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The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.

Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Background Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). Methods We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. Results Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. Conclusion Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. Statement of significance CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

BackgroundCheckpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME).MethodsWe developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI.ResultsUsing a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs.ConclusionTaken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer.Statement of significanceCPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.

Recruitment of suppressive CD4

The tumor microenvironment (TME) is characterized by deficiencies in oxygen and key nutrients, such as glucose and amino acids, resulting in an overall immune-suppressive environment. Key suppressive cell types in the TME include tumor, stromal and myeloid-derived suppressor cells (MDSC) which create a nutrient-poor environment that supports tumor growth and limits immune surveillance. General control nonderepressible 2 (GCN2), a stress response kinase, plays a key role in sensing and modulating the cellular response to amino acid deprivation. GCN2 activation in T cells triggers the integrated stress response pathway and promotes T cell anergy and apoptosis. We have developed small molecule GCN2 inhibitors (GCN2i) that are highly potent and selective in vitro. Culturing primary mouse or human immune cells under low nutrient conditions activates the GCN2 pathway limiting T cell proliferation and function. Treatment of these nutrient-deprived T cells with GCN2i resulted in rescue of CD8+ T cell proliferation and effector functions. In addition, GCN2 inhibition in MDSC alone fully reversed CD33+MDSC-induced T cell suppression and effector functions. Our GCN2 inhibitors are orally bioavailable with drug like in vivo ADME properties. Our GCN2i is currently being evaluated in vivo, in murine syngeneic tumor models. Our results demonstrate that inhibition of GCN2 is an attractive approach for relieving T cell suppression and promoting effector function, demonstrating GCN2 as a promising therapeutic target for the treatment of cancer. Note: This abstract was not presented at the meeting. Citation Format: Buvana Ravishankar, Lavanya Adusumilli, Deepa Pookot Pookot, Emily Huang, Raashi Sreenivasan, Lisa Marshall, Deepika Kaveri, Oezcan Talay, Silpa Suthram, Svetlana Miakicheva, Abood Okal, Mikhail Zibinsky, Jeffrey Jackson, Grant Shibuya, Paul Leger, Parcharee Tivitmahaisoon, Scott Jacobson, Steve Wong, Angela Wadsworth, Jerick Sanchez, Martin Brovarney, David Chian, Sachie Marubayashi, Aparna Jorapur, Delia Bradford, Christophe Colas, Gene Cutler, Jacob Schwartz, David Wustrow, Paul Kassner, Dirk Brockstedt. Targeting the stress response kinase GCN2 to restore immunity in the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1104.

Type 2 helper T cells (Th2)cells have been shown to express CCR4 receptor, and play a critical role in driving the pathogenesis of asthma and atopic dermatitis. FLX193 is a best-in-class, highly-potent and selective small molecule CCR4 antagonist under investigation for the treatment of allergic disorders. FLX193 blocked migration of CCR4+ Th2 cells (human and mouse) towards CCL17 and CCL22 in an in vitro chemotaxis assay. FLX193 is well-tolerated in animals at efficacious doses. In an Ovalbumin (OVA)-induced asthma model, FLX193 significantly reduced lymphocyte and eosinophil counts in the Bronchoalveolar lavage (BAL) fluid and showed a reduction of the effector Th2-relevant cytokines IL-5 and IL-13. FLX193 treatment also reduced the levels of CCL17 and CCL22 in the BAL fluid, indicating an overall reduction of inflammation. In addition, we used an atopic dermatitis mouse model to demonstrate that treatment with FLX193 decreased CCR4+ T-cell mediated inflammation. Hence, FLX193 shows promise in the treatment of atopic dermatitis.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with familial Parkinson’s disease (PD). The kinase activity of this complex protein is increased by pathogenic mutations. Inhibition of LRRK2 kinase activity has therefore emerged as a promising approach for the treatment of PD. Herein we report our findings on a series of 4-alkylamino-7-aryl-3-cyanoquinolines that exhibit kinase inhibitory activity against both wild type and G2019S mutant LRRK2. Activity was determined in both biochemical and cellular assays. Compound 14 was further evaluated in an in vivo pharmacodynamic study and found to significantly inhibit Ser935 phosphorylation after oral dosing.

