Journal: Journal for Immunotherapy of Cancer

Article Title: CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy

doi: 10.1136/jitc-2020-000905

Figure Lengend Snippet: Pep-20 binds to CD47 and blocks the CD47/SIRPα interaction. (A, B) Dose response curves of pep-20 binding to CD47. Binding assay of pep-20 to human (A) or mouse (B) CD47-IgV-Domain protein was examined by the MST. The human or mouse SIRPα protein served as positive control. (C, D) Dose response curves of pep-20 interfering CD47/SIRPα interaction. Flow cytometry analysis of human (C) or mouse (D) CD47-IgV-Domain-hIg fusion protein binding to CHO stably expressing human or mouse SIRPα cells in the presence of pep-20 with varying gradient concentrations. The data represented as the mean fluorescence intensity normalized to the maximum binding. (E to J) Phagocytosis assays were performed by co-culture of tumor cells with corresponding macrophages at a 1∶4 ratio in serum-free medium at 37℃ for 4 hours in low-attachment 96-well plates. GFP + MCF7 cells (E), GFP + HT29 cells (F) and GFP + Jurkat cells (G) were incubated with human peripheral blood-derived macrophages in the presence of 100 µM pep-20 or 20 µg/mL anti-human CD47 antibody (B6H12). CFSE + CT26 cells (H), GFP + MC38 cells (I) and GFP + B16-OVA cells (J) were incubated with mouse bone marrow-derived macrophages in the presence of 100 µM pep-20 or 20 µg/mL anti-mouse CD47 antibody (miap301). Phosphate-buffered saline was negative control in all assays. CFSE + or GFP + macrophages were detected by flow cytometry. Data are represented as means±SEM. Statistical significance was determined by unpaired Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. CHO, Chinese hamster ovary; CSFE, carboxyfluorescein succinimidyl ester; GFP, green fluorescent protein; MST, microscale thermophoresis.

Article Snippet: Human or mouse CD47-IgV-Domain-hIg fusion proteins (20 nM, Sino Biological, China), or biotinylated human CD47 protein (20 nM, ACROBiosystems, USA) was incubated with titrating concentrations of peptides or anti-human CD47 antibody (B6H12) in phosphate-buffered saline (PBS, pH 7.4) for 1 hour at 4°C.

Techniques: Binding Assay, Positive Control, Flow Cytometry, Protein Binding, Stable Transfection, Expressing, Fluorescence, Co-Culture Assay, Incubation, Derivative Assay, Negative Control, Microscale Thermophoresis

Journal: Journal for Immunotherapy of Cancer

Article Title: CD47/SIRPα blocking peptide identification and synergistic effect with irradiation for cancer immunotherapy

doi: 10.1136/jitc-2020-000905

Figure Lengend Snippet: Pep-20-D12 inhibits the growth of tumors and activates antitumor T-cell immune response via systemic administration. C57BL/6 mice were transplanted with 1×10 6 MC38 cells on the right flank, until the tumor volume reached 50 mm 3 . (A, B) Mice were treated i.p. with 2 mg/kg of pep-20-D12 every day for 2 weeks or 400 µg of anti-mouse CD47 antibody (miap301) every 3 days for a total of five times as the positive control, and normal saline and RatIg as the negative controls (n=8). (C) Tumors were detected for the percentage of tumor-infiltrating CD8 + T cells in total CD45 + cells (n=5). (D to F) Cells from tumor-infiltrating lymphocytes (D), draining lymph nodes (E) or spleens (F) were obtained and stimulated with 20 ng/mL of PMA and 1 µM ionomycin-containing protein transport inhibitor cocktail for 4 hours. IFN-γ-expressing CD8 + T cells were detected by flow cytometry. (n=5). Data are represented as means±SEM. Statistical significance was determined by unpaired Student’s t-test. *p<0.05; **p<0.01. IFN, interferon; i.p. intraperitoneally; n.s., no significance; PMA, phorbol12-myristate 13-acetate; s.c., subcutaneously.

Article Snippet: Human or mouse CD47-IgV-Domain-hIg fusion proteins (20 nM, Sino Biological, China), or biotinylated human CD47 protein (20 nM, ACROBiosystems, USA) was incubated with titrating concentrations of peptides or anti-human CD47 antibody (B6H12) in phosphate-buffered saline (PBS, pH 7.4) for 1 hour at 4°C.

Techniques: Positive Control, Expressing, Flow Cytometry