The allosteric modulation of complement C5 by knob domain peptides.

Bovines have evolved a subset of antibodies with ultra-long heavy chain complementarity determining regions that harbour cysteine-rich knob domains. To produce high-affinity peptides, we previously isolated autonomous 3-6 kDa knob domains from bovine antibodies. Here, we show that binding of four knob domain peptides elicits a range of effects on the clinically validated drug target complement C5. Allosteric mechanisms predominated, with one peptide selectively inhibiting C5 cleavage by the alternative pathway C5 convertase, revealing a targetable mechanistic difference between the classical and alternative pathway C5 convertases. Taking a hybrid biophysical approach, we present C5-knob domain co-crystal structures and, by solution methods, observed allosteric effects propagating >50 Å from the binding sites. This study expands the therapeutic scope of C5, presents new inhibitors, and introduces knob domains as new, low molecular weight antibody fragments, with therapeutic potential.

Antibodies are proteins produced by the immune system that can selectively bind to other molecules and modify their behaviour. Cows are highly equipped at fighting-off disease-causing microbes due to the unique shape of some of their antibodies. Unlike other jawed vertebrates, cows' antibodies contain an ultra-long loop region that contains a 'knob domain' which sticks out from the rest of the antibody. Recent research has shown that when detached, the knob domain behaves like an antibody fragment, and can independently bind to a range of different proteins. Antibody fragments are commonly developed in the laboratory to target proteins associated with certain diseases, such as arthritis and cancer. But it was unclear whether the knob domains from cows' antibodies could also have therapeutic potential. To investigate this, Macpherson et al. studied how knob domains attach to complement C5, a protein in the inflammatory pathway which is a drug target for various diseases, including severe COVID-19. The experiments identified various knob domains that bind to complement C5 and inhibits its activity by altering its structure or movement. Further tests studying the structure of these interactions, led to the discovery of a common mechanism by which inhibitors can modify the behaviour of this inflammatory protein. Complement C5 is involved in numerous molecular pathways in the immune system, which means many of the drugs developed to inhibit its activity can also leave patients vulnerable to infection. However, one of the knob domains identified by Macpherson et al. was found to reduce the activity of complement C5 in some pathways, whilst leaving other pathways intact. This could potentially reduce the risk of bacterial infections which sometimes arise following treatment with these types of inhibitors. These findings highlight a new approach for developing drug inhibitors for complement C5. Furthermore, the ability of knob domains to bind to multiple sites of complement C5 suggests that this fragment could be used to target proteins associated with other diseases.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.