Fig 2: Physicochemical properties of EGFR-targeted nano-TriNKE.(A) Representative FACS histograms of CD3- CD49b+ expanded murine NK cells (i) and expanded NK cells after incubation with FITC-labeled (ii) a-CD16 NPs, (iii) a-4-1BB NPs, (iv) a-EGFR NPs, (v) a-CD16/a-4-1BB NPs, and (vi) a-EGFR/a-CD16/a-4-1BB NPs. (B) Representative CLSM images of CD3- CD49b+ expanded murine NK cells after incubation with FITC-labeled (i) a-CD16 NPs, (ii) a-4-1BB NPs, (iii) a-EGFR NPs, (iv) a-CD16/a-4-1BB NPs, and (v) a-EGFR/a-CD16/a-4-1BB NPs. (C) Representative FACS histograms of EGFR-overexpressed HT29, MB468, and A431 cells after incubation with FITC-labeled a-EGFR NPs, a-CD16/a-4-1BB NPs, and a-EGFR/a-CD16/a-4-1BB NPs (n = 3). a.u., arbitrary unit; MFI, median fluorescence intensity. (D) Representative CLSM images of EGFR-overexpressed HT29, MB468, and A431 cells after incubation with FITC-labeled a-EGFR NPs, a-CD16/a-4-1BB NPs, and a-EGFR/a-CD16/a-4-1BB NPs (n = 3). (E) Direct in vitro toxicities of free EPI, nontargeted EPI NPs, and different antibody-functionalized EPI NPs against (i) HT29, (ii) MB468, and (iii) A431 cells, as assessed by MTS assay 3 days after initial treatment. (F) Representative CLSM images of a-?-H2AX–stained A431 cells after being treated with different EPI formulations for 18 hours.

Fig 3: Spatiotemporal coactivation of CD16 and 4-1BB costimulatory molecules on NK cells delays murine tumor growth in vivo.(A) Experimental scheme for B16F10 model in immunocompetent C57BL/6 mice. In the immune cell–depleted C57BL/6 mouse model, B cells, NK cells, CD4+ T cells, and CD8+ T cells were depleted by intraperitoneal injection of a-CD20, a-NK1.1, a-CD4, and a-CD8 (300 µg per injection) at 5, 8, 10, 12, 15, and 18 days after inoculation. Mouse immunoglobulin G2a (IgG2a) (300 µg per injection) was administered as an isotype control. a-CD16/a-4-1BB NPs (containing 100 µg of each antibody) were intravenously administered at 6, 7, and 8 days after inoculation. Immunotherapeutics containing 100 µg of a-CD16 and/or 100 µg of a-4-1BB (free or NP conjugated) were intravenously administered via the tail vein at 6, 7, and 8 days after inoculation. The xenograft tumors of mice in the immunostimulation groups were subjected to a single 5-Gy irradiation 4 hours before the administration of immunotherapeutics to up-regulate the NK cell–activating ligands in the cancer cells. (B) Average tumor growth curves [(i) and (ii)] and survival curves [(iii) and (iv)] of B16F10 tumor-bearing mice after receiving treatments with different immunotherapeutics (n = 6 mice per group). (C) Average tumor growth curves (i) and survival curves (ii) of B16F10 tumor-bearing immune cell–depleted mice after receiving treatments with a-CD16/a-4-1BB NPs (n = 7 mice per group). Data are presented as means ± SEM [B(i), B(ii)]. *P < 0.05. Statistical significances [B(i), B(ii), and C(i)] were calculated via one-way analysis of variance (ANOVA) with Tukey post hoc test. *P < 0.05. Statistical significances [B(iii), B(iv), and C(ii)] were calculated via log-rank (Mantel-Cox) test. *P < 0.05.

Fig 4: Mechanism of action and characterization of EGFR-targeted nano-TriNKEs.(A) The cartoon illustrates the mechanism of action of the EGFR-targeted nanoparticle-based trispecific NK cell engagers (nano-TriNKEs) (a-EGFR/a-CD16/a-4-1BB NPs) against EGFR-overexpressed cancer after systemic administration. (B) Representative transmission electron microscopy (TEM) images of EGFR-targeted drug-free and EPI-encapsulated nano-TriNKEs. (C) Representative number-average particle (DN) distribution curves of EGFR-targeted drug-free and EPI-encapsulated nano-TriNKEs (a-EGFR/a-CD16/a-4-1BB NPs), as determined by the NP tracking analysis (NTA) method on NP dispersion in PBS (1×, pH 7.4). The mean DN determined by the NTA method is larger than that determined by TEM because the PEG shell collapses to negligible thickness under ultrahigh vacuum conditions. (D) pH-dependent in vitro drug release kinetics of antibody-free EPI NPs and a-EGFR/a-CD16/a-4-1BB EPI NPs (n = 3). Averaged time-dependent UV-visible absorption spectra of 1 mg/ml of nonfunctionalized EPI NPs and a-EGFR/a-CD16/a-4-1BB EPI NPs determined at (i) pH 7.0 and (ii) pH 6.0. The encapsulated EPI retained in the NPs was quantified spectroscopically at 490 nm. (iii) EPI drug release profile of nonfunctionalized EPI NPs and a-EGFR/a-CD16/a-4-1BB EPI NPs at pH 6.0 and pH 7.0.

Fig 5: EGFR-targeted nano-TriNKEs activate NK cells to attack cancer cells in vitro.(A) In vitro cytotoxicities of NK cells pretreated with a-CD16, a-4-1BB, a-CD16 NPs, a-4-1BB NPs, and their 1:1 combinations, and a-CD16/a-4-1BB NPs. The effector cells–to–target cells (E/T) ratio was 1:1. The cytotoxicities were determined 24 hours after treatment. Data are presented as means ± SEM (n = 6). n.s., non-significant. (B) Representative phase-sensitive optical images of nonirradiated and 5 Gy irradiated B16F10 cells after incubation with NK cells pretreated with a-CD16 and a-4-1BB, a-CD16 NPs, a-4-1BB NPs, and a-CD16/a-4-1BB NPs. The E/T ratio was 1:1. Unbound NK cells were removed by washing before imaging. (C) In vitro cytotoxicities of NK cells against HT29-Luc2 cells. The cytotoxicities were quantified 24 hours after the treatment. The E/T ratio was 1:1. Data are presented as means ± SEM (n = 6). (D) Viabilities of HT29, MB468, and A431 cells recorded 3 days after being treated with drug-free or EPI-encapsulated a-EGFR/a-CD16/a-4-1BB NPs (containing 600 nM encapsulated EPI or the same amount of drug-free NPs) in the presence or absence of NK cells (at 1:1 E/T ratio). Data are presented as means ± SEM (n = 8). (E) Representative phase-sensitive optical images of a-CD16/a-4-1BB NPs plus a-EGFR NP– or a-EGFR/a-CD16/a-4-1BB NP–pretreated A431, MB468, and HT29 cells after a brief (10 min) incubation with NK cells. Unbound NK cells were removed by three washes.