Holly Koblish

Mechanism of tumor rejection with doublets of CTLA-4, PD-1/PD-L1, or IDO blockade involves restored IL-2 production and proliferation of CD8(+) T cells directly within the tumor microenvironment.

BackgroundBlockade of immune inhibitory pathways is emerging as an important therapeutic modality for the treatment of cancer. Single agent treatments have partial anti-tumor activity in preclinical models and in human cancer patients. Inasmuch as the tumor microenvironment shows evidence of multiple immune inhibitory mechanisms present concurrently, it has been reasoned that combination therapies may be required for optimal therapeutic effect.MethodsTo test this notion, we utilized permutations of anti-CTLA-4 mAb, anti-PD-L1 mAb, and/or the IDO inhibitor INCB23843 in the murine B16.SIY melanoma model.ResultsAll three combinations showed markedly improved tumor control over single treatments, with many mice achieving complete tumor rejection. This effect was seen in the absence of vaccination or adoptive T cell therapy. The mechanism of synergy was investigated to examine the priming versus effector phase of the anti-tumor immune response. Only a minimal increase in priming of anti-tumor T cells was observed at early time points in the tumor-draining lymph nodes (TdLN). In contrast, as early as three days after therapy initiation, a marked increase in the capacity of tumor-infiltrating CD8+ T cells to produce IL-2 and to proliferate was found in all groups treated with the effective combinations. Treatment of mice with FTY720 to block new T cell trafficking from secondary lymphoid structures still enabled restoration of IL-2 production and proliferation by intratumoral T cells, and also retained most of the tumor growth control.ConclusionsOur data suggest that the therapeutic effect of these immunotherapies was mainly mediated through direct reactivation of T cells in situ. These three combinations are attractive to pursue clinically, and the ability of intratumoral CD8+ T cells to produce IL-2 and to proliferate could be an important biomarker to integrate into clinical studies.

Discovery of potent competitive inhibitors of indoleamine 2,3-dioxygenase with in vivo pharmacodynamic activity and efficacy in a mouse melanoma model.

A hydroxyamidine chemotype has been discovered as a key pharmacophore in novel inhibitors of indoleamine 2,3-dioxygenase (IDO). Optimization led to the identification of 5l, which is a potent (HeLa IC(50) = 19 nM) competitive inhibitor of IDO. Testing of 5l in mice demonstrated pharmacodynamic inhibition of IDO, as measured by decreased kynurenine levels (>50%) in plasma and dose dependent efficacy in mice bearing GM-CSF-secreting B16 melanoma tumors.

Abstract 1336: Novel immunotherapeutic activity of JAK and PI3Kδ inhibitors in a model of pancreatic cancer

Immunotherapeutic agents are emerging as key components of efficacious multi-agent regimens in cancer. The majority of immunotherapeutic agents developed thus far either attempt to stimulate a more productive anti-tumoral immune response or to inhibit key proteins in the immunosuppressive tumoral milieu. In contrast, agents targeting signal transduction molecules have been largely developed for their ability to impact the proliferative potential of tumor cells directly. Notably, the JAK/STAT and PI3Kδ signaling pathways have been shown to contribute not only to tumor cell proliferation and survival but also to play a crucial role in regulating stromal cells, including immune cells, which are recruited to the tumor microenvironment. Activation of these pathways has been shown to result in the recruitment and expansion of predominantly negative regulatory cells such as myeloid derived suppressor cells and regulatory T cells, suggesting that inhibition of JAK/STAT and PI3Kδ signaling may promote antitumor immunity. Therefore, we have examined the immunotherapeutic potential of selective inhibitors of either JAK or PI3Kδ. We demonstrate that inhibitors of either JAK or PI3Kδ block tumor growth as single agents in the immuno-competent syngeneic PAN02 pancreatic model, which is not driven by oncogenic JAK or PI3K signaling. Tumor growth inhibition is not observed in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. These agents were tested pairwise with each other and in combination with other immune checkpoint modulators, including an anti-PD-L1 antibody and the IDO1 inhibitor INCB24360. The combinations of JAK inhibition with IDO1 inhibition, PI3Kδ inhibition or PD-1/PD-L1 blockade resulted in enhanced efficacy. Mechanistic studies revealed that the combination of JAK and IDO1 inhibition did not alter the number of infiltrating T cells within the tumor, but instead resulted in a more activated phenotype of the infiltrating T cells, leading to higher levels of IFNγ production. In contrast, combination treatment with regimens that included an inhibitor to PI3Kδ led to a marked increase in the numbers of T cell infiltrates, although the cells were not maximally activated. Further studies to understand the complex cellular responses elicited by these inhibitors may provide the mechanistic rationale to explore JAK or PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Michael Hansbury, Liang-Chuan S. Wang, Gengjie Yang, Taisheng Huang, Chu-Biao Xue, Yun-Long Li, Eddy Yue, Andrew Combs, Wenqing Yao, Reid Huber, Peggy Scherle. Novel immunotherapeutic activity of JAK and PI3Kδ inhibitors in a model of pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1336. doi:10.1158/1538-7445.AM2015-1336

