Abstract 616: The novel α-Gal-based immunotherapy AGI-134 invokes CD8+ T cell-mediated immunity by driving tumor cell destruction, phagocytosis and tumor-associated antigen cross-presentation via multiple antibody-mediated effector functions

Background: AGI-134 is a fully synthetic α-Gal (Galα1-3Galβ1-4GlcNAc-R) glycolipid that is being developed for the treatment of solid tumors. The α-Gal epitope is not expressed in humans, who, as a result of constant antigenic stimulation by α-Gal-bearing commensal bacteria, develop high titer natural antibodies to α-Gal. We have previously demonstrated that AGI-134 recruits anti-Gal antibodies to tumor cells in vitro, activating complement and driving phagocytosis by antigen presenting cells. AGI-134 also confers systemic protection from distal lesion development in a mouse model of melanoma and synergizes with anti-PD-1 1 . Here we present further data characterizing the multiple pathways activated by the anti-Gal subclasses to drive AGI-134-mediated anti-tumor immunity. Results: Using quantitative methods, we demonstrate that human anti-Gal is composed of a diverse repertoire of effector antibodies in a panel of serum samples, with IgM, IgG1 and IgG2 being the major subclasses. Polyclonal anti-Gal IgG purified from human IVIG was, like human serum, comprised mainly of IgG1 and IgG2. When AGI-134 treated cells were incubated with human serum, binding of all anti-Gal subclasses was detected by flow cytometry, demonstrating that all anti-Gal subclasses can interact with AGI-134 treated cells and are available to activate downstream effector functions. When human serum was depleted of IgG, AGI-134 stimulated complement dependent cytotoxicity (CDC) was still effective, indicating that anti-Gal IgM is primarily responsible for deposition of complement on AGI-134 treated cells. AGI-134 treated cells incubated with purified polyclonal anti-Gal IgG activated FcγRIIIA on a reporter cell line and promoted natural killer cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC). Opsonization of AGI-134 treated human cancer cells with human anti-Gal and complement stimulated their phagocytosis by human monocyte-derived macrophages. Ovalbumin-expressing cells treated with AGI-134 and then incubated with human serum to initiate CDC were specifically phagocytosed by murine CD8α+ dendritic cells and the immunodominant antigen of ovalbumin, SIINFEKL, was cross-presented to CD8+ T cells. In conclusion, AGI-134 stimulates adaptive anti-tumor immunity through immune activation and antigen cross-presentation, which is driven by the diverse repertoire of anti-Gal antibodies. 1. Shaw, S. et al. Abstract 4862: AGI-134: a fully synthetic alpha-Gal glycolipid that prevents the development of distal lesions and is synergistic with an anti-PD-1 antibody in a mouse melanoma model. [abstract]. AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4862. Citation Format: Jenny L. Middleton, Oliver Schulz, Amber Charlemagne, Sascha A. Kristian, Stephen Michael Shaw. The novel α-Gal-based immunotherapy AGI-134 invokes CD8+ T cell-mediated immunity by driving tumor cell destruction, phagocytosis and tumor-associated antigen cross-presentation via multiple antibody-mediated effector functions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 616. doi:10.1158/1538-7445.AM2017-616