Clinical and molecular effects of oral CCR4 antagonist RPT193 in atopic dermatitis: A Phase 1 study.

BACKGROUND: RPT193 is an orally administered small molecule antagonist of the human C-C motif chemokine receptor 4 (CCR4) that inhibits the migration and downstream activation of T-helper Type 2 (Th2) cells. We investigated single- and multiple-ascending doses of RPT193 in healthy subjects, and multiple doses of RPT193 in subjects with moderate-to-severe atopic dermatitis (AD). METHODS: This was a first-in-human randomized, placebo-controlled Phase 1a/1b monotherapy study (NCT04271514) to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and CCR4 surface receptor occupancy in eligible healthy subjects and subjects with moderate-to-severe AD. Clinical efficacy and skin biomarker effects of RPT193 monotherapy were assessed as exploratory endpoints in AD subjects. RESULTS: In healthy (n = 72) and AD subjects (n = 31), once-daily RPT193 treatment was generally well tolerated, with no serious adverse events reported and all treatment-emergent adverse events reported as mild/moderate. In AD subjects, numerically greater improvements in clinical efficacy endpoints were observed with RPT193 monotherapy versus placebo up to the end of the treatment period (Day 29), with statistically significant improvement, compared to Day 29 and placebo, observed 2 weeks after the end of treatment (Day 43) on several endpoints (p < .05). Moreover, significant changes in the transcriptional profile were seen in skin biopsies of RPT193-treated versus placebo-treated subjects at Day 29, which were also significantly correlated with improvements in clinical efficacy measures. CONCLUSIONS: To our knowledge, this is the first clinical study with an oral CCR4 antagonist that showed clinical improvement coupled with modulation of the cutaneous transcriptomic profile in an inflammatory skin disease.

Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

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.

EBV+ tumors exploit tumor cell-intrinsic and -extrinsic mechanisms to produce regulatory T cell-recruiting chemokines CCL17 and CCL22.

The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.

Safety and preliminary immunogenicity of JNJ-64041809, a live-attenuated, double-deleted Listeria monocytogenes-based immunotherapy, in metastatic castration-resistant prostate cancer.

BACKGROUND: The safety and immunogenicity of JNJ-64041809 (JNJ-809), a live-attenuated, double-deleted Listeria monocytogenes (LADD Lm)-based immunotherapy targeting 4 relevant prostate cancer antigens, was evaluated in a phase 1 study in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: Men with progressive mCRPC who had received ≥2 prior approved therapies were enrolled. Primary study objectives were to determine the recommended phase 2 dose (RP2D) and to evaluate the safety and immunogenicity of JNJ-809. RESULTS: A total of 26 patients received JNJ-809 (1 × 108 CFU (n = 6); 1 × 109 CFU (n = 20)). No dose-limiting toxicities were reported, and 1 × 109 CFU was selected as the RP2D. The most common adverse events (AEs) reported were chills (92%), pyrexia (81%), and fatigue (62%). The most frequent grade ≥3 AEs were lymphopenia (27%) and hypertension (23%). Serious AEs were reported in 27% of patients including 1 patient with grade 3 intestinal obstruction. JNJ-809 transiently induced peripheral cytokines, including interferon-γ, interleukin-10, and tumor necrosis factor-α. Of the 7 patients evaluable for T cell responses at the 1 × 109 CFU dose, evidence of post-treatment antigenic responses were observed in 6 to the Listeria antigen listeriolysin O and in 5 to ≥1 of the 4 encoded tumor antigens. Best overall response was stable disease in 13/25 response-evaluable patients. The study was terminated early as data collected were considered sufficient to evaluate safety and immunogenicity. CONCLUSIONS: JNJ-809 has manageable safety consistent with other LADD Lm-based therapies. Limited antigen-specific immune responses were observed, which did not translate into objective clinical responses.

Tumors establish resistance to immunotherapy by regulating Treg recruitment via CCR4.

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.

Novel, Selective Inhibitors of USP7 Uncover Multiple Mechanisms of Antitumor Activity In Vitro and In Vivo.

The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.

Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as a Single Agent and in Combination with Checkpoint Inhibitors.

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.

Discovery of Potent, Selective, and Orally Bioavailable Inhibitors of USP7 with In Vivo Antitumor Activity.

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.

Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer.

PURPOSE: Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated Listeria monocytogenes-expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B). PATIENTS AND METHODS: Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates. RESULTS: Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7-8.6] and 6.1 (95% CI, 3.5-7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55-1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8+ T cells and a decrease in CD68+ myeloid cells, were observed in long-term survivors in Arm A only. CONCLUSIONS: Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.

Single-Cell Immune Competency Signatures Associate with Survival in Phase II GVAX and CRS-207 Randomized Studies in Patients with Metastatic Pancreatic Cancer.

The identification of biomarkers for patient stratification is fundamental to precision medicine efforts in oncology. Here, we identified two baseline, circulating immune cell subsets associated with overall survival in patients with metastatic pancreatic cancer who were enrolled in two phase II randomized studies of GVAX pancreas and CRS-207 immunotherapy. Single-cell mass cytometry was used to simultaneously measure 38 cell surface or intracellular markers in peripheral blood mononuclear cells obtained from a phase IIa patient subcohort (N = 38). CITRUS, an algorithm for identification of stratifying subpopulations in multidimensional cytometry datasets, was used to identify single-cell signatures associated with clinical outcome. Patients with a higher abundance of CD8+CD45RO-CCR7-CD57+ cells and a lower abundance of CD14+CD33+CD85j+ cells had improved overall survival [median overall survival, range (days) 271, 43-1,247] compared with patients with a lower abundance of CD8+CD45RO-CCR7-CD57+ cells and higher abundance of CD14+CD33+CD85j+ cells (77, 24-1,247 days; P = 0.0442). The results from this prospective-retrospective biomarker analysis were validated by flow cytometry in 200 patients with pancreatic cancer enrolled in a phase IIb study (P = 0.0047). The identified immune correlates provide potential prognostic or predictive signatures that could be employed for patient stratification.