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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.

Hematopoietic progenitor kinase 1 (HPK1) is an intracellular protein kinase that negatively regulates T cell signaling and proliferation. Upon T cell receptor (TCR) activation, active HPK1 phosphorylates the adaptor protein SLP76 in the TCR complex, recruiting the negative regulator 14-3-3 and targeting components of the TCR signaling complex for degradation. HPK1 thus limits the TCR signaling important for mounting an effective immune response against tumor cells. We are employing structure-guided drug design to develop potent small-molecule inhibitors of HPK1. Our compounds potently inhibit HPK1 in biochemical assays, reduce levels of phosphorylated SLP76 and concomitantly increase IL-2 production by Jurkat T cells. Importantly, our HPK1 inhibitors enhance cytokine production by human and mouse primary T cells above that observed with TCR activation alone. Treatment of mice with orally available HPK1 inhibitors results in increased activation of antigen-specific CD8+ T cells in vivo and decreased tumor growth as single agent and in combination with clinically relevant checkpoint inhibitor antibodies. Our work confirms the importance of HPK1 for T cell function and supports HPK1 as a promising next generation immuno-oncology target. Citation Format: George Katibah, Yamini Ohol, Cyril Bucher, Lavanya Adusumilli, Deepika Kaveri, Omar Robles, Michael Sun, Cynthia Cho, Heather Milestone, Rachel Ames, Scott Jacobson, Dan Nebalasca, Justy Gomez- Guagua, Jerick Sanchez, Molly Grandcolas, Steve Wong, Martin Brovarney, Chandru Ramana, Thant Zaw, Lan Nguyen, Parcharee Tivitmahaisoon, Andrew Ng, Anqi Ma, Blanca Gomez, Michelle Ko, Paul Leger, Jeffrey Jackson, Grant Shibuya, Anton Shakhmin, Delia Bradford, Mengshu Xu, Mikhail Zibinsky, Daniel Poon, David Wustrow, Paul Kassner, Dirk Brockstedt. Development of small-molecule HPK1 inhibitors to unleash tumor-specific T cell responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1646.

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.

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.

The CD20-specific monoclonal antibody rituximab (MabThera(®), Rituxan(®)) is widely used as the backbone of treatment for patients with hematologic disorders. Intravenous administration of rituximab is associated with infusion times of 4-6 hours, and can be associated with infusion-related reactions. Subcutaneous administration of rituximab may reduce this and facilitate administration without infusion-related reactions. We sought to determine the feasibility of achieving equivalent efficacy (measured by endogenous B-cell depletion) and long-term durability of CD20 target coverage for subcutaneously administered rituximab compared with intravenous dosing. In these preclinical studies, male cynomolgus monkeys were treated with either intravenous rituximab or novel subcutaneous formulation of rituximab containing human recombinant DNA-derived hyaluronidase enzyme. Peripheral blood samples were analyzed for serum rituximab concentrations, peripheral B-cell depletion, and CD20 target coverage, including subset analysis according to CD21+ status. Distal lymph node B-cell depletion and CD20 target coverage were also measured. Initial peak serum concentrations of rituximab were significantly higher following intravenous administration than subcutaneous. However, the mean serum rituximab trough concentrations were comparable at 2 and 7 days post-first dose and 9 and 14 days post-second dose. Efficacy of B-cell depletion in both peripheral blood and distal lymph nodes was comparable for both methods. In lymph nodes, 9 days after the second dose with subcutaneous and intravenous rituximab, B-cell levels were decreased by 57% and 42% respectively. Similarly, levels of peripheral blood B cells were depleted by >94% for both subcutaneous and intravenous dosing at all time points. Long-term recovery of free unbound surface CD20 levels was similar, and the duration of B-cell depletion was equally sustained over 2 months for both methods. These results demonstrate that, despite initial peak serum drug level differences, subcutaneous rituximab has similar durability, pharmacodynamics, and efficacy compared with intravenous rituximab.

