Fig. 1

Discovery of QP5038 as potent QPCTL inhibitor. a Design of novel and potent QPCTL inhibitors. QPCT protein structure (PDB: 6GBX) is downloaded from PDB protein structure database. b Fluorescent assay determination of IC₅₀ values of inhibitors against QPCTL. Data represent n = 3 biological replicates and mean ± SD. c Cell surface binding of anti-human CD47 antibody clone hCD47-B6H12, hCD47-CC2C6 and human hSIRPα-Fc to HEK293T cells after treatment with 100 nM QPCTL inhibitors for 48 h, as determined by flow cytometry. d Cell surface binding of anti-human CD47 antibody clone hCD47-B6H12, hCD47-CC2C6 and human hSIRPα-Fc to Raji cells after treatment with 100 nM QPCTL inhibitors for 48 h as determined by flow cytometry. In c and d, values indicated mean fluorescence intensity (MFI) relative to cells stained with DMSO. Data represent n = 3 biological replicates and mean ± SD of triplicates. Statistically significant differences were determined by one-way ANOVA, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. e Dose-dependent inhibition of pGlu-CD47 following treatment with QPCTL inhibitors for 48 h in HEK293T cells. f Dose-dependent inhibition of cell surface binding of human hSIRPα-Fc to HEK293T cells following treatment with QPCTL inhibitors for 48 h. In e and f, data represent n = 3 biological replicates and mean ± SD of triplicates. g Cell surface binding of anti-human CD47 antibody clone hCD47-CC2C6 to different cells, such as myeloma (H929), colon cancer (HCT116), hepatocellular carcinoma (Huh7), ovarian adenocarcinoma (SKOV3), lymphoma (SU-DHL-8), bladder cancer (T24), lung cancer (H1299), and breast cancer (MCF-7), after treatment with 500 nM QPCTL inhibitors for 48 h, as determined by flow cytometry. Data are representative of three independent experiments. Statistically significant differences were determined by unpaired two-tailed t-test, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. h, i Phagocytosis of control-treated (DMSO) (-) or QPCTL inhibitors-treated (+) B16F10 cells in the presence or absence of the anti-mouse TRP1 antibody TA99 by mouse macrophages following treatment with 10 μM inhibitors for 48 h. j, k Phagocytosis of control-treated (DMSO) (-) or QPCTL inhibitors-treated (+) Raji cells in the presence or absence of the anti-human CD20 antibody rituximab (Ritux) by mouse macrophages following treatment with 10 μM inhibitors for 48 h. Phagocytosis was determined by the number of the CFSE ⁺ labelled F4/80⁺ macrophages vs the total tumor cells, and data are mean values of three biological experiments in h, i, j and k. The presented data is a representative image from three independent experiments with similar results in i and k. Statistically significant differences were determined by one-way ANOVA, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 in h, i, j and k. l Anti-tumor efficacy of QP5038 with once daily dosing at 25 mg/kg in the presence or absence of the anti-PD-1 antibody. The total study length was 24 days. Statistically significant differences were determined by two-way ANOVA, ∗∗∗∗p < 0.0001. m Quantification of xenografted tumor weight when mice are sacrificed. The data were presented as the mean ± SD and statistically significant differences were determined by two-way ANOVA, ∗∗p < 0.005. n Anti-tumor efficacy of QP5038 in the presence or absence of the T cell depletion antibody. Statistically significant differences were determined by two-way ANOVA, ∗∗∗∗p < 0.0001, ns not significant