designed the study, performed experiments, analyzed and interpreted data, and wrote the manuscript. C. Bebernitz performed experiments, analyzed data, and interpreted data. A. Lopez performed experiments. S. Rafiq designed experiments, interpreted data and wrote manuscript. R. Brentjens designed the study, interpreted data, and wrote the manuscript.
Mcl-1, an anti-apoptotic Bcl-2 family member, is a known resistance factor to many cancer therapies, and a historically difficult target to drug. Recently however, we have made significant progress optimizing the potency and characterizing the mechanism of action of a novel class of selective Mcl-1 inhibitors. Here, we characterize the activity of one of our lead compounds and confirm on-target mechanistic activity in vitro and in vivo. Using an Mcl-1 binding assay we demonstrate very potent activity (IC50 < 3nM), while maintaining selectivity over other anti-apoptotic Bcl-2 family proteins, Bcl-2 (IC50 > 33 µM) and Bcl-xL (IC50 > 33 µM). In an Mcl-1 dependent multiple myeloma cell line, MOLP-8, we demonstrate rapid Mcl-1/Bak complex disruption with 10 nM inhibitor by 15 min followed by caspase 9-dependent apoptosis by 1 hr (EC50= 50 nM), consistent with an Mcl-1 dependent mechanism of action. Importantly, by depleting Bak from cells using siRNA, we also confirm that the observed apoptosis is through a Bak-dependent mechanism. Moreover, our Mcl-1 inhibitor exhibits broad submicromolar activity across a panel of multiple myeloma cell lines. In the absence of a validated selective Mcl-1 reference compound to benchmark in vivo activity, we engineered MOLP-8 cells to express the pro-apoptotic protein, Noxa, using a Tet-on expression system and grew these as subcutaneous tumors on the flanks of SCID mice. Within 24 hours of switching the mice to a diet containing doxycycline, we observe significant upregulation of Noxa, leading to an increase in cleaved caspase 3 (CC3) and cleaved PARP in tumor lysates, resulting in rapid tumor regression. In the same MOLP-8 xenograft model, we demonstrate that our Mcl-1 inhibitor induces rapid dissociation of Bak from Mcl-1 in tumors leading to accumulation of CC3 and cleaved PARP by 1 hr. Complete tumor regression was observed after a single 60 mg/kg or 100 mg/kg dose, while partial regression was observed after a single 30 mg/kg dose, and tumor growth inhibition was observed after a single 10 mg/kg dose. All doses were well tolerated with no significant body weight loss. Together, these data reinforce the potential utility of selective Mcl-1 inhibitors in hematological malignancies. Disclosures Belmonte: Astrazeneca: Employment, Equity Ownership. Adam:Astrazenenca: Employment, Equity Ownership. Borrelli:AstraZeneca: Employment, Equity Ownership. Bhavsar:AstraZeneca: Employment, Equity Ownership. Bebernitz:AstraZeneca: Employment, Equity Ownership. Hird:AstraZeneca: Employment, Equity Ownership. Secrist:AstraZeneca: Employment, Equity Ownership.
