Abstract 292: A dual CTLA4 and PD1 microbially derived inhibitor shows efficacy in anti-PD1 mAb unresponsive tumor models through a unique allosteric mechanism of action
Authors: Gutierrez Gabriel M. , Phares Timothy W. , Kotraiah Vinayaka , Buontempo Peter , Pannucci James , Keltner Llew 
Published: July 2021

Abstract
Background Maintenance of a symbiotic relationship with microbes has led to the evolution of a complex host immune recognition system. How microbes achieve homeostasis with their hosts is an unexploited source of novel chemistry and targets for cancer immunotherapy. Peptides are a proven and powerful medium through which microbes communicate amongst themselves and with the host immune system [Hilchie et al. Nat Chem Biol. 2013 and Hancock Nat Rev Microbiol 2012]. Accordingly, we have developed a platform to mine bioactive peptides from microbial databases/libraries. As proof of concept of our screening technology we targeted PD1 to (A) determine if the PD1 “rheostat” is a target of the microbial immune manipulation, and (B) characterize the activity of the peptide(s) towards developing a novel checkpoint inhibitor therapeutic modality.
Methods A microbiome database was queried against previously identified PD1 binding peptides [Kotraiah V Front. Immunol. 2020] and identified peptides were computationally modeled to determine binding site. Reporter assays and ex vivo models were used to measure binding and quantify receptor antagonism. Syngeneic tumor models responsive and unresponsive to anti-PD1 monoclonal antibodies were used to test the efficacy of the peptides. Further, an infectious disease therapeutic model was used to measure and characterize the T-cell response of the peptides in vivo.
Results A 22-amino acid peptide, BtLD01, from Bacillus thuringiensis was computationally shown to target a novel allosteric binding site on PD1 which is shared by the related CD28 family receptor, CTLA4. Indeed, BtLD01 antagonizes PD1 and CTLA4 simultaneously as measured in a cell-based reporter assay. Using ex vivo assays we show that BtLD reverses exhaustion-like phenotype in human PBMC recall and that they can stimulate T-Cell proliferation in the presence of PD-L1 blockade. Intriguingly, BtLD checkpoint inhibition is not dependent on PD-L1 blockade but by potentially recruiting or stabilizing the PD1:PD-L1 complex. In vivo studies with sygeneic, mouse tumor models displayed activity in both anti-PD1 mAb responsive and unresponsive tumors. Further, in a lethal malaria model of T-cell activation, we saw that BtLD01 not only protects mice and forms a memory recall response, but also suppresses Tregs.
Conclusions Novel Microbially-Derived BtLD peptides target PD1/CTLA4 receptors. The unique allosteric binding site allows peptides to function non-competitively with PD-L1. This novel combined checkpoint inhibition mechanism of activating Tcells and suppressing Tregs along with the rapid and transient modulating nature of peptide may provide a safe and potent means to treat tumors refractory to anti-PD1 mAb therapy alone in clinic.
Citation Format: Gabriel M. Gutierrez, Timothy W. Phares, Vinayaka Kotraiah, Peter Buontempo, James Pannucci, Llew Keltner. A dual CTLA4 and PD1 microbially derived inhibitor shows efficacy in anti-PD1 mAb unresponsive tumor models through a unique allosteric mechanism of action [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 292.