Catherine A. Evans

Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate

Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.

Regulation of T cell alloimmunity by PI3Kγ and PI3Kδ

Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ−/− and PI3KδD910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ−/−, but not PI3ΚδD910A/D910A, recipients exhibit indefinite prolongation of heart allograft survival. PI3KδD910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation.Phosphatidylinositol-3-kinases (PI3K) γ and δ are key regulators of T cell signaling. Here the author show, using mouse heart allograft transplantation models, that PI3Kγ or PI3Kδ deficiency prolongs graft survival, but selective inhibition of PI3Kγ or PI3Kδ reveals alternative transplant survival outcomes post CTLA4-Ig treatment.

Abstract B029: The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Introduction: The phosphoinositide-3-kinase (PI3K) lipid kinases are a family of kinase isoforms that transduce signals in response to various stimuli in different cell types. The PI3K-γ isoform is expressed in immune cells and has limited, if any, expression in epithelial cancer cells. Genetic deletion and kinase-dead knock-in studies highlight a key role for PI3K-γ in the development and function of myeloid-derived cells that constitute a key component of the immune suppressive tumor microenvironment (Joshi Mol Canc Res 2014; Schmid Canc Cell 2011). Targeting PI3K-γ in these tumor-associated myeloid cells could therefore inhibit the immune suppressive tumor microenvironment, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results: Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD value of 0.29 nM for PI3K-γ with >58-fold weaker binding affinity for the other Class I PI3K isoforms. Enzymatic assays utilizing physiological ATP concentrations (3 mM) confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over other Class I PI3K isoforms. Cellular assays designed to assess individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). Further selectivity screening revealed that IPI-549 is selective for PI3K-γ over other protein and lipid kinases, receptors, ion channels, and transporters. In vitro functional assays demonstrated that IPI-549 blocked bone marrow derived M2 murine macrophage polarization in response to IL-4 and MCSF1, but did not inhibit ConA-induced T-cell activation. These data indicate the potential for IPI-549 to block immune suppressive macrophage development but not T-cell activity. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of the PI3K-γ isoform relative to the other Class I PI3K isoforms. In an in vivo PI3K-γ-dependent neutrophil migration murine model, IPI-549 blocked neutrophil migration in a dose dependent manner. To evaluate the effect of PI3K-γ inhibition on tumor growth in an immunocompetent animal, IPI-549 was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple syngeneic models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects immune suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell activity. Studies in nude or CD8 T-cell depleted mice demonstrated the T-cell dependence of IPI-549 mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors showed increased tumor growth inhibition compared to either monotherapy. Conclusions: IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. IND-enabling studies with IPI-549 are ongoing to support its initial clinical exploration in the setting of solid tumors. Citation Format: Jeffery Kutok, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Karen McGovern, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Katherine Walsh, Kerry White, David Winkler, Vito Palombella. The potent and selective phosphoinositide-3-kinase-gamma inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B029.

Abstract A192: The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment

Introduction The phosphoinositide-3-kinase (PI3K) lipid kinases transduce signals in response to various stimuli in different cell types. PI3K-γ is expressed in immune cells and has limited expression in epithelial cancer cells. Genetic inactivation of PI3K-γ highlights its role in the development and function of myeloid-derived cells that constitute a key component of the suppressive tumor microenvironment (Schmid 2011). Targeting PI3K-γ in tumor-associated myeloid cells could potentially relieve tumor immune tolerance, enabling the immune system to attack tumor cells more effectively. To date, potent and selective PI3K-γ inhibitors with drug-like properties have not been available to test this hypothesis. We now report the structure, biochemical, cellular, and in vivo properties of a potent and selective, small molecule inhibitor of PI3K-γ, IPI-549, and provide data to support the therapeutic potential of breaking tumor immune tolerance through PI3K-γ inhibition. Results Discovery efforts identified a highly selective inhibitor of PI3K-γ, IPI-549, with pharmaceutical properties suitable for further development. Binding studies with IPI-549 revealed a KD of 0.29 nM for PI3K-γ and enzymatic assays confirmed the selectivity of IPI-549 for PI3K-γ (>200-fold) over the other Class I PI3K isoforms. Comparison of IPI-549 to previously reported PI3K-γ inhibitors in this enzymatic confirmed its unique potency and selectivity for PI3K-γ. Cellular assays for individual Class I PI3K isoform activity demonstrated that IPI-549 is highly potent and specific for PI3K-γ (IC50 of 1.2 nM; >140-fold selectivity). In kinase screens, IPI-549 is selective for PI3K-γ over other kinases, receptors, ion channels, and transporters. In vitro assays demonstrated that IPI-549 blocked immune suppressive M2 murine macrophage polarization in response to IL-4 and MCSF1. Pharmacokinetic studies in mice demonstrated IPI-549 to be orally bioavailable with a long plasma half-life enabling selective inhibition of PI3K-γ relative to other Class I PI3K isoforms. To characterize IPI-5499s ability to inhibit PI3K-γ in vivo, mice with air pouches treated with IPI-549 showed dose responsive inhibition of PI3K-γ-dependent neutrophil migration. The effect of IPI-549 on tumor growth was tested in murine syngeneic solid tumor models. Mice treated with IPI-549 demonstrated significant tumor growth inhibition in multiple models. Studies to elucidate the mechanism of tumor growth inhibition indicated that IPI-549 affects suppressive myeloid cell numbers and/or function, leading to an increase in cytotoxic T-cell numbers and activity, as assessed by marker studies. Nude or CD8 T-cell depleted mice studies demonstrated a T-cell dependence of IPI-549-mediated tumor growth inhibition. Finally, in vivo studies with IPI-549 in combination with immune checkpoint inhibitors or following chemotherapy showed increased tumor growth inhibition compared to monotherapies. Conclusions IPI-549 is a potent and selective inhibitor of PI3K-γ with pharmaceutical properties that allow for the selective inhibition of PI3K-γ in vivo. Our findings provide evidence that targeted inhibition of PI3K-γ by IPI-549 can restore antitumor immune responses and inhibit solid tumor growth in preclinical models. Citation Format: Karen McGovern, Janid Ali, Erin Brophy, Alfredo Castro, Jonathan DiNitto, Catherine Evans, Kerrie Faia, Stanley Goldstein, Nicole Kosmider, Andre Lescarbeau, Tao Liu, Christian Martin, Somarajan Nair, Melissa Pink, Jennifer Proctor, Matthew Rausch, Sujata Sharma, John Soglia, Jeremy Tchaicha, Martin Tremblay, Vivian Villegas, Kerry White, David Winkler, Vito Palombella, Jeffery Kutok. The potent and selective phosphoinositide-3-kinase-γ inhibitor, IPI-549, inhibits tumor growth in murine syngeneic solid tumor models through alterations in the immune suppressive microenvironment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A192.