Abstract Hematopoietic progenitor kinase 1 (HPK1) has been shown to act as a negative regulator of T cell receptor signaling and of subsequent effector T cell function. Inhibiting HPK1 to enhance T cell activity has emerged as a promising strategy for cancer immunotherapy. Upon T cell receptor engagement, the kinase domain of HPK1 is activated and targets components of the T cell receptor (TCR) signaling pathway for degradation, including SLP76. However, the molecular events connecting HPK1 kinase activity to observed T cell functions downstream of proximal TCR signaling are not well understood. Through transcriptional profiling of HPK1-kinase-dead (HPK1-KD) versus wild-type CD8+ T cells following an acute, attenuated Listeria monocytogenes infection, we observed increased expression of many genes associated with T cell activation and effector function, including changes in expression of several key transcription factors and their targets. Upon activation, HPK1-KD T cells, as well as T cells treated with our small-molecule HPK1 inhibitor, produce higher levels of a wide range of effector cytokines in vitro and in vivo. Treatment of mice with the HPK1 inhibitor also inhibited tumor growth in several syngeneic tumor models. These data expand our understanding of the role of HPK1 in inhibiting CD8+ T cell effector programs, and, importantly, the impact of HPK1 inhibitors on T cell function. Citation Format: Rachel Y. Ames, Rongqi Zhao, Parker Mace, Adam Grant, Guorui Xie, Heather Milestone, Molly Grandcolas, Eva Fang, Nicolae Kiosea, Stephen Wong, Martin Brovarney, Scott Jacobson, Danilo Nebalasca, Jorge Arguello, Blanca Gomez, Hiranmayee Kandala, Michelle Y. Ko, Lan Nguyen, Omar Robles, Grant Shibuya, Anton A. Shakhmin, Parcharee Tivitmahaisoon, Vi-Anh Vu, Ashkaan Younai, Mikhail Zibinsky, Babu Subramanyam, Daniel Poon, Mohsen Sabouri Ghomi, David J. Wustrow, Paul D. Kassner, George E. Katibah, Dirk G. Brockstedt. HPK1 inhibits CD8+ T cell effector gene expression following T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2654.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.

The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (T-reg) as well as other circulating and tissue-resident T cells. T-reg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. T-reg 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 T-reg 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 T-reg 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.

OBJECTIVE: To compare the potential for oral XP23829 and DMF to cause persistent GI irritation in animals after repeated dosing. BACKGROUND: XP23829 and DMF are both prodrugs of monomethyl fumarate (MMF). DMF was shown to be effective in treatment of relapsing-remitting multiple sclerosis (MS) and psoriasis, but can cause gastrointestinal (GI) adverse effects. XP23829 has similar efficacy to DMF in animal models of MS and psoriasis, but shows less gastric irritation in acute (4 day) studies in rats. DESIGN/METHODS: XP23829 was administered to CD rats (15/sex/dose) at 0 (vehicle), 150, 250, or 500 mg/kg/day and monkeys (3/sex/group) at 0 (vehicle), 25, 75, or 200 mg/kg/day for 4 weeks in GLP toxicity studies. DMF was administered to parallel groups at similar MMF exposures. GI toxicity was assessed by gross necropsy and histopathology. Toxicokinetics were assessed in parallel cohorts. Tissue distribution of 14C-labeled DMF and XP23829 (both labeled in fumarate) were compared in rats by whole body autoradiography. RESULTS: DMF caused dose-dependent GI irritation in rats and monkeys after 4 weeks. In rats, 300 mg/kg/day DMF caused ulceration, necrosis and loss of mucosa of glandular stomach. At similar MMF exposure, XP23829 showed no adverse effects on glandular stomach. In monkeys, DMF showed greater gastric mucosal hyperplasia than XP23829. Both prodrugs were converted rapidly to MMF after absorption; DMF and XP23829 were not detected in the blood. Radioactivity in stomach mucosa was observed 24 hours after dosing 14C-labeled DMF but not for XP23829. CONCLUSIONS: DMF caused significantly greater local gastric irritation after 4 weeks in rats and monkeys. DMF, but not XP23829, remains localized in gastric mucosa 24 hours after oral dosing of rats. XP23829 may have the potential for a lower risk of GI side effects in clinical use compared to DMF. Supported by: XenoPort, Inc. Disclosure: Dr. McCullough has received personal compensation for activities with XenoPort, Inc. Dr. McCullough has received research support from XenoPort, Inc. Dr. Annamalai has received personal compensation for activities with XenoPort, Inc as an employee. Dr. Annamalai has received research support from XenoPort, Inc. Dr. Wustrow has received personal compensation for activities with XenoPort Inc and Cleave Bioscience. Dr. Wustrow holds stock and/or stock options in Xenoport Inc. Dr. Wustrow has received research support from XenoPort, Inc., and Cleave Bioscience. Dr. Cundy has received personal compensation for activities with Xenoport, Inc. Dr. Cundy has received research support from Xenoport, Inc.

AbstractCompound ((R)‐I) shows a 25 to 50fold greater potency than R‐baclofen at human and rodent GABAB receptors in vitro.

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.