022 Jan-Dec;14(1):2076295. doi: 10.1080/19420862.2022.2076295.
Cysteine-rich knob domains can be isolated from the ultralong heavy-chain complementarity-determining region (CDR) 3, which are unique to a subset of bovine antibodies, to create antibody fragments of ~4 kDa. Advantageously, the N- and C- termini of these small binding domains are in close proximity, and we propose that this may offer a practical route to engineer extrinsic binding specificity into proteins. To test this, we transplanted knob domains into various loops of rat serum albumin, targeting sites that were distal to the interface with the neonatal Fc receptor. Using knob domains raised against the clinically validated drug target complement component C5, we produced potent inhibitors, which exhibit an extended plasma half-life via attenuated renal clearance and neonatal Fc receptor-mediated avoidance of lysosomal catabolism. The same approach was also used to modify a V, targeting a framework loop situated at the opposing end of the domain to the CDRs, to produce a small, single-chain bispecific antibody and a dual inhibitor of Complement C3 and C5. This study presents new protein inhibitors of the complement cascade and demonstrates a broadly applicable method to engineer target specificity within polypeptide chains, using bovine knob domains.
富含半胱氨酸的 knob 结构域可以从超长重链互补决定区（CDR）3 中分离出来，该结构域是牛抗体亚群所特有的，可以构建约 4 kDa 的抗体片段。有利的是，这些小结合域的 N 端和 C 端非常接近，我们提出这可能为将外在结合特异性引入蛋白质提供一种实用途径。为了验证这一点，我们将 knob 结构域移植到大鼠血清白蛋白的各种环中，靶向与新生儿 Fc 受体的界面较远的部位。我们使用针对临床验证的药物靶标补体成分 C5 的 knob 结构域产生了有效的抑制剂，这些抑制剂通过减弱肾脏清除和新生儿 Fc 受体介导的避免溶酶体代谢来延长血浆半衰期。同样的方法也用于修饰 V H ，靶向位于该结构域与 CDRs 相对端的框架环，产生一个小的、单链双特异性抗体和补体 C3 和 C5 的双重抑制剂。本研究提供了补体级联的新的蛋白质抑制剂，并展示了一种在多肽链中构建靶标特异性的广泛适用的方法，使用牛 knob 结构域。
