Journal: Cell Reports Medicine
Article Title: Glycoengineering-based anti-PD-1-iRGD peptide conjugate boosts antitumor efficacy through T cell engagement
doi: 10.1016/j.xcrm.2024.101590
Figure Lengend Snippet: Antitumor efficacy of αPD-1-(iRGD) 2 depends on pre-existing intra-tumoral CD8 + T cells (A) Schematic of the T cell migration inhibition regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1-(iRGD) 2 (5 mg/kg), and FTY720 (MCE, #HY-12005, 20 μg) every 3 days. (B) Flow cytometry analysis of T cell abundance in peripheral blood to validate the T cell migration inhibition of FTY720. (C) Tumor growth profile of (A). (D) Schematic of the CD8 + T cell depletion regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1-(iRGD) 2 (5 mg/kg), and αCD8 (BioXCell, #BE0117, 200 μg) every 3 days. (E) Flow cytometry analysis of CD8 + T cell abundance in tumor to validate CD8 + T cell depletion. (F) Tumor growth profile of CD8 + T cell depletion assay. (G) Schematic of the treatment regimen in MFC mouse gastric tumor model. Briefly, mice were treated with 1 × 10 6 MFC cells and injected intraperitoneally with PBS (100 μL control), αPD-1 (0.1 mg/kg) alone, or with free iRGD (4 μmol/kg) and αPD-1-(iRGD) 2 (0.1 mg/kg). (H) Weight of murine subcutaneous tumors resected at the end of (G). (I) Tumor growth profile of (G). (J) Flow cytometry results indicating the abundance of CD8 + T cell (Biolegend, #100702) from resected tumor bulk at the endpoint of (G). (K) Flow cytometry quantification of PD-1 (Biolegend, #135206) expression on CD8 + T cell from resected tumor bulk at the endpoint of (G). (L) Flow cytometry quantification of CD39 (Biolegend, #143806) expression on PD-1 + CD8 + T cells from resected tumor bulk at the endpoint of (F). (M and N) Flow cytometry results indicating the abundance of CD4 + T cell (Biolegend, #100408) (M) and Treg (Biolegend, #320014 and #100412; eBioscience, #12-0251-83) (N) from resected tumor bulk at the endpoint of (G). (O) Flow cytometry analysis of propidium-iodide-positive tumor cells preloaded with GP33 (Genescript Biotech Corporation, 500 nM) or SIINFEKL peptide (Genescript Biotech Corporation, 500 nM) after simultaneously coculturing with OT-I cells. The ratio of unprimed, GP33-primed, SIINFEKL-primed B16F10 cells, and OT-I cells was 1:1:1:10. Concentration of αPD-1 and αPD-1-(iRGD) 2 was 10 μg/mL, and that of iRGD was 100 μg/mL (P) Flow cytometry analysis of activation markers (CD25 and CD69) on OT-I cells and spleen T cells simultaneously cocultured with SIINFEKL peptide (500 nM) preloaded tumor cells. The ratio of OT-I cells, non-specific spleen T cells, and SIINFEKL-primed B16 cells was 5:5:1. (Q) Histogram of the percentage of conjugated cells when 2 × 10 5 Dye 670-stained NY-ESO-1 157-165 -primed HLA-A∗0201-Raji cells were cocultured 1 h with mixed 2 × 10 4 CFSE-stained 1G4-PD-1-Jurkat cells and 2 × 10 4 Dye 450-stained PD-1-Jurkat cells. The concentration of αPD-1-(iRGD) 2 was 10 μg/mL, while blinatumomab (MCE, #HY-P9963) was 1 μg/mL (R) Histogram of CD69 (Biolegend, #319102) expression on 1G4-PD-1-Jurkat and PD-1-Jurkat under the same condition in (P). (S) Flow cytometry chart of (Q). (T) Flow cytometry chart of (R). Data represent mean ± SEM. For (B) and (O)–(T), n = 3. For (C), (E), (F), and (H)–(N), n = 5. For (B), (E), (H), and (J)–(M), one-way ANOVA test and Tukey’s multiple comparisons test. For (C), (F), (I), and (O)–(R), two-way ANOVA test and Tukey’s multiple comparisons test. n.s., not significant; ∗ p < 0.5; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.
Article Snippet: Recombinant human PD-1 (HEK293, His) , MCE , HY-P7396.
Techniques: Migration, Inhibition, Injection, Control, Flow Cytometry, Depletion Assay, Expressing, Concentration Assay, Activation Assay, Staining
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