USP7 is a deubiquitinase (DUB) that has attracted much attention recently due to its multiple roles in promoting cancer progression. By removal of ubiquitin from protein substrates, USP7 stabilizes oncogenes such as MDM2 and Myc, destabilizes and inactivates the key tumor suppressors p53 and PTEN, and imparts resistance to DNA-damaging chemotherapy by enhancing DNA repair responses. USP7 plays an important role in suppression of immune responses in the tumor microenvironment by stabilizing the transcription factor FOXP3 and thereby enhancing the suppressive function of regulatory T cells. Thus, inhibition of USP7 is an appealing therapeutic strategy because it has the potential to impact important oncology targets such as transcription factors that have been widely viewed as undruggable. We employed structure-based and other medicinal chemistry techniques to enable the design of potent and selective USP7 inhibitors. Using a high-throughput assay of DUB activity employing rhodamine-labeled ubiquitin, we optimized several series of reversible USP7 inhibitors to sub-100 pM potency and selectivity of >10,000-fold over all other DUBs. Cellular activity was demonstrated using a luciferase reporter gene assay of p53 activation, revealing compounds with EC50 values ranging down to 20 nM. To assess the role of USP7 inhibition in enhancement of immune responses, we determined relief of suppression of effector T cells in vitro. Effector T cells (CD8+) were co-cultured with regulatory T cells (CD4+ FOXP3+) and antigen-presenting cells for 4 days, after which CD8+ cell proliferation was determined by flow cytometry. Treatment with USP7 inhibitors during co-culture resulted in relief of regulatory T cell suppression of CD8+ cell proliferation. In vivo enhancement of immune responses was assessed in rodent models of inflammation and tumor growth. Direct effects on tumor cell growth and viability were explored by profiling cytotoxicity of USP7 inhibitors as single agents and in combination with chemotherapeutic agents in a broad range of cancer cell lines. In preparation for future clinical development, compounds were modified to obtain desirable in vitro and in vivo ADME and toxicity profiles. Following extensive pre-clinical optimization, we have in hand orally bioavailable compounds with high permeability, low clearance, and minimal off-target activity. Citation Format: Betty Abraham, Lavanya Adusumilli, Berenger Biannic, Delia Bradford, Martin Brovarney, David Chian, Gene Cutler, Xinping Han, Dennis Hu, Scott Jacobson, Sherra Johnson, Paul Kassner, Deepika Kaveri, John Ketcham, Andrea Kim, Paul Leger, Lisa Marshall, Sachie Marubayashi, Jack Maung, Jenny McKinnell, Cesar Meleza, Yamini Ohol, Akinori Okano, Leanne Peiser, Deepa Pookot, Payal Rana, Jacob Schwarz, Nick Shah, Grant Shibuya, Michael Sun, Silpa Suthram, Oezcan Talay, Angela Wadsworth, David Wustrow, Kyle Young, Andrew Napper. Discovery and optimization of potent and selective inhibitors of USP7 to enhance anti-tumor immunity and target tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2915.

Naturally suppressive CD4+ Foxp3+ Treg are essential for immune tolerance. Although Treg-mediated suppression of effector cells is important to control inflammation and prevent autoimmune diseases, the presence of Treg in the tumor microenvironment (TME) has been shown to dampen anti-tumor immune responses. Human Treg express CCR4, the receptor for the chemokines CCL17 and CCL22. These chemokines are produced by tumor cells, tumor-associated macrophages and dendritic cells, as well as by effector T cells (Teff). Preclinical and clinical data supports a role for CCR4-mediated recruitment and accumulation of Treg in the TME which can be associated with poor prognosis. Further, recent longitudinal studies in patients receiving IO agents demonstrate an influx of Treg in responding patients which may dampen optimal anti-tumor responses. Therefore, CCR4 is an ideal target to selectively block Treg recruitment into the TME. We have developed structurally unique series of small molecule antagonists of CCR4. These antagonists have cellular potencies in multiple assays (e.g. chemotaxis of primary human Treg in 100% serum) in the low double-digit nM range. Representative compounds are selective against other chemokine receptors, GPCRs and ion channels, including the hERG channel, and lack inhibition of common human CYP450 enzymes. Moreover, compounds have excellent in vitro and in vivo ADME properties, consistent with convenient oral dosing. In preclinical syngeneic tumor models, these CCR4 antagonists block Treg migration and support expansion of activated Teff. In contrast to the non-selective approach of depleting anti-CCR4 antibodies, our compounds reduce Treg in the tumor, but not in peripheral tissues such as blood, spleen or skin. In preclinical efficacy studies, CCR4 antagonists potentiate the anti-tumor effects of various checkpoint inhibitors and immune stimulators such as anti-PD-L1 and anti-CD137 antibodies. We observe enhanced tumor growth inhibition and increased tumor regressions when these agents are combined with CCR4 antagonists, without any gross toxicity. Further characterization of these CCR4 antagonists and their anti-tumor effects will be described. Citation Format: Oezcan Talay, Lisa Marshall, Cesar Meleza, Maureen K. Reilly, Omar Robles, Mikhail Zibisky, Abood Okal, Lisa Seitz, Jenny McKinnell, Scott Jacobson, Erin Riegler, Emily Karbaz, David Chian, Angela Wadsworth, Paul Kassner, David Wustrow, Jordan S. Fridman. Potent and selective C-C chemokine receptor (CCR4) antagonists potentiate anti-tumor immune responses by inhibiting regulatory T cells (Treg) [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 4600. doi:10.1158/1538-7445.AM2017-4600

