Abstract 1723: Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy

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Abstract Background: Activation of the innate immune system and natural killer (NK) cells has been a key effort in cancer immunotherapy research. Several NK-cell-targeted therapeutics are under clinical investigation. Current research is focused on bispecific antibodies that can bind to and target both tumor cells and NK cells. However, NK activation remains a key challenge. One approach is to simultaneously co-activate more than one NK cell stimulatory molecule, such as 4-1BB and CD16 (FcγRIII), to increase immune activation. To achieve this, we developed a new nanoparticle (NP)-based trispecific NK cell engager (nano-TriNKE) platform that can target epidermal growth factor receptor (EGFR) overexpressing tumors and promote the recruitment and activation of NK cells to eradicate these cancer cells. Moreover, the nanoengager platform allows the targeted delivery of chemotherapeutics such as epirubicin (EPI) to further improve the therapeutic efficacy. Methods: EGFR-targeted drug-free/EPI-encapsulated nano-TriNKEs were prepared by conjugating cetuximab (anti-EGFR), anti-4-BB, and anti-CD16 agonistic antibodies to PEG-PLGA NPs. Their binding specificities were evaluated in expanded murine NK cells, EGFR-overexpressed A431 (epidermoid carcinoma), MB468 (mammary gland adenocarcinoma), and HT29 (colorectal adenocarcinoma) cells in vitro. The in vitro NK cell anticancer activities in nano-TriNKE-pretreated A431, MB468, and HT29 cells were evaluated via viability assays. We also performed comprehensive in vivo efficacy studies in A431, MB468, and HT29 tumor model to investigate the anticancer activities of EGFR-targeted drug-free/EPI-encapsulated nano-TriNKEs. Further biodistribution and mechanistic studies were performed to verify the anticancer activities of nano-TriNKEs. Results: In vitro binding assay confirmed that the EGFR-targeted nano-TriNKEs bind selectively to EGFR-overexpressed cancer cells. In vitro anticancer activity studies demonstrated that effective NK cell activation can be achieved by spatiotemporal co-activation of CD16 and 4-1BB stimulatory molecules on NK cells with nanoengagers, and that the nanoengagers are more effective than free antibodies. Comprehensive in vivo efficacy studies in A431, MB468, and HT29 tumor models demonstrated that the EGFR-targeted nano-TriNKEs can augment both NK activating agents and chemotherapy as highly effective anticancer agents, providing robust chemoimmunotherapy against a broad range of EGFR-overexpressed cancers. Conclusions: Our data demonstrated that nano-TriNKEs are more effective in NK activation than free agonistic antibodies, and that the nanoengager platform provides more effective biological targeting, which is critical in NK-mediated cancer immunotherapy. Citation Format: Kin Man Au, Steven Park, Andrew Wang. Trispecific natural killer cell nanoengagers for targeted chemoimmunotherapy [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 1723.