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.

Phospholipid-based deformable nanovesicles (DNVs) that have flexibility in shape offer an adaptable and facile method to encapsulate diverse classes of therapeutics and facilitate localized transdermal delivery while minimizing systemic exposure. Here we report the use of a microfluidic reactor for the synthesis of DNVs and show that alteration of input parameters such as flow speeds as well as molar and flow rate ratios increases entrapment efficiency of drugs and allows fine-tuning of DNV size, elasticity, and surface charge. To determine the ability of DNV-encapsulated drug to be delivered transdermally to a local site, we synthesized, characterized, and tested DNVs carrying the fluorescently labeled hydrophilic bisphosphonate drug AF-647 zoledronate (AF647-Zol). AF647-Zol DNVs were lyophilized, resuspended, and applied topically as a paste to the calvarial skin of mice. High-resolution fluorescent imaging and confocal microscopy revealed significant increase of encapsulated payload delivery to the target tissue—cranial bone—by DNVs as compared to nondeformable nanovesicles (NVs) or aqueous drug solutions. Interestingly, NV delivery was not superior to aqueous drug solution. Our studies show that microfluidic reactor-synthesized DNVs can be produced in good yield, with high encapsulation efficiency, reproducibility, and stability after storage, and represent a useful vehicle for localized transdermal drug delivery.

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.

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