Fig 1: Mechanistic insight: EGFR-targeted nano-TriNKEs improve chemoimmunotherapy by recruiting NK cells to the tumor and increasing dsDNA breaks.(A) Biodistribution of Cy5-labeled and EPI-encapsulated nanoengagers in an A431 tumor model in Nu mice recorded 40 hours after intravenous tail vein administration of different immunotherapeutics/chemoimmunotherapeutics (n = 5 for all control and experimental groups, except n = 6 for the groups administered with Cy5-labeled α-EGFR/α-CD16/α-4-1BB NPs, α-EGFR/α-CD16/α-4-1BB EPI NPs, and α-EGFR/α-CD16/α-4-1BB NPs plus free EPI, and n = 4 for the group administered with α-EGFR EPI NPs). Data represent the means ± SEM. Statistical significances were calculated via two-way ANOVA with a Tukey post hoc test. *P < 0.05. (B) Representative ex vivo fluorescent images of A431 tumor preserved after different treatments. (C) Representative immunofluorescence images of α-NK1.1– and α-EGFR–costained A431 tumor sections preserved 40 hours after treatment. The immunofluorescence images show the colocalization of NK cells (red) with the EGFR-positive cancer cells in tumors treated with the EGFR-targeted nanoengagers. Dox, doxorubicin. (D) Representative immunofluorescence images of α-γ-H2AX–stained A431 tumor sections preserved 40 hours after treatment. (E) Serum TNF-α and INF-γ levels recorded for A431 tumor–bearing Nu mice 40 hours after intravenous administration of different immunotherapeutics/chemoimmunotherapeutics.
Fig 2: Characterization of the EGFR-targeted 4-1BB-agonistic trimerbody. a Arrangement of 1D8N/CEGa1 trimerbody in solution by SAXS. Rigid-body fitting of the model corresponding to 1D8NCEGa1 inside the SAXS envelope (colored in pale gray). Each chain has been colored in blue, magenta, or cyan. b Sensorgrams (black curves) and the results of fitting to a 1:1 model (red curves) obtained using biolayer interferometry for the interaction of 1D8N/CEGa1 (2 and 4 nM) with immobilized m4-1BB and the interaction of 1D8N/CEGa1 (0.5 and 1 nM) with immobilized hEGFR. c Simultaneous binding to both m4-1BB and hEGFR was demonstrated for 1D8N/CEGa1 but not 1D8N18. Biosensors were coated with m4-1BB, after which 4 nM of 1D8N/CEGa1 (black curves) or 1D8N18 (blue curves) were added. d The binding of anti-4-1BB antibodies to m4-1BB on the cell surface of stimulated mouse CD8a+ T cells measured by FACS. e, –f Mouse CD8a+ T cells were plated with immobilized anti-CD3 mAb and hEGFR or BSA in the presence of 1D8N18, 1D8N/CEGa1, or 1D8 IgG, and proliferation (e) and IFN-γ secretion (f) were determined after 48 h. EGFR-negative 3T3 cells or EGFR-positive 3T3hEGFR cells were co-cultured with mouse CD8a+ T cells in the presence of anti-CD3 mAb and 1D8N18, 1D8N/CEGa1, or 1D8 IgG. IFN-γ secretion analyzed after 48 h (g) and cell viability after 72 h (h). Data are represented as fold change relative to the values obtained from anti-CD3 mAb stimulated cells. Rat IgG2a and MFE-23N18 were used as negative controls. Data are mean ± SD (n = 3), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, Student's t test
Fig 3: Pharmacokinetic properties and tumor imaging of the EGFR-targeted 4-1BB-agonistic trimerbody. Pharmacokinetic studies after a single i.v. dose (1 mg/Kg) of 1D8N18 or 1D8N/CEGa1 in CD-1mice (a) or of 3H3 IgG or 1D8N/CEGa1 in BALB/c mice (b). Pharmacokinetic study after a single i.p. dose (1 mg/Kg) of 3H3 IgG or 1D8N/CEGa1 in BALB/c mice (c). d In vivo fluorescence imaging of A431 tumor-bearing nude mice 24 h after i.v. injection of PBS or 100 µg of Cy5-labeled cetuximab, CF647-labeled 3H3 IgG or CF647-labeled 1D8N/CEGa1. e Tumor to normal tissue (T/N) ratios. The color scale bar represents the fluorescence intensity recorded as photons per second per centimeter squared per steradian (p/s/cm2/sr). Data are mean ± SD (n = 3), **P ≤ 0.01, ***P ≤ 0.001, Student's t test
Fig 4: 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) α-CD16 NPs, (iii) α-4-1BB NPs, (iv) α-EGFR NPs, (v) α-CD16/α-4-1BB NPs, and (vi) α-EGFR/α-CD16/α-4-1BB NPs. (B) Representative CLSM images of CD3− CD49b+ expanded murine NK cells after incubation with FITC-labeled (i) α-CD16 NPs, (ii) α-4-1BB NPs, (iii) α-EGFR NPs, (iv) α-CD16/α-4-1BB NPs, and (v) α-EGFR/α-CD16/α-4-1BB NPs. (C) Representative FACS histograms of EGFR-overexpressed HT29, MB468, and A431 cells after incubation with FITC-labeled α-EGFR NPs, α-CD16/α-4-1BB NPs, and α-EGFR/α-CD16/α-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 α-EGFR NPs, α-CD16/α-4-1BB NPs, and α-EGFR/α-CD16/α-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 α-γ-H2AX–stained A431 cells after being treated with different EPI formulations for 18 hours.
Fig 5: Characterization of anti-4-1BB trimerbodies. a Sensorgrams (black curves) and fitting curves for 1D8 antibodies (2 and 4 nM), obtained by biolayer interferometry with surface-immobilized m4-1BB. b The binding to 4-1BB on the cell surface of stimulated mouse CD8a+ T cells measured by FACS. c–e Costimulatory activity of anti-4-1BB antibodies. Mouse CD8a+ T cells were stimulated with immobilized anti-CD3 mAb in the presence of m4-1BBL, 1D8N5, 1D8N18, or 1D8 IgG, and proliferation (c) and secretion of IFN-γ (d) were measured after 48 h and cell viability (e) after 72 h. Data are reported as fold change relative to the values obtained from anti-CD3 mAb-stimulated CD8a+ T cells. Rat IgG2a and MFE-23N18 were used as controls. Data are mean ± SD (n = 3), *P ≤ 0.05, **P ≤ 0.01, Student's t test. f RICS analyses performed in living HEK293m4-1BB-S cells at regions containing clusters formed upon 1D8 IgG or 1D8N18 addition and at regions where clusters where not present (insert and zoomed-in regions ii, and i, respectively). Representative maximum intensity projection maps showing the RICS analyzed regions of interest. Values in the zoomed-in regions show the diffusion coefficient of bound antibody. The color heat map indicates in blueish tones the lower intensity and in redder tones the higher intensity. g Statistical analysis of the quantified diffusion coefficient obtained from 5 to 7 independent live cell experiments and 3–5 different regions of interest per cell (N-CR non-clustered region, CR clustered region). Data are presented as median (center line), upper and lower quartiles (boxes), and minimum-maximum (whiskers). h Arrangement of 1D8N18 trimerbody in solution, as determined by SAXS. Rigid-body overlaying of the ab initio-determined SAXS envelope for 1D8N18. The generated model (where each chain is colored in blue, magenta, or cyan) fits into the envelope (colored in pale gray)
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