Abstract B062: Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system

Abstract

Next generation sequencing technologies, coupled with personal neoantigen identification approaches, have significantly improved the capability to develop patient-specific T-cell-based therapies targeting tumors. Current peptide neoantigen vaccine approaches are promising, but do not adequately stimulate and expand patient T cells to the levels required to achieve robust efficacy. To address these limitations, Rubius Therapeutics has developed allogeneic artificial antigen presenting cells (aAPCs), which express the required signals for complete T cell activation: antigenic peptide-MHC complex, costimulatory ligand and cytokine. By engineering red cells to express immunomodulatory signals 1, 2 and 3, these aAPCs dramatically expand antigen-specific T cells in vivo and promote T cell memory and effector function. To use the aAPC approach with personal neoantigens, Rubius Therapeutics has developed a loadable MHC system that enables the rapid generation of aAPCs. Unloaded MHC class I molecules typically do not express robustly on the cell surface, as innate biophysical instability and internal cellular quality control mechanisms prevent MHC molecules that lack a loaded peptide from displaying on the cell surface. We demonstrate here that the empty MHC class I complex can be stably presented on the red cell surface. This was achieved by fusing wild-type MHC class I [human] HLA-A2 and β2 microglobulin to the glycophorin A transmembrane domain. Of note, disulfide engineering of HLA-A2 did not change expression when compared to wild-type constructs on the red cells. Fluorescently labelled peptides were used to measure the kinetics of peptide loading, which demonstrated that disulfide-engineering dramatically increased binding rates. Further, peptide competition experiments indicated slightly increased affinity for peptide with disulfide engineered HLA-A2. Functional testing revealed that addition of exogenous HPV E7 peptide and co-incubation of loaded cells with Jurkat-Lucia NFAT cells expressing HPV E7-specific TCR cells demonstrated TCR-specific activity. Additional TCR activity assays showed that peptide loaded onto empty wild-type HLA-A2 was stable up to 3 days, while disulfide-engineered HLA-A2 activity was abrogated after 1 day. Finally, this concept was extended to other HLA genes, demonstrating that our approach could achieve expression of a variety of HLA alleles on the red cell surface, including MHC class II alleles in the HLA-DR and HLA-DP gene families. Collectively, these results demonstrate that Rubius’ loadable aAPC system is highly generalizable and can be applied to produce aAPC populations presenting multiple antigenic peptides across a range of functional alleles on the red cell surface. Rubius’ allogeneic aAPC system represents a novel approach to generate effective personal neoantigen-specific therapies in a wide patient population with requisite ease of manufacturability Citation Format: Christopher L Moore, Sneha Pawar, Mellissa Nixon, Timothy J Lyford, Douglas C McLaughlin, Shamael R Dastagir, Abigail Bracha, Lori Melancon, Christopher L Carpenter, Thomas J Wickham, Tiffany F Chen. Enabling the rapid generation of allogeneic artificial antigen presenting cell (aAPC) Red Cell Therapeutics with a loadable MHC system [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B062. doi:10.1158/1535-7163.TARG-19-B062

Erythrocytes as Carriers: From Drug Delivery to Biosensors

“…Currently, the first phase of clinical trials of RTX-134, erythrocytes carrying the AvPAL gene inside cells (the phenylalanine-ammonia lyase gene Anabaena variabilis), is being conducted to treat adult phenylketonuria (NCT04110496) [239]. At conferences in Philadelphia and Boston in 2019, Zhang [240] and Moore [241] proposed interesting ideas for creating artificial antigen-presenting cells, the genetically modified erythrocytes (RCT-aAPC), which expresses immunomodulating signals that are directed against the tumor. Such cells, on the one hand, are loaded with tumor-specific antigen and costimulatory molecules, and, on the other hand, express proteins of the main histocompatibility class I complex on the surface to create an effective tumor-specific T-cell response.…”