<p>(A) Media from in vitro co-culture of CAR T cells with EL4mCD19 tumor cells was assessed for IL12p70 on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of two independent experiments. (B) EL4mCD19 tumor bearing C57BL/6 mice, pretreated with 250mg/kg per mouse of cyclophosphamide day -3, treated i.v. with CAR T cells day 0. Significance determined bylong-rank Mantel-Cox Test, with 95% confidence interval (n=3 per group). *p=0.0224 m1928� compared to m1928� + cyclophosphamide and *p=0.0295 m19� compared to m19� + cyclophosphamide (C) Flow cytometry demonstrating CD25 expression on CAR T cells prior to injection in terms of percentage and representative flow cytometry plot (ns by ordinary one-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments (n=3 per group). (D) CAR T cells were co-cultured with EL4mCD19 tumor cells and assessed for cytotoxicity 4 hours later, using a luciferase killing assay. m1928�, m19�IL-12, and m1928�IL-12 CAR T cells showed significantly increased cytotoxicity compared to m19� CAR T cells at E:T ratios of 2:1, 1:1, and 0.5:1. There was no difference in tumor lysis between 19�12 and m1928�IL-12 CAR T cells (*p=<0.001 by two-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments. Serum was obtained through retro-orbital bleeds and assessed for IL12p70 through luminex on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of three independent</p>
<p>(A) CAR T cells were co-cultured with EL4mCD19 tumor cells and then assessed for cytotoxicity 4 hours later, using a luciferase killing assay. There was no difference in tumor lysis between CAR T cells and vex-GFP tagged CAR T cells (two-way ANOVA). Data shown is representative of two independent experiments. (B) Flow cytometry gating strategy to determine CD8+ and CD4+ ratio and PD-1, TIM-3, and LAG-3 inhibitory receptor expression.</p>
<p>(A) CAR T cells, day 0 before infusion, were characterized by flow cytometry. Data shown is mean +/- s.e.m. of three independent experiments. "ns" (not significant) by unpaired t test. (B) Representative flow cytometry plots of CAR T cell CD8+ and CD4+ ratios day 5 after infusion referenced in Figure 2D. (C) Representative flow cytometry plots of PD-1, LAG-3, and TIM-3 inhibitory receptor expression of CD8+ CAR T cells referenced in Figure 2E. (D) CAR T cells, day 5 after infusion, were characterized by flow cytometry. m1928�IL-12 CAR T cells, gated on CD8+ T cells, expressed higher levels of double positive inhibitory receptors, LAG-3;TIM-3 (*p=0.0397) and LAG-3;PD-1 (*p=0.001), and showed a trend toward higher levels of TIM-3;PD-1 (*p=0.0563). Data obtained is mean +/- s.e.m. of three independent experiments analyzed by unpaired t test.</p>
<p>Sequences of CAR T cells with mCD8 signal peptide annotated in orange, scFv annotated in red, myc tag annotated in highlighted gray, mCD28 transmembrane and intracellular annotated in purple, mZeta annotated in green, IRES annotated in pink, mIL12 annotated in black, and m4-1BB annotated in yellow.</p>
<p>(A) B cells were assessed for CD80 expression by flow cytometry through peripheral blood. *p=0.048 unpaired t test of BM and *p=0.0233 unpaired t test of spleen. Data shown is mean +/- s.e.m. of two independent experiments. (B) Luminex data showing IL-4 secreting from EL4mCD19 tumor cells. Data shown is mean +/- s.e.m. of two independent experiments. *p=0.002 by unpaired t test. (C) Flow cytometry demonstrating lack of CD28 expression on CD28-/- T cells. Data shown is representative of two independent experiments. (D) CAR T cells were co-cultured with EL4mCD19 tumor cells and then assessed for cytotoxicity 4 hours later, using a luciferase killing assay. There was no difference in tumor lysis between CD28-/- or wildtype transduced CAR T cells. Data shown is mean +/- s.e.m. of two independent experiments.</p>
<p>(A) Media from in vitro co-culture of CAR T cells with EL4mCD19 tumor cells was assessed for IL12p70 on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of two independent experiments. (B) EL4mCD19 tumor bearing C57BL/6 mice, pretreated with 250mg/kg per mouse of cyclophosphamide day -3, treated i.v. with CAR T cells day 0. Significance determined bylong-rank Mantel-Cox Test, with 95% confidence interval (n=3 per group). *p=0.0224 m1928� compared to m1928� + cyclophosphamide and *p=0.0295 m19� compared to m19� + cyclophosphamide (C) Flow cytometry demonstrating CD25 expression on CAR T cells prior to injection in terms of percentage and representative flow cytometry plot (ns by ordinary one-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments (n=3 per group). (D) CAR T cells were co-cultured with EL4mCD19 tumor cells and assessed for cytotoxicity 4 hours later, using a luciferase killing assay. m1928�, m19�IL-12, and m1928�IL-12 CAR T cells showed significantly increased cytotoxicity compared to m19� CAR T cells at E:T ratios of 2:1, 1:1, and 0.5:1. There was no difference in tumor lysis between 19�12 and m1928�IL-12 CAR T cells (*p=<0.001 by two-way ANOVA). Data shown is mean +/- s.e.m. of three independent experiments. Serum was obtained through retro-orbital bleeds and assessed for IL12p70 through luminex on day 7 after CAR T cell treatment. Data shown is mean +/- s.e.m. of three independent</p>
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