INCB040093 is a novel PI3Kδinhibitor for the treatment of B cell lymphoid malignancies

Phosphatidylinositol 3-kinase delta (PI3Kδ) is a critical signaling molecule in B cells and is considered a target for development of therapies against various B cell malignancies. INCB040093 is a novel PI3Kδ small-molecule inhibitor and has demonstrated promising efficacy in patients with Hodgkin’s lymphoma in clinical studies. In this study, we disclose the chemical structure and the preclinical activity of the compound. In biochemical assays, INCB040093 potently inhibits the PI3Kδ kinase, with 74- to >900-fold selectivity against other PI3K family members. In vitro and ex vivo studies using primary B cells, cell lines from B cell malignancies, and human whole blood show that INCB040093 inhibits PI3Kδ-mediated functions, including cell signaling and proliferation. INCB040093 has no significant effect on the growth of nonlymphoid cell lines and was less potent in assays that measure human T and natural killer cell proliferation and neutrophil and monocyte functions, suggesting that the impact of INCB040093 on the human immune system will likely be restricted to B cells. INCB040093 inhibits the production of macrophage-inflammatory protein-1β (MIP-1beta) and tumor necrosis factor-β (TNF-beta) from a B cell line, suggesting a potential effect on the tumor microenvironment. In vivo, INCB040093 demonstrates single-agent activity in inhibiting tumor growth and potentiates the antitumor growth effect of the clinically relevant chemotherapeutic agent, bendamustine, in the Pfeiffer cell xenograft model of non-Hodgkin’s lymphoma. INCB040093 has a favorable exposure profile in rats and an acceptable safety margin in rats and dogs. Taken together, data presented in this report support the potential utility of orally administered INCB040093 in the treatment of B cell malignancies.

