Discovery and optimization of a synthetic small protein domain targeting antibodies

Ana Margarida Goncalves Carvalho Dias^1,2Manuel Matos^1,2Cátia Soares^1,2Carolina Natal^1,2Ana Sofia Pina^1,2Ana Cecília Roque^1,2*

• ¹Associate Laboratory Institute for Health and Bioeconomy, Caparica, Portugal
• ²Unidade de Ciencias Biomoleculares Aplicadas, Caparica, Portugal

Antibodies and their derivatives represent a vital class of molecules in contemporary biotechnological and therapeutic applications. Thus, the quest for chemically robust affinity ligands that recognize antibodies remains a significant endeavour. In this study, an in-house phage display library based on the WW domain scaffold (WWp5_4) was used to identify a binder towards human polyclonal IgG. The lead ligand E6 exhibited a dissociation constant of 133nM. To further enhance the developability of the WW ligands derived from the library, we rationally reduced the liabilities in the scaffold framework to improve chemical stability and solubility, and two mutants were generated (WWp5_4_M2 and E6_M2). Subsequently, the E6 mutated sequence was used to design a head-to-tail dimeric sequence (E6_M2_T2). The resultant molecules exhibited 5-times higher expression yields compared to their native sequences, and the dimeric sequence exhibited better solubility and an estimated dissociation constant of 62 nM for human IgG. Our research highlights the role of small folded protein domains, such as WW domains, as affinity ligands for antibodies and other molecular targets, paving the way for diverse applications in biotechnology and bioengineering.