Antigen-specific immunotherapy of type 1 diabetes, typically via delivery of a single native β cell antigen, has had little clinical benefit to date. With increasing evidence that diabetogenic T cells react against multiple β cell antigens, including previously unappreciated neo-antigens that can be emulated by mimotopes, a shift from protein- to epitope-based therapy is warranted. To this end, we aimed to achieve efficient co-presentation of multiple major epitopes targeting both CD4+ and CD8+ diabetogenic T cells. We have compared native epitopes versus mimotopes as well as various targeting signals in an effort to optimize recognition by both types of T cells in vitro. Optimal engagement of all T cells was achieved with segregation of CD8 and CD4 epitopes, the latter containing mimotopes and driven by endosome-targeting signals, after delivery into either dendritic or stromal cells. The CD4+ T cell responses elicited by the endogenously delivered epitopes were comparable with high concentrations of soluble peptide and included functional regulatory T cells. This work has important implications for the improvement of antigen-specific therapies using an epitope-based approach to restore tolerance in type 1 diabetes and in a variety of other diseases requiring concomitant targeting of CD4+ and CD8+ T cells.
Checkpoint inhibitors and T-cell therapies have highlighted the critical role of T cells in anti-cancer immunity. However, limitations associated with these treatments drive the need for alternative approaches. Here, we engineer red blood cells into artificial antigen-presenting cells (aAPCs) presenting a peptide bound to the major histocompatibility complex I, the costimulatory ligand 4-1BBL, and interleukin (IL)-12. This leads to robust, antigen-specific T-cell expansion, memory formation, additional immune activation, tumor control, and antigen spreading in tumor models in vivo. The presence of 4-1BBL and IL-12 induces minimal toxicities due to restriction to the vasculature and spleen. The allogeneic aAPC, RTX-321, comprised of human leukocyte antigen-A*02:01 presenting the human papilloma virus (HPV) peptide HPV16 E711-19, 4-1BBL, and IL-12 on the surface, activates HPV-specific T cells and promotes effector function in vitro. Thus, RTX-321 is a potential ‘off-the-shelf’ in vivo cellular immunotherapy for treating HPV + cancers, including cervical and head/neck cancers.
T cell-based therapies have demonstrated efficacy in a small subset of cancers; however, they have the potential to proliferate uncontrollably and manufacturing these therapies at scale has proven difficult. To address this limitation, Rubius Therapeutics has genetically engineered red cells to create allogeneic artificial antigen presenting cells (RCT-aAPCs) that express MHC class I loaded with a tumor specific antigen, together with costimulatory molecules that recapitulate normal APC-T cell interactions. These RCT-aAPC cells are designed to expand and activate tumor-specific T cells already present within the patient, thus eliminating the need to individually manufacture patient-derived T cells. As a proof of principle, red cells were engineered to express mouse MHC class I H-2Kb loaded with OVA 257-264 peptide and murine 4-1BBL. These cells induced in vitroT cell proliferation of OVA antigen-specific OT1 cells, whereas red cells expressing only MHC I or 4-1BBL did not induce proliferation. The RCT-aAPC expanded OT1 cells demonstrated an activated phenotype with increased CD44 expression, secretion of both IFNγ and IL2, as well as antigen-specific tumor killing of EG7.OVA tumor cells. To test in vivo efficacy, a mouse surrogate RCT-aAPC was created using murine red blood cells chemically conjugated with H-2Kb OVA and the m4-1BBL molecule. CellTrace Violet (CTV)-labeled OT1 cells were adoptively transferred into B6 Cd45.1 mice followed by intravenous dosing of the RCT-aAPC several hours later. Significant OT1 proliferation was observed 3-4 days post-dosing as measured by CTV dilution. Administration of a second RCT-aAPC dose at this time drove >200-fold expansion of OT1 cells with a memory-like phenotype in the peripheral blood and secondary lymphoid organs. Using a similar dosing strategy, administration of RCT-aAPC to mice bearing EG7.OVA tumors caused 60% tumor growth inhibition by Day 7 after dosing, which corresponded with the increased expansion of the OT1s. Treatment with RCT-aAPC significantly prolonged survival compared to the control group (p-val = 0.0024). After interacting with RCT-aAPC, antigen-specific T cells, traffic to the lymph nodes and tumor as demonstrated by OT1 presence at these sites. Based on the proof of concept using a murine system, human RCT-aAPCs expressing [human] 4-1BBL and [human] HLA-A2 loaded with an HPV E7 peptide were developed to expand and activate HPV E7-specific T cells. These RCT-aAPC cells activated TCR signaling in primary HPV E7-specific T cells as measured by upregulation of Nur77 expression and in engineered HPV E7-specific TCR Jurkat lines, measured using an NFAT luciferase reporter assay. Further validation of RCT-aAPC is ongoing and will be the focus for future clinical development in patients with HPV-positive cancers. Citation Format: Xuqing Zhang, Shamael R. Dastagir, Naren Subbiah, Mengyao Luo, Vikram Soman, Sneha Pawar, Douglas C. McLaughlin, Nicholas Bayhi, Viral Amin, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Tiffany F. Chen. Engineered red-cell therapeutics (RCT) as artificial antigen presenting cells promotein vivoexpansion and anti-tumor activity of antigen specific T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3260.
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
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