Abstract PO044: RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses
Authors: Luo Mengyao , Dastagir Shamael S. , Zhang Xuqing , Schmidt Andrea , Marques Beatriz , Lyford Timothy J. , Blanco Billy , Turka Laurence A. , Wickham Thomas J. , Chen Tiffany F. 
Published: February 2021

Abstract
The high-risk human papillomavirus (HPV) strain 16 accounts for ~ 70% of all cervical cancers and 80% of head and neck cancers associated with HPV infection., Despite available therapies, there remains a critical need for new treatment options for advanced HPV 16-associated cancers. To address this, we have genetically engineered red blood cells to create an allogeneic artificial antigen presenting cell (APC), RTX-321, that expresses an HPV E7 peptide bound to MHC I (HLA-A*02:01), 4-1BBL and IL-12 on the cell surface to mimic the biology of T cell APC interactions. RTX-321 is designed to enhance both the quantity and quality of endogenous tumor-specific T cells. In this study, we evaluated the mechanisms through which RTX-321 promotes anti-tumor immune responses, particularly its ability to engage and activate HPV 16-specific CD8+ T cells in the context of mixed immune populations in peripheral blood mononuclear cells (PBMCs) and to modulate other key immune cell types in PBMCs. Primary CD8+ T cells were engineered to express the HPV E7 TCR, resulting in HPV E7-specific CD8+ T cells (E7 TCR-T). To determine whether RTX-321’s immune modulatory activities are dependent on the presence of antigen-specific T cells, we examined the effect of RTX-321 on PMBCs admixed with E7 TCR-T as compared to PBMCs alone. RTX-321 selectively expanded HPV E7-specific CD8+ T cells from the PBMC mixture in a dose-dependent manner. RTX-321 preferentially upregulated activation markers and effector molecules, and promoted effector memory on HPV E7-specific CD8+ T cells, compared to HPV independent CD8+ T cells. In addition to these HPV antigen-specific responses, modulations on other immune cells were observed with RTX-321 treatment. RTX-321 induced activation marker upregulation on general CD8+ T cells, as well as NK cell expansion, activation and effector molecule upregulation, independent of the presence of E7 TCR-T. In PBMCs, RTX-321 increased proinflammatory cytokine and chemokine secretion, including IFNγ, TNFα and CXCL10. The addition of E7 TCR-T cells further increased the production of IFNγ and TNFα in PBMCs, suggesting its potential role in further promoting the immune response by secreting effector molecules. Overall, our data indicate that RTX-321 not only engages HPV 16 antigen-specific CD8+ T cells, but also other key immune cell populations of the adaptive and innate immune systems to promote a broad and robust anti-tumor response. An Investigational New Drug application for RTX-321 for the treatment of patients with HPV 16-positive solid tumors is planned by the end of 2020.
Citation Format: Mengyao Luo, Shamael S. Dastagir, Xuqing Zhang, Andrea Schmidt, Beatriz Marques, Timothy J. Lyford, Billy Blanco, Laurence A. Turka, Thomas J. Wickham, Tiffany F. Chen. RTX-321, an allogeneic red blood cell-based artificial antigen presenting cell, expressing MHC I-peptide, 4-1BBL and IL-12, engages primary human HPV-specific T cells and boosts other general immune responses [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO044.