Andres Bermudez

Therapeutic allergen vaccine immunogens can trigger IgE-mediated mast cell activation, resulting in allergic reactions. Here, we report on a mode of hypoallergenic immunogen design that enables immunization against IgE-reactive peptide B cell epitopes by optimizing the distance between epitopes. Using DNA-based model immunogens, we show that mast cells and B cells exhibit idiosyncratic sensitivity to interepitope spacing, with mast cell activation being dampened by high interepitope spacing while B cells remain responsive to identical immunogen configurations. To exploit this finding, we construct hypoallergenic immunogens based on supramolecular peptide nanofibers with ultralow epitope density that, when used as an allergen vaccine, raise protective allergen-neutralizing IgG antibody responses. This study provides a proof-of-concept for a mode of hypoallergenic immunogen design based on nanoscale control of the distances between IgE-reactive epitopes, which may enable allergen vaccination against IgE-reactive epitope targets in the absence of allergic reactogenicity.

Supplementary Figure S8. (A and B) Representative experiment and summary results showing the effect of blood and tumor FcR+ effectors on the growth of GFP+A549 tumor cell spheroids in the presence of cetuximab (1ug/ml). Experiments were performed as described in supplemental figure 5 (A and B). Original magnification x10. Scale bar, 400 µm. Data are represented as mean ± SEM. Wilcoxon matched pairs test (n=6). Indicated FcR+ effectors were freshly isolated for all experiments. (C) Flow cytometry histogram showing the expression of EGFR on the surface of H1975 tumor cell line in comparison with A431 and A549 tumor cells. (D and E) Cumulative flow cytometry results showing the different levels of trogocytosis and killing activity mediated by blood and tumor FcR+ effectors co-cultured with PKH67+ H1975 tumor cells at a 50:1 E:T ratio in the presence of cetuximab or isotype control Abs for 12 hrs. All comparisons used one-way ANOVA with Turkey’s multiple comparisons tests. All data represented as mean ± SEM. FcR+ effectors were freshly isolated for all experiments. IC-isotype control Ab.

Supplementary Figure S4. (A) Cumulative results showing the kinetics of GFP+A431 tumor cell growth when co-cultured with PBN isolated from lung cancer patients at 50:1 E:T ratio in the presence of cetuximab (IgG) and IgA anti-EGFR Ab (IgA) using the IncuCyte® Live Cell Analysis System. One-way ANOVA with Turkey’s multiple comparisons test. Number of patients included in each analysis is indicated on the graphs. All data represented as mean ± SEM. (B) Cumulative results showing the kinetics of GFP+A431 tumor cell growth when co-cultured with blood and tumor FcR+ effectors for 48 hrs at 50:1 E:T ratio in the presence of IgA2 isotype control Abs in the IncuCyte® Live Cell Analysis System. One-way ANOVA with Dunnett's multiple comparisons test. All data represented as mean ± SEM. (C) Representative dotplots showing ADT mediated by TAM pretreated with cytohalasin D (1uM) or blocking anti-CD64 F(ab')2 and anti-CD32 F(ab')2 fragments (5ug/ml). TAM were co-cultured with PKH67+ A431 cells at a ratio 3:1 in the presence of cetuximab (1ug/ml) for 2 hrs. (D) Cumulative flow cytometry results showing the trogocytosis mediated by indicated blood and tumor myeloid effectors co-cultured with PKH67+A431 cells for 12 hrs at a 50:1 E:T ratio in the presence of IgG1 and IgA2 isotype control Abs (1ug/ml). Indicated FcR+ effectors were freshly isolated for all experiments.

Supplementary Figure S8. (A and B) Representative experiment and summary results showing the effect of blood and tumor FcR+ effectors on the growth of GFP+A549 tumor cell spheroids in the presence of cetuximab (1ug/ml). Experiments were performed as described in supplemental figure 5 (A and B). Original magnification x10. Scale bar, 400 µm. Data are represented as mean ± SEM. Wilcoxon matched pairs test (n=6). Indicated FcR+ effectors were freshly isolated for all experiments. (C) Flow cytometry histogram showing the expression of EGFR on the surface of H1975 tumor cell line in comparison with A431 and A549 tumor cells. (D and E) Cumulative flow cytometry results showing the different levels of trogocytosis and killing activity mediated by blood and tumor FcR+ effectors co-cultured with PKH67+ H1975 tumor cells at a 50:1 E:T ratio in the presence of cetuximab or isotype control Abs for 12 hrs. All comparisons used one-way ANOVA with Turkey’s multiple comparisons tests. All data represented as mean ± SEM. FcR+ effectors were freshly isolated for all experiments. IC-isotype control Ab.

