Objective

Bicycles^® are a new class of drugs - fully synthetic, constrained bicyclic peptides that combine the attributes of three therapeutic modalities (antibodies, small molecules, and peptides). The Bicycle peptides are characterised by high affinity and selective binding to the target protein. The compact molecular structure of the peptides results in efficient tumour distribution and rapid renal elimination. We optimised Bicycle peptides to develop novel and unique immunotherapeutic agonists. The costimulatory molecule CD137 (4-1BB) belongs to the TNF receptor superfamily and forms trimeric receptor complexes on immune cell surfaces. Agonistic anti-CD137 antibodies have shown potent anti-tumour activity in preclinical models. The effects are mainly mediated by cytotoxic T cells that generate long-lasting memory responses.  Two human anti-CD137 antibodies Urelumab (BMS) and Utomilumab (Pfizer) are currently undergoing clinical testing.

 

We utilized phage display to screen for CD137 binders using a library of 10¹⁵ Bicycles^®. Initial hits underwent primary affinity maturation followed by chemical optimisation. The lead peptide BCY3814 was further characterised and bound to the CD137 ligand-binding site with K_D ~30 nM. CD137 activation requires receptor cross-linking and multivalent binders would be required to mimic the biology of the natural trimeric ligand. We generated more than 60 different bi-, tri- and tetra-valent variants of BCY3814 with chemical linkers and hinges of various lengths with the total molecular weight spanning 4-15 kDa. We systematically explored different attachment points, which enabled development of multimers with a broad range of cellular activities in CD137 reporter assay. Selected CD137 multimers are being tested in vivo to profile their properties.  In conclusion, novel Bicycle technology resulted in generation of potently agonistic, compact and fully synthetic CD137 multimeric agonists.