Abstract 3257: Activation of CD137 using multivalent and tumor targeted Bicyclic peptides
Authors: Hurov Kristen E. , Upadhyaya Punit , Kublin Jessica , Zhou Xueyuan , Kristensson Julia , Lani Rachid , Mudd Gemma , Rietschoten Katerine van , An Frank , Lahdenranta Johanna , Chen Liuhong , Bennett Gavin , McDonnell Kevin , Park Peter , Keen Nicholas 
Published: August 2019

Abstract
CD137 (4-1BB/TNFRSF9) is a costimulatory T-cell receptor belonging to the TNF receptor superfamily. Agonistic anti-CD137 antibodies have shown potent, often curative anti-tumor activity in preclinical models. Two human anti-CD137 antibodies, urelumab and utomilumab are currently undergoing clinical testing. Urelumab has shown several single-agent, partial responses, but its use has been hampered by hepatoxicity, while utomilumab has shown little or no single agent activity.
Bicycles® are a new class of drugs - fully synthetic, constrained bicyclic peptides that combine the attributes of antibodies, small molecules, and peptides by delivering high affinity, selectivity, and rapid clearance. Their small size (1.5-2 kDa) delivers advantages in tumor penetration, and renal elimination may avoid the liver and GI toxicity often associated with other drug modalities, including certain antibodies. We hypothesized that fully synthetic Bicycle CD137 agonists with rapid clearance, minimal liver exposure and no Fc receptor interaction may induce CD137 mediated anti-tumor activity while avoiding liver toxicity.
A high affinity lead BCY3814 (KD ~30 nM) that binds to the CD137 ligand-binding site was identified. CD137 activation requires receptor crosslinking, thus multivalent binding would be expected to recapitulate the action of the natural trimeric ligand. We envisioned that CD137 agonism could be achieved directly by using multimeric CD137 Bicycles or in a tumor targeted fashion with bispecific or “heterotandem” Bicycles. The synthetic simplicity and highly modular nature of the Bicycle® platform enabled us to rapidly explore both formats.
To generate a “pure” CD137 agonist we synthesized &gt;50 different multimeric variants of BCY3814 with chemical linkers of various lengths and rigidity and using different sites of attachments, while maintaining a compact size (&lt;15 kDa). Tumor targeted CD137 agonists were generated as heterotandems, whereby BCY3814 is conjugated to a tumor antigen targeting Bicycle. In this design, the CD137 Bicycle only induces CD137 agonism after the molecule binds to a tumor cell with high receptor expression.
We discovered Bicycle multimers that exhibit a range of potencies in a cell-based CD137-dependent reporter assay, activate human T cells in vitro as indicated by increased cytokine release, and show biological activity in vivo. Bicycle heterotandems targeting Nectin-4 and EphA2 exhibited highly potent and tumor cell specific activity in both the cell-based reporter assay and the human T cell assay. Selected CD137 multimers and heterotandems are being tested further in humanized mouse models for T cell activation, anti-tumor activity, and liver safety. These molecules are promising, novel cancer immunotherapy candidates and importantly, they pave the way for development of synthetic agonists of other TNF receptors that can be targeted to the local tumor microenvironment.
Citation Format: Kristen E. Hurov, Punit Upadhyaya, Jessica Kublin, Xueyuan Zhou, Julia Kristensson, Rachid Lani, Gemma Mudd, Katerine van Rietschoten, Frank An, Johanna Lahdenranta, Liuhong Chen, Gavin Bennett, Kevin McDonnell, Peter Park, Nicholas Keen. Activation of CD137 using multivalent and tumor targeted Bicyclic peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3257.