Abstract LB-082: In vivo efficacy and pharmacodynamic analysis of RTX-321, an engineered allogeneic artificial antigen presenting red cell therapeutic
Authors: Mellissa Joy Nixon, Xuqing Zhang, Albert Lee, Mengyao Luo, Annie Khamhoung, Andrea Schmidt, Douglas McLaughlin, Shamael R. Dastagir, Viral Amin, Chris Moore, Jennifer Mellen, Chris Carpenter, Tom Wickham, Tiffany Chen
Journal: Cancer Research (2020)

Abstract Human papilloma virus (HPV)-infection is strongly associated with the risk of cancer, with the high-risk strain HPV16 accounting for ~80% of head and neck cancers. HPV16+ malignancies remain a critical area of high unmet need for new and innovative treatment options. To address this, Rubius Therapeutics has genetically engineered red cells to create an allogeneic artificial antigen-presenting cell (RCT-aAPC) for the potential treatment of HPV16+ cancers. RTX-321 is engineered to express an HPV16 antigen bound to MHC I, 4-1BBL and IL-12 on the cell surface to mimic T cell-APC interactions. RTX-321 is designed to activate and expand tumor-specific T cells present within the patient, bypassing the dependence on host APCs typically required for cancer vaccines and eliminating the need for manufacturing patient-derived T cells as with CAR-T therapies. Previously, a surrogate murine red cell (mRBC-321) with surface conjugated murine MHC I-OVA, 4-1BBL and IL-12 demonstrated in vivo tumor killing in an antigen-specific tumor model (EG7.OVA) with adoptive cell transfer of naïve OT1 cells. We now demonstrate through confocal live cell imaging a direct and tight interaction between mRBC-321 and activated OT1 cells. In addition, mRBC-321 preferentially accumulates in the spleen, directly interacting with OT1 cells, which induces activation and eventual expansion of OT1 cells in the spleen. TCR-sequencing in this tumor model reveals increased TCR clonality (p=0.02) in tumors treated with mRBC-321, accompanied by tumor growth inhibition (TGI) compared to controls, indicating an antigen-specific tumor response. Ongoing analysis will examine TCR diversity in the blood during tumor re-challenge (EG7.OVA or parental EL4) in cured mice to determine the degree of immunological memory and epitope spreading induced by mRBC-321. Follow-up efficacy studies were conducted without OT1 transfer to evaluate endogenous antigen-specific T cells in mediating TGI. Mice treated with mRBC-321 showed significant TGI (p&amp;lt;0.001) and increased overall survival (p&amp;lt;0.001) compared to controls. Immunophenotyping by flow cytometry revealed key mRBC-321 mediated pharmacodynamic changes including increased activation/effector markers PD-1, KLRG-1 and effector memory phenotypes on CD8+ T cells (p&amp;lt;0.05). Additionally, PBMCs from CMV+ donors were used to determine relevant antigen-specific pharmacodynamic readouts. Co-culture of these PBMCs and RCT-aAPC (MHC-I-CMV+4-1BBL+IL12) induced CD137 shedding, IFNγ secretion and expansion of antigen specific T cells with increased CD25, 4-1BB and HLA-DR expression compared to control (p&amp;lt;0.05). Together, these data confirm that RCT-aAPC can induce activation of antigen-specific CD8 T cells which can translate to tumor regression. Rubius plans to file an Investigational New Drug application for RTX-321 in HPV16+ advanced solid tumors in 2020. Citation Format: Mellissa Joy Nixon, Xuqing Zhang, Albert Lee, Mengyao Luo, Annie Khamhoung, Andrea Schmidt, Douglas McLaughlin, Shamael R. Dastagir, Viral Amin, Chris Moore, Jennifer Mellen, Chris Carpenter, Tom Wickham, Tiffany Chen. In vivo efficacy and pharmacodynamic analysis of RTX-321, an engineered allogeneic artificial antigen presenting red cell therapeutic [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-082.