Abstract 572: Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model

Purpose: To determine if epacadostat, an oral indoleamine 2,3-dioxygenase (IDO1) inhibitor has therapeutic benefit against GBM when administered as single agent and with PD-1 blocking antibody. Methods: An initial survival experiment was performed to assess efficacy and was followed by an identical repeat experiment for validation. 1X10 5 luciferized GL261 cells, a murine GBM tumor line derived from intracerebral methylcholanthrene implantation, were stereotactically implanted intracranially in albino syngeneic C57BL/6 mice. Mice with increasing bioluminescence on days 3 and 6 were randomized (n=8/group) to receive treatment beginning on day 6: anti-PD-1 (332.8H3, mouse IgG1; 500 μg intraperitoneal (IP) on day 6, 250 μg q 3 days X 7); epacadostat (Incyte Corporation, orally dosed at 300 mg/kg/day for 5 days on/2days off for 3 weeks); anti-PD-1 + epacadostat; and control therapy (isotype IgG antibody IP and 0.5% methocel in water). Tumor response assessments were performed by quantifying bioluminescence and survival. A re-challenge experiment was performed in long-term survivors to assess for tumor immune responses capable of preventing relapse. All long-term surviving mice (defined as ≥ 100 days) from the efficacy experiment were injected with 1X10 5 GL261 cells in the contralateral hemisphere and followed for survival. Results: In both preclinical efficacy experiments, median survival in the epacadostat monotherapy group did not differ from controls (approximately 30 days). Four of 8 mice (50%) treated with anti-PD-1 were long-term survivors in both efficacy experiments. In the epacadostat plus anti-PD-1 combination group, 81% of the mice were long-term survivors (7 of 8 in experiment 1 and 6 of 8 in experiment 2). Of note, none of the long-term surviving mice developed evidence of tumor; thus the median survival among the anti-PD-1 and epacadostat plus anti-PD-1 combination groups were both > 100 days. In the re-challenge study, all of the mice who underwent GL261 re-inoculation survived > 100 more days with no evidence of tumor recurrence. Conclusions: IDO1 inhibition with epacadostat increased the eradication rate of anti-PD-1 therapy in an orthotopic syngeneic GBM model and long term survivors rejected tumor following orthotopic re-challenge. Further combinatorial studies incorporating IDO inhibitor therapy for GBM, including mechanistic studies, are warranted. Citation Format: David A. Reardon, Prafulla C. Gokhale, Sarah R. Klein, Kristen L. Jones, Paul T. Kirschmeier, Maria Speranza, Holly Koblish, Peggy Scherle, Lance Leopold, Robert Newton, Gordon J. Freeman. Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 572. doi:10.1158/1538-7445.AM2017-572

Abstract 4904: The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models

Inhibitors of the BET family of bromodomain proteins have been shown to be growth inhibitory across a spectrum of tumor types due to their ability to regulate the expression of key survival and cell fate determining genes such as c-myc. In addition to their role in cancer, studies using genetic knockdown and small molecule inhibitors have demonstrated that targeting BET proteins controls the expression of pro-inflammatory cytokine genes in macrophages and is therapeutic in models of acute inflammation. These data suggest that in addition to their tumor intrinsic effects, BET inhibitors may also regulate the cytokine milieu within the tumor microenvironment and have immunomodulatory activity in cancer. To study this aspect, we evaluated INCB054329, a novel and selective BET inhibitor currently in Phase 1 trials, alone and in combination either with epacadostat, a highly selective IDO1 inhibitor, or with PD-1/PD-L1 axis blockade in syngeneic tumor models using immunocompetent animals. When used alone, INCB054329 suppressed a panel of cytokines and chemokines in a whole blood assay, confirming that INCB054329 can antagonize a pro-inflammatory response. The potency of INCB054329 in reducing the levels of these inflammatory mediators in the whole blood assay was similar to that for inhibition of c-myc, suggesting that the effects were on-target. INCB054329 was capable of inhibiting the growth of multiple syngeneic tumor models in immunocompetent mice, whereas only modest tumor growth inhibition was observed in immunodeficient mice and a lack of activity was observed in vitro, supporting the immunomodulatory activity of the compound. Because maximal in vivo tumor growth inhibition required an intact immune system, we investigated the impact of INCB054329 on various immune cell subsets, both in vitro and in vivo. Of note, increases in effector T cell populations were observed and efforts are ongoing to further characterize the tumor infiltrating immune cells following INCB054329 treatment. The mechanistic complimentarity of this novel BET inhibitor-mediated immunomodulation was also evaluated in combination with other therapeutically relevant mechanisms, including IDO1 inhibition and PD-1 axis blockade. Enhanced efficacy was observed with all INCB054329-containing regimens. These data demonstrate for the first time that BET inhibition can suppress tumor growth through both tumor-intrinsic and immune modulatory mechanisms, and support the potential of epigenetic-based, immunotherapy combinations as a novel approach to cancer therapy. Citation Format: Holly K. Koblish, Michael Hansbury, Leslie Hall, Liang-Chuan Wang, Yue Zhang, Maryanne Covington, Timothy Burn, Mark Rupar, Christine Gardiner, Thomas Condamine, Kerri Lasky, Matthew C. Stubbs, Eddy Yue, Richard Sparks, Richard Sparks, Thomas Maduskuie, Andrew P. Combs, Gregory Hollis, Reid Huber, Phillip CC Liu, Peggy Scherle. The BET inhibitor INCB054329 enhances the activity of checkpoint modulation in syngeneic tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4904.

