Lymphatic Delivery of Virus-Like Particles in Combination with Mono- or Combinational Checkpoint Blockade Immunotherapy Enhances Tumor Response and Antigen-Specific Functionality of T Cells in Tumor Microenvironment.

Abstract T cell priming against tumor antigens (tAgs) is essential for checkpoint blockade immunotherapy (CBI). When these therapies are delivered systemically (intravenously, i.v), Ag-indiscriminate T cell priming and proliferation can occur, reducing anti-tumor immunity and increasing the potential for immune related adverse events (irAEs). Herein, we use lymphatic delivery (intradermally, i.d) of αCTLA4 combined with virus-like particles (VLPs) to increase tAg functionality of tumor infiltrating lymphocytes (TILs) in mono- and combinational CBI regimens. Beginning at 7 days post implant (p.i) of B16F10 in the right hindlimb, αCTLA4 alone or in combination with αPD1 was administered i.v or i.d at the right base of the tail to reach popliteal or inguinal tumor draining lymph nodes (tdLNs) at intervals of 2 days for three doses. Near-infrared fluorescence imaging confirmed delivery to tdLNs. VLP presenting Melan-A as tAg was administered i.d at days 7 and 14 p.i. Tumor volumes were measured daily and at sacrifice on day 17 p.i. Animals with exponential tumor growth at time of sacrifice were deemed to be non-responders. PBMCs and tumor tissues were collected and analyzed via flow cytometry for CD4+ and CD8+ cells as well as for functionality to tAg as reflected from INFγ and TNFα production. αCTLA4 delivered i.d (N=5) exhibited significantly (p<0.05) reduced growth rates compared to when αCTLA4 was delivered i.v (N=5). VLP administration further depressed growth rates (N=20, p<0.05). Additionally, dosing of αCTLA4 i.d and VLP i.d resulted in 50% more responders than when αCTLA4 was dosed i.v. Dose of αCTLA4, αPD1, and VLP i.d further reduced tumor growth compared to animals receiving VLP i.d and combinational CBI i.v (N=20, p<0.05). Finally, there were no responders in animals receiving αCTLA4 i.v alone (N=5) or in combination with αPD1 i.v and VLP i.d, highlighting the importance of lymphatic delivery on immune priming. We found increased tAg specific CD8+ TNFα+ TILs when αCTLA4 was administered i.d than i.v in both mono- and combinational CBIs (p<0.05). TAg specific CD8+TNFα+ T-cells in PBMCs were also higher in responders than non-responders who received αCTLA4 i.d. In non-responders of αCTLA4 monotherapy, IFNγ production was higher in CD4+ and CD8+ TILs than in responders. Taken together these data support the hypothesis that regional lymphatic delivery of αCTLA4 alone or in combination with αPD1 in tdLNs that are recipient of tAgs enhances anti-tumor immunity. Lymphatic delivery of αCTLA4 and VLP induces robust antitumor activity. Future work involves evaluating how manipulation of tdLNs and non-tdLNs can shape the immune status of the tumor microenvironment. Whether irAEs can be alleviated with lymphatic delivery remains to be evaluated in our pre-clinical models. Supported by CPRIT RP19009. Citation Format: Carolina Mantilla Rojas, Dongliang Liu, Fred Christian Velasquez, Janelle Morton, Wen-Jen Hwu, Qizhi Yao, Eva Sevick-Muraca. Lymphatic delivery of virus-like particles in combination with mono- or combinational checkpoint blockade immunotherapy enhances tumor response and antigen-specific functionality of T cells in tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1688.