Asthma, chronic obstructive pulmonary disease (COPD) and acute lung injury/acute respiratory distress syndrome (ALI/ARDS) are characterized by neutrophilic inflammation and elevated levels of leukotriene B4 (LTB4). However, the exact role of LTB4 pathways in mediating pulmonary neutrophilia and the potential therapeutic application of LTB4 receptor antagonists in these diseases remains controversial. Here we show that a novel dual BLT1 and BLT2 receptor antagonist, RO5101576, potently inhibited LTB4-evoked calcium mobilization in HL-60 cells and chemotaxis of human neutrophils. RO5101576 significantly attenuated LTB4-evoked pulmonary eosinophilia in guinea pigs. In non-human primates, RO5101576 inhibited allergen and ozone-evoked pulmonary neutrophilia, with comparable efficacy to budesonide (allergic responses). RO5101576 had no effects on LPS-evoked neutrophilia in guinea pigs and cigarette smoke-evoked neutrophilia in mice and rats. In toxicology studies RO5101576 was well-tolerated. Theses studies show differential effects of LTB4 receptor antagonism on neutrophil responses in vivo and suggest RO5101576 may represent a potential new treatment for pulmonary neutrophilia in asthma.

Abstract 3980 Rituximab (MabThera)® is a chimeric mouse/human monoclonal antibody recognizing the B cell specific trans-membrane protein CD20. Rituximab's CD20 specific binding facilitates the prolonged depletion of both malignant and endogenous CD20+ B cells. Rituximab is currently FDA approved for monotherapy and combinatorial therapies in the treatment of multiple forms of Non Hodgkin's Lymphoma (NHL), Chronic Lymphocytic Leukemia (CLL) and Diffuse Large B cell Lymphomas (DLBCL), as well as for the treatment of moderate to severe rheumatoid arthritis patients who are refractory to TNF antagonists (in combination with methotrexate, MTX). As with most large protein based biologics, the proto-typical route of administration of rituximab is by intravenous (IV) infusion. Following delivery to the circulation, rituximab distributes into both primary and secondary lymphoid tissues throughout the body. This distribution is essential to full target coverage of CD20+ cells and therefore critical for the efficacy in targeting those cells for depletion. For other monoclonal antibodies in other clinical settings, the conversion from IV to SC administration has resulted in an improved tolerability with less infusion-related reactions, shorter administration times, an increased patient-convenience and an improved cost-effectiveness. These advantages are anticipated for the SC administration of rituximab as well. Here we describe the results of an IV/SC pharmacokinetic/pharmacodynamic (PKPD) cynomolgus monkey study comparing the traditional IV infusion of rituximab to a novel SC dosing route of rituximab administration using an SC formulation of rituximab containing recombinant human hyaluronidase (rHuPH20). rHuPH20 transiently degrades the local interstitial matrix component, hyaluronan, at the SC injection site, acting as a permeation enhancer and allowing SC administration of high volumes. Cynomolgus monkeys were treated twice, one week apart, with the rituximab SC formulation or with IV rituximab (2 × 10 mg/kg IV or SC). CD20 target coverage and depth of distal lymph node B-cell depletion was measured 7 days later, while the prolonged target coverage and duration of peripheral blood B-cell depletion was evaluated over an extended two month period. The results indicate similar rituximab trough concentrations in serum as well as similar B-cell depletion efficacy in both peripheral blood and distal secondary lymphoid tissue after IV and SC dosing. In conclusion, the results of the study suggest that the SC versus IV dosing routes do not influence the non-clinical efficacy in this B cell depletion model given that similar trough concentrations of rituximab are reached with both administration routes. Disclosures: Del Nagro: Genentech/Roche: Employment. Mao:Roche: Employment. Dabbagh:Roche: Consultancy, Employment. Birnboeck:Roche: Consultancy, Employment. Richter:Roche: Employment.