AUTHOR=Adams Ralph , Joyce Callum , Kuravskiy Mikhail , Harrison Katriona , Ahdash Zainab , Balmforth Matthew , Chia Kelda , Marceddu Cinzia , Coates Matthew , Snowden James , Goursaud Emmanuel , Ménochet Karelle , Elsen Jean van den , Payne Richard J. , Lawson Alastair D. G. , Scott-Tucker Anthony , Macpherson Alex 

TITLE=Serum albumin binding knob domains engineered within a VH framework III bispecific antibody format and as chimeric peptides

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1170357

DOI=10.3389/fimmu.2023.1170357

ISSN=1664-3224

ABSTRACT=<sec><title>Background</title><p>Serum albumin binding is an established mechanism to extend the serum half-life of antibody fragments and peptides. The cysteine rich knob domains, isolated from bovine antibody ultralong CDRH3, are the smallest single chain antibody fragments described to date and versatile tools for protein engineering.</p></sec><sec><title>Methods</title><p>Here, we used phage display of bovine immune material to derive knob domains against human and rodent serum albumins. These were used to engineer bispecific Fab fragments, by using the framework III loop as a site for knob domain insertion. </p></sec><sec><title>Results</title><p>By this route, neutralisation of the canonical antigen (TNFα) was retained but extended pharmacokinetics <italic>in-vivo</italic> were achieved through albumin binding. Structural characterisation revealed correct folding of the knob domain and identified broadly common but non-cross-reactive epitopes. Additionally, we show that these albumin binding knob domains can be chemically synthesised to achieve dual IL-17A neutralisation and albumin binding in a single chemical entity. </p></sec><sec><title>Conclusions</title><p>This study enables antibody and chemical engineering from bovine immune material, via an accessible discovery platform.</p></sec>