There are many pathways that activate the Type I interferon (IFN) response to combat tumor progression including the cytosolic nucleic acid sensing pathways. PARP7 is a monoART enzyme that was reported to negatively regulate the Type I IFN response by interacting with TBK1 during viral infection. We and others have demonstrated that PARP7 is a novel negative regulator of cytosolic nucleic acid sensing in tumor cells. RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. We have shown that administration of RBN-2397 to CT26 tumor-bearing mice significantly decreased tumor growth and led to complete and durable regressions in a subset of mice. The antitumor effects correlated with activation of p-STAT1 and increased expression of ISGs including CXCL10, MX1, and CCL5 in tumors. A growing body of work has shown that DNA damaging agents such as chemotherapies activate the Type I IFN response by generating accumulation of nucleic acids, including DNA, in the cytosol or tumor microenvironment. These aberrant cytosolic nucleic acids are recognized by the innate immune response via cGAS leading to activation of the STING/TBK pathway and subsequent production of IFN-β and expression of ISGs (interferon induced genes). Given that PARP7 suppresses the Type I IFN response, we hypothesized that the immune-related mechanism observed with chemotherapy would be inhibited in tumors with high PARP7 expression. Combining chemo with RBN-2397 could potentiate Type I IFN response activation in PARP7 expressing tumors and enhance immune mediated anti-tumor responses. We tested the ability of platinum (cisplatin, oxaliplatin, and carboplatin) and taxane (docetaxel and paclitaxel) chemotherapy agents to activate the Type I IFN response in the CT26 mouse colon cancer cell line in vitro by gene expression analysis of several ISGs (CXCL10, CCL5, MX1, IFIT1, and IFN-β). Single agent chemo led to modest increases in the expression of ISGs. The combination of cisplatin or oxaliplatin with RBN-2397 significantly potentiated the effect of RBN-2397 and led to a synergistic increase of the ISGs in CT26 cells after 24 hours of treatment. Similar effects were obtained with the combination of docetaxel or paclitaxel with RBN-2397. To evaluate if the combinatorial effect observed in vitro translated into increased efficacy in vivo, we first identified doses of single agent cisplatin or docetaxel that increased expression of ISGs and enhanced cytosolic-DNA in CT26 tumors. Next, we verified the tolerability of validated doses in combination with RBN-2397. Finally, we performed an efficacy study in CT26 tumor bearing mice and observed that the combination of cisplatin and RBN-2397 increased tumor growth inhibition and survival relative to single agent activity. This study provides rationale for exploration of DNA damaging agents in combination with RBN-2397. Citation Format: Kaiko Kunii, Jeff J. Song, Danielle Zimmerman, Chang Liu, Kristy G. Kuplast-Barr, Jonathan J. Novak, Sunaina P. Nayak, Joseph M. Gozgit, Melissa M. Vasbinder, Kristen McEachern, Kevin W. Kuntz, Heike Keilhack, Jennifer R. Molina. Potentiation of Type I interferon signaling leads to in vivo efficacy achieved with combination of chemotherapy and the PARP7 inhibitor RBN-2397 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3260.

