Abstract 6695: Short linear peptide modulators of checkpoint receptor activity rescue suppression and enhance newly activated T cells

We characterized peptide-based checkpoint (CP) inhibitors (Microtides™) for their ability to activate T cell responses in ex vivo human PBMC (hPBMC) assays and in vivo mouse models of T cell immunity. These peptides were discovered by screening diverse libraries and do not share homology with sequences of the CP ligands. Peptides that inhibit LAG3 or dually inhibit PD1 and CTLA4 were tested in ligand-CP receptor functional binding assays. Select peptides were then evaluated in antigen recall assays using hPBMCs. In mouse vaccine models, the peptides enhanced vaccine-induced CD8 T cells, resulting in more effective tumor control. As monotherapeutics administered systemically in vivo, the peptides were able to inhibit melanoma B16-F10 lung metastases. We characterized PD1/CTLA4 inhibitors (LD series) and LAG3 inhibitors of FGL1 and MHCII binding (LG series). These 18-20 amino acid long linear peptides inhibit CP activity in vitro with potencies in the micromolar range, however when used in vivo, and despite their short half-life, both LD and LG peptides were able to enhance vaccine-induced T cells in a mouse model with a single sub-cutaneous dose, likely due to extended occupancy on the target CP receptor. Consistent with this observation, a single high dose of LD peptide administered intravenously did not lead to an increase in inflammatory cytokines in the serum of injected mice. In vitro, the peptides reversed PDL-1 suppression of T cell proliferation. The LD and LG peptides showed efficacy that was comparable to pembrolizumab in the hPBMC antigen recall assay. The efficacy of LD peptides was comparable to an established mouse anti-PD1 antibody in the B16-F10 lung metastasis model. Targeting multiple receptors is ongoing using bispecific peptides (Bitides™) which were made by linking the LD and LG peptides via a standard PEG linker. Our data indicate that Bitides retain in vitro LAG3 inhibitory activity. Testing their PD1/CTLA4 inhibitory activity is currently underway. These data provide proof of concept and illustrate the advantages and versatility of Microtides for targeting CP receptors via systemic or local administration. We demonstrated enhancement of CD8 responses in vitro and in a therapeutic setting in vivo. The LD and LG peptides, with their demonstrated activity and short half-life, allow us to activate cellular immunity while significantly reducing the potential for adverse events that is often associated with the antibody-based CP inhibitors or mixtures thereof. These peptides can be as standalone therapeutics or can be used as immune modulators in combination with other therapies (eg vaccines). They can be genetically encoded within CART and oncolytic viral platforms to effectively target the tumor microenvironment. Citation Format: Vinayaka Kotraiah, Timothy Phares, Cecille Browne, Marc Mansour, James Pannucci, Gabe Gutierrez. Short linear peptide modulators of checkpoint receptor activity rescue suppression and enhance newly activated T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6695.