PEGylated and CD47-conjugated nanoellipsoidal artificial antigen-presenting cells minimize phagocytosis and augment anti-tumor T-cell responses

Purpose: Antigen-presenting cells (APCs) are powerful tools to expand antigen-specific T cells ex vivo and in vivo for tumor immunotherapy, but suffer from time-consuming generation and biosafety concerns raised by live cells. Alternatively, the cell-free artificial antigen-presenting cells (aAPCs) have been rapidly developed. Nanoscale aAPCs are recently proposed owing to their superior biodistribution and reduced embolism than conventional cell-sized aAPCs, but pose the challenges: easier cellular uptake and smaller contact surface area with T cells than the cell-sized counterparts. This study aimed to fabricate a new "stealth" nano-aAPCs with microscale contact surface area to minimize cellular uptake and activate antigen-specific T cells by combination uses of ellipsoidal stretch, PEGylation, and self-marker CD47-Fc conjugation. Methods: The spherical polylactic-co-glycolic acid nanoparticles were fabricated using a double-emulsion method, and then stretched twofold using film-stretching procedure followed by PEGylation and co-coupling with CD47-Fc, H-2K(b)/TRP2(180-188)-Ig dimers, and anti-CD28. The resulting PEGylated and CD47-conjugated nanoellipsoidal aAPCs (EaAPC(PEG/CD47)) were co-cultured with macrophages or spleen lymphocytes and also infused into melanoma-bearing mice. The in vitro and in vivo effects were evaluated and compared with the nanospherical aAPCs (SaAPC), nanoellipsoidal aAPCs (EaAPC), or PEGylated nanoellipsoidal aAPC (EaAPC(PEG)). Results: EaAPC(PEG/CD47) markedly reduced cellular uptake in vitro and in vivo, as compared with EaAPC(PEG), EaAPC, SaAPC, and Blank-NPs and expanded naive TRP2(180-188)-specific CD8(+) T cells in the co-cultures with spleen lymphocytes. After three infusions, the EaAPC(PEG/CD47) showed much stronger effects on facilitating TRP2(180-188)-specific CD8(+) T-cell proliferation, local infiltration, and tumor necrosis in the melanoma-bearing mice and on inhibiting tumor growth than the control aAPCs. Conclusion: The superimposed or synergistic effects of ellipsoidal stretch, PEGylation, and CD47-Fc conjugation minimized cellular uptake of nano-aAPCs and enhanced their functionality to expand antigen-specific T cells and inhibit tumor growth, thus suggesting a more valuable strategy to design "stealth" nanoscale aAPCs suitable for tumor active immunotherapy.