Figure 5.
INCA033989 mouse surrogate induces hematologic and molecular effects in the blood and bone marrow by targeting disease-initiating stem cells. A preclinical mouse model with competitive transplantation using 20% of CALRdel52/del52 LoxP/SCL-Cre-ERT bone marrow cells and 80% of wt/GFP+ bone marrow cells. Two weeks after CALRdel52/del52 induction, blood parameters and chimerism were assessed, and the mice were randomized. Mice were treated with isotype or INCA033989 mouse surrogate (10 mg/kg) twice a week, and the hematologic and molecular responses were assessed every 2 weeks for 10 weeks. (A) Platelet counts measured in blood. The gray area depicts the normal range of platelet counts. Data are presented as mean ± SEM (n = 6 mice per group). Significance was calculated using a parametric t test with Welch correction. ∗∗P < .01. (B) mutCALR chimerism was monitored by flow cytometry in platelets (CD41+). Data are presented as mean ± SEM (n = 6 mice per group). Significance was calculated by parametric t test with Welch correction. ∗∗∗P < .001. (C-D) Bone marrow evaluation at 10 weeks after treatment initiation. (C) The bone marrow was analyzed for mutCALR chimerism in progenitors by flow cytometry using a GFP marker. Data are presented as the mean ± SEM (n = 4 mice per group). Significance was determined using 2-way ANOVA with the Tukey multiple comparison test. (D) Pictures represent the histopathological analyses of bone marrow from 1 out of 4 mice per group treated with isotype or INCA033989 mouse surrogate after von Willebrand factor staining. (E-F) Relapse study in mice treated with IgG or INCA033989 mouse surrogate for 10 weeks, in which treatment was discontinued during the following 10 weeks. Platelet counts (E) and proportion of mutCALR-positive platelets (F) are shown. Data are mean ± SEM (n = 4 mice per group). Significance was calculated by parametric t test with Welch correction. ∗∗P < .01. (G-H) Secondary engraftments were performed with bone marrow cells isolated from mice treated with IgG or INCA033989 mouse surrogate, and the platelet counts (G) and platelet chimerism (H) were followed without any treatment over 16 weeks. Data are presented as the mean ± SEM (n = 10 mice per group from 2 donor mice). Significance was calculated by parametric t test with Welch correction. ∗P < .05; ∗∗P < .01. BMT, bone marrow transplant; LK, Lin−Sca+; LSK, Lin−Sca+Kit+; LT-HSC, long-term HSC; SLAM, signaling lymphocyte activation molecule; ST-HSC, short-term HSC.

INCA033989 mouse surrogate induces hematologic and molecular effects in the blood and bone marrow by targeting disease-initiating stem cells. A preclinical mouse model with competitive transplantation using 20% of CALRdel52/del52 LoxP/SCL-Cre-ERT bone marrow cells and 80% of wt/GFP+ bone marrow cells. Two weeks after CALRdel52/del52 induction, blood parameters and chimerism were assessed, and the mice were randomized. Mice were treated with isotype or INCA033989 mouse surrogate (10 mg/kg) twice a week, and the hematologic and molecular responses were assessed every 2 weeks for 10 weeks. (A) Platelet counts measured in blood. The gray area depicts the normal range of platelet counts. Data are presented as mean ± SEM (n = 6 mice per group). Significance was calculated using a parametric t test with Welch correction. ∗∗P < .01. (B) mutCALR chimerism was monitored by flow cytometry in platelets (CD41+). Data are presented as mean ± SEM (n = 6 mice per group). Significance was calculated by parametric t test with Welch correction. ∗∗∗P < .001. (C-D) Bone marrow evaluation at 10 weeks after treatment initiation. (C) The bone marrow was analyzed for mutCALR chimerism in progenitors by flow cytometry using a GFP marker. Data are presented as the mean ± SEM (n = 4 mice per group). Significance was determined using 2-way ANOVA with the Tukey multiple comparison test. (D) Pictures represent the histopathological analyses of bone marrow from 1 out of 4 mice per group treated with isotype or INCA033989 mouse surrogate after von Willebrand factor staining. (E-F) Relapse study in mice treated with IgG or INCA033989 mouse surrogate for 10 weeks, in which treatment was discontinued during the following 10 weeks. Platelet counts (E) and proportion of mutCALR-positive platelets (F) are shown. Data are mean ± SEM (n = 4 mice per group). Significance was calculated by parametric t test with Welch correction. ∗∗P < .01. (G-H) Secondary engraftments were performed with bone marrow cells isolated from mice treated with IgG or INCA033989 mouse surrogate, and the platelet counts (G) and platelet chimerism (H) were followed without any treatment over 16 weeks. Data are presented as the mean ± SEM (n = 10 mice per group from 2 donor mice). Significance was calculated by parametric t test with Welch correction. ∗P < .05; ∗∗P < .01. BMT, bone marrow transplant; LK, Lin−Sca+; LSK, Lin−Sca+Kit+; LT-HSC, long-term HSC; SLAM, signaling lymphocyte activation molecule; ST-HSC, short-term HSC.

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