Background: PARP7 is a stress-induced monoART that suppresses the cellular type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor that induces IFN and an adaptive immune response. The tumor-intrinsic immunomodulatory mechanism of RBN-2397 and preliminary antitumor activity in patients (pts) was demonstrated during dose escalation (Falchook, ASCO 2021; Kuplast-Barr, AACR 2022). Methods: Pts with solid tumors were treated with RBN-2397 at the RP2D of 200 mg BID in 3 expansion cohorts: squamous cell carcinoma of the lung (SCCL), head and neck squamous cell carcinoma (HNSCC), and hormone receptor-positive breast cancer (HR+ BC). Objectives of the expansion phase included safety, pharmacokinetics, pharmacodynamics, and antitumor activity. Results: As of 2 July 2022, 31 pts have been treated: SCCL (n=13), HNSCC (n=10), and HR+ BC (n=8). RBN-2397-related AEs (all grades >10%) included dysgeusia (42%, n=13), nausea (26%, n=8), fatigue (23%, n=7), with Grade 3 events of nausea and pleural infection (each n=1) and ALT/AST increase (n=2), and no Grade 4 events. No significant chronic toxicities were observed. The disease control rate in response-evaluable pts was 44% in SCCL (stable disease [SD] in 4/9 pts), 71% in HNSCC (RECIST partial response [PR] for 12+ months in 1/7; SD in 4/7), and 29% in HR+ BC (SD in 2/7). Biomarker analyses confirmed PARP7 mRNA expression in all baseline biopsies, with H-scores higher in tumor cells than in stromal cells (n=26, H-score range 66-256, P<0.0001). Four of 17 evaluable pts showed PARP7 focal copy number gains, and 2 had copy number gains that tracked with chromosome 3 copy number. One pt with HPV-negative HNSCC (nonsmoker; paranasal sinus primary) has an ongoing durable RECIST PR (−41%) for 12+ months. Tumor analysis of this responder confirmed PARP7 expression at baseline and SMARCB1 deletion, and demonstrated an excluded immune phenotype by IHC and MIBI-SCOPE. Analyses of paired tumor biopsies confirmed induction of adaptive immunity with ≥2-fold increases in CD8+ T cells and/or granzyme B expression in 10 (63%) of 16 pts across tumor types. Increases in immune checkpoint expression (PD-1 and LAG3 on T cells; PD-L1 on tumor cells) from 30% to 150% were observed in 3 (60%) of 5 pt tumor samples evaluated using MIBI-SCOPE, indicating the potential to prime tumors for immune checkpoint inhibitor therapy. These changes were independent of PARP7 copy number or PARP7 mRNA expression level at baseline. Conclusions: RBN-2397 was well tolerated at biologically active drug exposures, with preliminary antitumor activity observed. Paired tumor biopsy translational studies demonstrated the immunomodulatory mechanism of RBN-2397 and support the ongoing trial of RBN-2397 in combination with pembrolizumab (NCT05127590). Citation Format: Timothy A. Yap, Andres Cervantes, Gerald S. Falchook, Manish R. Patel, Dejan Juric, Saiama N. Waqar, Erin L. Schenk, Geoffrey Shapiro, Valentina Boni, Cesar A. Perez, Barbara Burtness, Yana G. Najjar, Fabricio Racca, Katerin Rojas, Kristy Kuplast-Barr, Kristen McEachern, Manoj Samant, Viviana Bozón, Sudha Parasuraman, Melissa Johnson. First-in-class first-in-human phase 1 trial and translational study of the mono(ADP-ribose) polymerase-7 (PARP7) inhibitor RBN-2397 in patients with selected advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT109.

Supplementary Materials and Methods; Supplementary Figure S1. Binding kinetics of hedgehog antibodies to human and mouse hedgehog proteins using surface plasmon resonance. Supplementary Figure S2. Hedgehog ligands stimulate mGLI1 reporter activity and osteoblast differentiation in a dose-dependent manner. Supplementary Figure S3. Selective inhibition of mGLI1 or mPTC1 induction in C3H10T1/2 cells by hedgehog antibodies. Supplementary Figure S4. MEDI-5304 inhibits GLI1 expression in Colo205 xenograft stromal cells but not tumor cells. Supplementary Figure S5. Hedgehog pathway component RNA expression in primary pancreatic tumor explant model P479 and effects of MEDI-5304 on tumorspheres derived from pancreatic explant model 947. Supplementary Figure S6. Pharmacokinetics of MEDI-5304 in rats. Supplementary Figure S7. Exploratory toxicology of MEDI-5304 in rats. Supplementary Figure S8. Pharmacokinetics of MEDI-5304 from preliminary toxicology study in cynomolgus monkeys. Supplementary Figure S9. Pharmacodynamics of MEDI-5304 in cynomolgus monkeys.