Abstract C103: The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models

Understanding the in vivo responses to immunoregulatory agents provides a basis for building more efficacious combination regimens. Pharmacologic inhibition of the oncogenic PI3Kδ pathway has been shown to be active in patients with hematopoietic malignancies. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, through the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses, suggesting that it may have additional utility in this patient population. We identified a similar immunomodulatory role for the PI3Kδ-selective inhibitor INCB050465 in a preclinical model of pancreatic cancer, where an increase in the number of CD8+ T cells, a decrease in the number of suppressor cells and efficacy were seen. Therefore we explored the potential of INCB050465 in additional preclinical solid tumor models, alone and in combination with other immunotherapeutic agents. INCB050465 inhibited tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition was not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate immune-mediated mechanisms, tumors were analyzed for modulation of gene expression and immune phenotype after mice received short-term treatment. INCB050465 was shown to significantly downregulate the T cell gene signature in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. As seen previously, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T-cell-inflamed model by reducing both Treg and M2 macrophages, which promotes re-activation of both CD4+ and CD8+ T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell response over time. In summary, pharmacological inhibition of PI3Kδ can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and complex cellular responses may provide rationale to identify individuals who may benefit from PI3Kδ inhibitor-based immunotherapy combinations in the clinic. Citation Format: Holly K. Koblish, Liang-Chuan Wang, Michael Hansbury, Yue Zhang, Gengjie Yang, Timothy Burn, Paul Waeltz, Mark Rupar, Eddy Yue, Brent Douty, Thomas Maduskuie, Nikoo Falahatpisheh, Yun-long Li, Andrew Combs, Gregory Hollis, Reid Huber, Peggy Scherle. The combination of PI3kδ-selective inhibition and immunomodulation shows efficacy in solid tumor models. [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 C103.

Abstract 5416: Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia

The PIM family of serine-threonine protein kinases (PIM1, PIM2 and PIM3) was initially identified as preferential integration sites of the Moloney murine leukemia virus in Eμ-myc mice, resulting in perinatal lymphomagenesis. Molecular characterization has revealed that PIM kinases drive cell proliferation and survival in a number of hematological malignancies beyond lymphomas by mediating responses to cytokines, growth factors and cellular stress. Overexpression of various PIM kinase family members in these malignancies has been associated with poor overall survival and with resistance to chemotherapeutic agents. Therefore, development of a pan-PIM inhibitor may be useful in the treatment of hematological malignancies, both as a single agent and in combination with chemotherapy or targeted agents. The in vitro and in vivo activity of INCB053914, a pan-PIM kinase inhibitor, was determined in a panel of acute myelogenous leukemia (AML) cell lines. Greater than half of all AML cell lines tested were sensitive to single agent INCB053914, with anti-proliferative IC50 potencies Citation Format: Holly Koblish, Niu Shin, Leslie Hall, Sybil O9Connor, Qian Wang, Kathy Wang, Lynn Leffet, Maryanne Covington, Krista Burke, Jason Boer, Kevin Bowman, Ke Zhang, Hao Feng, Chu-Biao Xue, Yun-Long Li, Wenqing Yao, Reid Huber, Kris Vaddi, Peggy Scherle. Activity of the pan-PIM kinase inhibitor INCB053914 in models of acute myelogenous leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5416. doi:10.1158/1538-7445.AM2015-5416

Abstract B191: Preclinical assessment of targeting the PI3K/AKT pathway in combination with other signal transduction pathway inhibitors