Supplementary Figure S7. (A) Cumulative flow cytometry results showing the ability of blood and tumor FcR+ effectors to kill PKH-67+A431 tumor cells in the presence of nonspecific human IgG1 or IgA2 isotype control Abs (1ug/ml) at a 2:1 E:T ratio in a 12 hrs FACS-based assay. (B and C) Cumulative results showing the kinetics of GFP+A431 tumor cell growth when co-cultured with blood and tumor FcR+ effectors for 48 hrs at a 2:1 E:T ratio in the presence of IgG1 or IgA2 isotype control Abs (1ug/ml) in the IncuCyte® Live Cell Analysis System. (D) Cumulative flow cytometry results showing the ability of blood and tumor FcR+ effectors to kill PKH-67+A549 tumor cells in the presence of nonspecific human IgG1 or IgA2 isotype control Abs (1ug/ml) at a 50:1 E:T ratio in a 12 hrs FACS-based assay. (E and F) Cumulative results showing the kinetics of GFP+A549 tumor cell growth when co-cultured with blood and tumor FcR+ effectors for 48 hrs at a 50:1 E:T ratio in the presence of IgG1 or IgA2 isotype control Abs (1ug/ml) in the IncuCyte® Live Cell Analysis System. All comparisons used one-way ANOVA with Dunnett’s multiple comparisons test. All Data are represented as mean ± SEM. Indicated FcR+ effectors were freshly isolated for all experiments.

Supplementary Figure S5. (A) Representative dotplots showing the levels of trogocytosis and killing activity mediated by TAM co-cultured with cetuximab-opsonized PKH67+A431 cells at a 50:1 E:T ratio for 12 hrs in the presence of blocking anti-CD32a Ab (clone 2B6) or blocking anti-CD32a Ab (clone 6G11). (B and C) Summary results showing the levels of trogocytosis (B) and killing activity (C) mediated by blood monocytes and TAM co-cultured with cetuximab-opsonized PKH67+A431 cells at a 50:1 E:T ratio for 12 hrs in the presence of blocking anti-CD32a Ab (clone2B6) or anti-CD32a Ab (clone 6G11). Blocking anti-CD32a Ab were added at concentration (10ug/ml). Summary graphs represent the total tumoricidal activity of effectors that was calculated as described in material and methods. IC Ab-isotype control Abs. (D) Representative experiment and cumulative results showing the kinetics of GFP+A431 tumor cell growth with blood monocytes or TAM at a 50:1 E:T ratio in the presence of cetuximab and blocking anti-CD32a Ab (clone2B6) or blocking anti-CD32a Ab (clone 6G11) in the IncuCyte® Live Cell Analysis System. Percentage of tumor cell growth inhibition/stimulation was calculated at 60 hrs as described earlier. Number of patients included in each analysis is indicated on the graphs. One-way ANOVA with Friedman multiple comparisons test. All data represented as mean ± SEM.

Suppl Movie 4, monocyte lethal trogocytosis

Supplementary Movie S1. TAM-mediated phagocytosis of rituximab-opsonized Daudi tumor cells. PKH26-labeled TAM (red) were incubated with PKH67-labeled Daudi tumor cells (green) at a 3:1 E:T ratio for 2 hrs in the presence of rituximab (1ug/ml). Time-lapse microscopy was performed using a Leica DMI4000 inverted microscope. The images were taken every 5 min with Hamamatsu ImagEM 16-bit cooled EMCCD camera at 20x magnification. Supplementary Movie S2. TAM-mediated trogocytosis of cetuximab-opsonized A431 tumor cells. PKH26-labeled TAM (red) were incubated with PKH67-labeled A431 tumor cells (green) at a 3:1 E:T ratio for 2 hrs in the presence of cetuximab (1ug/ml). Time-lapse microscopy was performed using a Leica DMI4000 inverted microscope. The images were taken every 5 min with Hamamatsu ImagEM 16-bit cooled EMCCD camera at 20x magnification. Supplementary Movie S3. Sublethal trogocytosis performed by PBN during their interaction with cetuximab–opsonized A431 tumor cells. PKH26-labeled PBN (red) were incubated with PKH67-labeled A431 targets (green) at a 10:1 E:T ratio for 2 hrs in the presence of cetuximab (1ug/ml). Time-lapse microscopy was performed using a Leica DMI4000 inverted microscope. The images were taken every 5 min with Hamamatsu ImagEM 16-bit cooled EMCCD camera at 20x magnification. Supplementary Movie S4. Lethal trogocytosis performed by blood monocytes during their interaction with cetuximab–opsonized A431 tumor cells. PKH26-labeled monocytes (red) were incubated with PKH67-labeled A431 targets (green) at a 10:1 E:T ratio for 2 hrs in the presence of cetuximab (1ug/ml). Time-lapse microscopy was performed using a Leica DMI4000 inverted microscope. The images were taken every 5 min with Hamamatsu ImagEM 16-bit cooled EMCCD camera at 20x magnification.

Supplementary Figure S2. (A-D) The correlation analyses of the ability of blood and tumor-derived FcR+ effectors to regulate A431 tumor cell growth in the presence of anti-EGFR Abs with key clinical parameters of lung cancer patients including tumor type, stage, size, and smoking history. The effect of FcR+ effectors on A431 tumor cell growth was evaluated in the IncuCyte® Live Cell Analysis System. AC-adenocarcinoma, SCC-squamous cell carcinoma. Pearson test. Number of patients included in each analysis is indicated on the graphs.