Abstract Introduction: PARP7, encoded by the TIPARP gene, is a monoART involved in cellular stress responses and with immunomodulatory functions in cancer. The PARP7 gene is amplified in a subset of squamous cell carcinomas and ongoing clinical studies are assessing its role as anti-cancer therapeutic target (NCT04053673, NCT05127590) Here, we analyzed the frequency of PARP7 copy number alterations and its association with tumor immune microenvironment (TIME) features and outcomes in non-small cell lung cancer (NSCLC) and head & neck squamous-cell carcinoma (HNSCC) cohorts. Methods: The PARP7 gene copy number and amplification was analyzed using a dual probe fluorescence in situ hybridization (FISH) assay in two retrospective cohorts of NSCLC (Cohort #1, n=124) and HNSCC (Cohort #2, n=83) represented in tissue microarray format. In a subset of cases, the PARP7 copy number was also studied using whole exome DNA sequencing. The TIME was assessed on consecutive tumor sections using a multiplexed quantitative immunofluorescence panel including the markers DAPI, cytokeratin, CD4, CD8 and PD-L1 coupled to computational pathology analysis. The association between the markers, with clinicopathologic variables and survival was studied. Results: Increased PARP7 copy number (>2 copies/cell) was identified in 55% of NSCLCs (85% of them of squamous-cell histology) and 76% of HNSCCs. A high copy number (>3 copies/cell) was identified in 22% cases from both tumor type cohorts. After adjustment of the PARP7 gene copy number by the chromosome 3 centromeric probe (CEN3) signal, PARP7 amplification (>1.5 PARP7/CEN3 signal) was detected in 22% of NSCLC cases and 34% of HNSCC. A higher number of cases with PARP7 copy number gains were identified using FISH than with whole exome DNA sequencing. No significant associations between the PARP7 gains/losses and major clinicopathologic variables were found. However, higher PARP7 copy number was associated with reduced CD8+ or CD4+ tumor infiltrating lymphocytes (TILs) and a numerically lower tumor PD-L1 protein levels in HNSCC. Elevated PARP7 copy number was associated with shorter overall survival in both studied tumor types. Conclusion: Elevated PARP7 gene copy number identifies a subset of lung and head & neck squamous-cell carcinomas with unfavorable TIME features and adverse prognosis. Detection of PARP7 gene abnormalities using FISH in tumor biopsy samples is sensitive and has potential as a predictive biomarker. Citation Format: Viviana A. Ahumada, Kristen McEachern, Kristy Kuplast-Barr, Kurt Alex Schalper. Clinical significance of PARP7 (TIPARP) gene copy number alterations in human non-small cell and head & neck carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2508.

Background: PARP7 is a mono-ART that is upregulated in response to cellular stress (e.g., viral infection, cigarette smoke), and suppresses the Type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor, inducing cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Moreover, RBN-2397 induces CD8 T cell-dependent tumor-specific immune memory in an immunocompetent mouse cancer model [1]. RBN-2397 is currently being tested in an ongoing Phase I clinical study (NCT04053673) [2]. Here we present evidence of proof of mechanism in the paired biopsies of tumors from Phase 1 patients. Methods: Plasma CXCL10 from patients was measured by MSD while ISG expression in PBMCs was measured by NanoString. Baseline and on-treatment patient tumor biopsies were analyzed by NanoString, CD8/GZMB IHC, and MIBI-TOF to characterize immune changes in the tumor microenvironment. Results: In peripheral blood from patients treated with RBN-2397, neither plasma nor PBMC CXCL10 increased more than 2-fold over baseline. Expression of 42 ISGs was not consistently induced in a dose-dependent manner in PBMCs. However, in tumor types of interest (e.g., cancers of the upper aerodigestive tract), CXCL10 expression increased, with similar effects observed for a subset of ISGs in multiple evaluable paired biopsy samples. Confirming preclinical studies [1], increases in CD8 T cell infiltration along with induction of granzyme B expression were observed in several evaluable paired patient tumor biopsies by immunohistochemistry. Using the MIBI-TOF technology, we observed up to 50-fold increases in intratumoral activated T cells as well as monocytes and M1 macrophages, most strikingly in two NSCLC patients. Conclusions: In patients treated with RBN-2397 pharmacodynamic effects were preferentially observed in tumor tissue relative to the periphery, including an increase in immune infiltration into the tumor microenvironment. These data provide evidence for induction of an adaptive immune response and confirm the tumor-intrinsic, immunomodulatory mechanism of action of RBN-2397 in patients. References: 1. Gozgit et al. PARP7 negatively regulates the Type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell. 2021 2. Falchook et al. A First-In-Human Phase 1 Study of a Novel PARP7 Inhibitor RBN-2397 in Patients with Advanced Solid Tumors. ASCO 2021 oral presentation Citation Format: Kristy Kuplast-Barr, Melissa L. Johnson, Manish R. Patel, Timothy A. Yap, Gerald S. Falchook, Patricia LoRusso, Ryan Abo, Chang Liu, Erika L. Manyak, Lisa Cleary, Viviana Bozon, Sudha Parasuraman, Heike Keilhack, Kristen McEachern. RBN-2397, a novel, potent, and selective PARP7 inhibitor, induces tumor-intrinsic type I interferon responses and adaptive immunity in patient tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1836.