Inhibitors targeting the phosphoinositide 3-kinase (PI3K)/serine-threonine protein kinase B (AKT) pathway have been developed and have shown efficacy in inhibiting tumor growth both in preclinical models and in the clinic. The effectiveness of PI3K/AKT inhibitors, however, is attenuated by the activation of alternative signaling pathways. Conversely, the PI3K/AKT pathway is considered to be a resistance mechanism for other therapeutic or cytotoxic agents. Therefore, understanding these mechanisms may help to inform the most optimal combinations for advancement into clinical studies. Here, we explored the potential of targeting the JAK/STAT or PIM signaling pathways in combination with a novel, potent and selective AKT inhibitor, INCB047775. Consistent with published data, pharmacological inhibition of AKT by INCB047775 caused the upregulation of PIM kinases. The combined therapies of PIM inhibition and AKT inhibition exhibited significant synergistic anti-tumor effects. Combination of PIM inhibition with AKT inhibition resulted in synergistic induction of apoptosis in cancer cells. Similarly, the combination of JAK tyrosine kinase inhibition with INCB047775 exerted synergistic inhibition of the proliferation of multiple myeloma cell lines. Similar data were observed with an inhibitor of PI3Kδ in combination with both PIM and JAK inhibition, demonstrating significant crosstalk between the PI3K/AKT pathway and other critical signaling pathways. These data provide the rationale for combinatorial therapies with inhibitors targeting JAK or PIM kinases with PI3K/AKT pathway inhibitors in the clinic. Citation Format: Sang Hyun Lee, Holly Koblish, Xiangdong Liu, Haifen Ye, Jun Li, Xuesong Liu, Melody Diamond, Yue Zhang, Leslie Hall, Valerie Dostalik, Margaret Favata, Elham Behshad, Richard Wynn, Yun-long Li, Andrew Combs, Chu-Bio Xue, Wenqing Yao, Gregory Hollis, Reid Huber, Peggy Scherle. Preclinical assessment of targeting the PI3K/AKT pathway in combination with other signal transduction pathway inhibitors. [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 B191.

Pharmacological inactivation of PI3Kδ in the tumor microenvironment enhances efficacy of other immunotherapeutic agents

Pharmacological inhibition of the oncogenic PI3Kδ pathway has been shown to be efficacious in patients with hematopoietic malignancies. However, its therapeutic application in patients with solid tumors has not yet been tested. Recently, genetic inactivation of PI3Kδ in mice was shown to delay the growth of solid tumors, resulting from the inactivation of Treg-mediated suppression of cytotoxic CD8+ T cell responses. Therefore we explored the immunotherapeutic potential of our PI3Kδ-selective compound, INCB050465, in multiple preclinical tumor models. We demonstrate that INCB050465 can block tumor growth in multiple established tumor models which are not dependent upon oncogenic PI3K signaling. Tumor growth inhibition is not observed in these models in immunocompromised mice, demonstrating that the anti-tumor effects of these agents require an intact immune system. To further investigate the immune-mediated mechanisms, tumors exposed to vehicle or INCB050465 were harvested and analyzed for modulation of gene expression and immune phenotype. INCB050465 was shown to significantly downregulate T cell gene signatures in tumors, and this was primarily due to depletion of CD4+CD25+FoxP3+ regulatory T cells. In contrast, the number of CD8+ T cells was shown to be higher in INCB050465-treated tumors. We next examined INCB050465 in combination with other immune modulators. The combination of PI3Kδ and JAK inhibition resulted in enhanced activity in a T cell-inflamed model by reducing both Treg and M2 macrophages, which then allowed the re-activation of both CD4 and CD8 T cells. In addition, PI3Kδ inhibition and PD-L1 blockade resulted in enhanced efficacy by depleting Treg and prolonging T cell responses over time. In summary, inactivation of PI3Kδ with a pharmacological inhibitor can enhance anti-tumor immunity by depleting Treg while increasing the numbers of cytotoxic CD8+ T cells. These data support clinical evaluation of the mechanism, and further studies to understand the molecular basis of efficacy and associated cellular responses may provide a rationale to identify individuals who may benefit most from PI3Kδ inhibitor-based immunotherapy combinations in the clinic.