Several neurological diseases, including Parkinson disease and dementia with Lewy bodies, are characterized by the accumulation of alpha-synuclein phosphorylated at Ser-129 (p-Ser-129). The kinase or kinases responsible for this phosphorylation have been the subject of intense investigation. Here we submit evidence that polo-like kinase 2 (PLK2, also known as serum-inducible kinase or SNK) is a principle contributor to alpha-synuclein phosphorylation at Ser-129 in neurons. PLK2 directly phosphorylates alpha-synuclein at Ser-129 in an in vitro biochemical assay. Inhibitors of PLK kinases inhibited alpha-synuclein phosphorylation both in primary cortical cell cultures and in mouse brain in vivo. Finally, specific knockdown of PLK2 expression by transduction with short hairpin RNA constructs or by knock-out of the plk2 gene reduced p-Ser-129 levels. These results indicate that PLK2 plays a critical role in alpha-synuclein phosphorylation in central nervous system.

The IDO1 pathway has been proposed to mediate immunosuppressive effects in the tumor microenvironment through its role in the catabolism of tryptophan, resulting in effects on the differentiation and proliferation of T cells. IDO1 inhibition has shown promising clinical benefit as well as exacerbated toxicity in the treatment of melanoma, when combined with the anti-CTLA-4 antibody ipilimumab. We have discovered a novel class of highly selective small molecule inhibitors of IDO1 which surpass the potency of the compounds currently in clinical development. These compounds potently inhibit IDO1 activity in IFN-γ stimulated HeLa cells with single digit nM potency. Importantly, they also retain their potency in the presence of human serum, with IC50 values ranging between 5 and 15 nM in this more physiologically relevant media. Consistent with the role of IDO1+ dendritic cells in the suppression of T cell proliferation, this series of molecules is capable of restoring the proliferative capacity of human T cells (which is inhibited by allogeneic IDO1+ dendritic cells) with EC50 values of 2-3 nM. The molecules exhibit preclinical PK characteristics that are suitable for assessing the contribution of IDO1 to tumor growth in murine models, both alone and in combination with other therapeutic agents. The compounds have high metabolic stability against cultured human hepatocytes and exhibit preclinical PK and ADME characteristics consistent with once-daily dosing in humans. The full preclinical profile of one of these molecules, selected for clinical evaluation, will be the focus of this presentation. In conclusion, we have discovered a novel class of small molecule inhibitors of IDO1, which provides a preclinical basis for the clinical evaluation of a next generation IDO1 inhibitor in combination with other therapeutic agents. Citation Format: Jay P. Powers, Matthew J. Walters, Rajkumar Noubade, Stephen W. Young, Lisa Marshall, Jan Melom, Adam Park, Nick Shah, Pia Bjork, Jordan S. Fridman, Hilary P. Beck, David Chian, Jenny V. McKinnell, Maksim Osipov, Maureen K. Reilly, Hunter P. Shunatona, James R. Walker, Mikhail Zibinsky, Juan C. Jaen. Potent and selective next generation inhibitors of indoleamine-2,3-dioxygenase (IDO1) for the treatment of 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 4290. doi:10.1158/1538-7445.AM2015-4290