Abstract P260: Development and validation of a novel T-cell modulating, microbiome-based peptide for combination with immunotherapy

The gut microbiome is an important determinant for the success of anti-tumor therapies including chemotherapeutics and anti-checkpoint inhibitors. In this study we wanted to leverage Second Genome’s large and curated microbiome database coupled with its proprietary bioinformatics and machine learning tools to discover bioactive peptides from Bifidobacteria that have the potential to drive response to immunotherapy. The genome of Bifidobacterium (B.) breve and B. longum were analyzed for proteins which were potentially secreted, and had unknown functions. 50 peptides were chemically synthesized and then screened in cell based assays for T cell activation and cytokine secretion. In the present study we describe one such novel B. breve-derived 42-aa peptide (SG-3-0020). The peptide stimulated secretion of effector cytokines by in vitro-cultured T cells (IFNg, TNF-a, IL-10 and IL-2) and increased the expression of PD-1 on both CD4+ and CD8+ T cells when stimulated with low-dose anti-CD3 antibody. To identify the binding partners and mechanism of action of the peptide, Mass Spec and Single-cell RNA-seq was used. Mass spec analysis showed that SG-3-05308, a variant of SG-3-0020 binds to a transmembrane glycoprotein of the immunoglobulin superfamily. Silencing this gene via CRISPR-Cas significantly decreased PD1 levels, cell proliferation and IFNg production in human pan T cells. Single cell RNA-seq data showed that SG-3-0020 activates NF-kB signaling and modulates calcium signaling in T cells. The potency of SG-3-0020 was further optimized for binding to activated T cells by using alanine scanning, saturation, and combinatorial mutation libraries using phage display. The results of protein engineering demonstrated that the 13 amino acids from the C-terminus of SG-3-0020 were not critical for binding and a 29-aa long (lacking the C-term) peptide stimulated increased IFNg production in human T cells across multiple human donors in a dose dependent manner. The peptide with the highest potency SG-3-05429 was selected for further understanding of how it interacts with the identified glycoprotein target and activates downstream T cell signaling pathways . Collectively, these data suggest that SG-3-0020’s ability to up-regulate key co-stimulatory and checkpoint molecules on T cells provides a strong rationale for its potential future use in combination with IO. These results validate the capability of the Second Genome drug discovery platform to identify novel microbial peptides/proteins of potential therapeutic relevance in IO and demonstrate a unique approach that can identify microbial derived bioactive molecules involved in modulating immune cell effector functions and/or immune cell differentiation. Citation Format: Michelle Lin, Archana S. Nagaraja, Dhwani Haria, Yuliya Katlinskaya, Divya Ravichandar, Preston Williams, Roberta Hannibal, Todd Z. DeSantis, Bum-Yeol Hwang, Michi Wilcoxon, Toshi Takeuchi, Karim Dabbagh, Preeti Lal, Helena Kiefel. Development and validation of a novel T-cell modulating, microbiome-based peptide for combination with immunotherapy [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P260.