■双特异性抗体的治疗潜力正在得到广泛认可,已经描述了一百多种格式。对于许多应用,寻求增强的组织渗透,因此,低分子量的双特异性药物可能提供增强效力的途径。在这里,我们报告了分子量分别低至14.5和22kDa的基于双特异性和三特异性抗体的构建体的设计。
■自主牛超长CDRH3(旋钮结构域肽)模块已被设计为具有源自SinNombre正坦病毒核衣壳蛋白和人Beclin-1的人工卷曲螺旋茎,并串联以产生双和三特异性抗体为基础的构建体,分子量极低。
■带有卷曲螺旋柄的旋钮结构域肽保留较高,独立的抗原结合亲和力,表现出卓越的热稳定性,并且可以容易地头尾连接,产生最小的所述多特异性抗体形式。所得到的构建体能够在没有干扰的情况下同时结合它们的所有靶标。
■与现有的双特异性格式相比,降低分子量的旋钮结构域融合物可以增强组织渗透并促进与常规抗体无法接触的隐蔽表位的结合。此外,它们可以作为重组产物容易地以高产率生产,并且没有重链-轻链错配问题。一起来看,我们的方法为简约的双细节和多细节的模块化构建提供了一条有效途径,从而进一步拓宽旋钮结构域肽的治疗范围。
The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively.
Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights.
Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference.
Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.