Targeting cytosolic nucleic acid sensing pathways to activate the Type I interferon (IFN) response is an emerging therapeutic strategy being explored in oncology. The PARP family consists of seventeen enzymes that regulate fundamental biological processes including response to cellular stress. PARP7 (TIPARP) is a stress-induced mono-ART that catalyzes the transfer of a single unit of ADP-ribose onto substrates (MARylation) to regulate their function and plays a role in suppressing the Type I IFN response in tumor cells (Gozgit 2021 Cancer Cell). RBN-2397 is the first potent and selective small molecule inhibitor of PARP7 catalytic function. To investigate the cell autonomous effects of PARP7 inhibition, we performed a cell line screen to identify PARP7 dependent cancer cell lines. We found that treatment of a subset of lines across several cancers led to a robust decrease in cell viability. Additionally, dosing of tumor bearing mice led to complete regressions in NCI-H1373 lung cancer xenografts. To investigate the mechanism of action (MOA) leading to decreased cell viability, we treated NCI-H1373 cells with RBN-2397 and found accumulation of cells in the G0/G1 phase of the cell cycle indicative of a cell cycle arrest. This arrest in NCI-H1373 cells was associated with the induction of senescence and increased mRNA expression of senescence associated secretory phenotype (SASP) genes. To evaluate the in vivo MOA, we performed an NCI-H1373 xenograft study and collected tumors after 7 days of RBN-2397 treatment. PARP7 inhibition led to decreased expression of Ki67, and increased expression of P21 and cleaved caspase-3, suggesting decreased proliferation and increased apoptosis. Increased expression of SASP genes was also observed in RBN-2397 treated tumors. Finally, we investigated transcriptional changes after RBN-2397 treatment by RNA sequencing. In addition to the effects observed in Type I IFN signaling, we also observed differential expression of genes associated with other pathways including autophagy and energy metabolism. Further evaluation of key autophagy proteins revealed that RBN-2397 affects autophagy flux and leads to a decrease in the oxygen consumption rate of cells and reduced ATP production from the mitochondria, suggesting that a change in energy metabolism may be related to the tumor intrinsic effect of RBN-2397. In summary, we show treatment of cancer cells with RBN-2397 not only leads to activation of tumor cell IFN signaling, but also causes G1 arrest and senescence, and changes in cancer cell autophagy and energy metabolism. In vivo, RBN-2397 treatment leads to complete tumor regressions in xenografts accompanied by decreased proliferation and increased apoptosis of tumor cells. RBN-2397 is currently being evaluated in the clinic as single agent in selected cancer types (NCT04053673) and in combination with anti-PD-1 therapies. Citation Format: Jennifer R. Molina, Joseph M. Gozgit, Melissa M. Vasbinder, Ryan P. Abo, Kaiko kunii, Kristy G. Kuplast-Barr, Bin Gui, Sunaina P. Nayak, Elena Minissale, Kerren K. Swinger, Tim J. Wigle, Alvin Z. Lu, Danielle J. Blackwell, Christina R. Majer, Yue Ren, Ellen Bamberg, Mario Niepel, Jan-Rung Mo, William D. Church, Ahmed S. Mady, Jeff Song, Zacharenia A. Varsamis, Luke Utley, Patricia E. Rao, Timoty J. Mitchison, Kevin W. Kuntz, Victoria M. Richon, Kristen McEachern, Heike Keilhack. PARP7 inhibitor RBN-2397 increases tumoral IFN signaling leading to various tumor cell intrinsic effects and tumor regressions in mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2154.

Background PARP7 is a mono-ART that is upregulated in response to cellular stress (e.g., viral infection, cigarette smoke), and suppresses the Type I interferon (IFN) response following cytosolic nucleic acid sensing. RBN-2397 is a first-in-class PARP7 inhibitor, inducing cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Moreover, RBN-2397 induces CD8 T cell-dependent tumor-specific immune memory in an immunocompetent mouse cancer model. 1 RBN-2397 is currently being tested in an ongoing Phase I clinical study ( NCT04053673 ). 2 Here we aimed to compare biomarker results from preclinical models and patient samples. Methods In preclinical models, interferon-stimulated gene (ISG) expression was assessed by qPCR, NanoString, or ELISA. Plasma CXCL10 from patients was measured by MSD while ISG expression in PBMCs was measured by NanoString. Baseline and on-treatment patient tumor biopsies were analyzed by NanoString, CD8/GZMB IHC, and MIBI-TOF to characterize immune changes in the tumor microenvironment. Results RBN-2397 potently restored tumoral Type I IFN signaling in preclinical models as demonstrated by increases in ISGs, namely CXCL10, which were not observed in non-tumor tissue (e.g. spleen, PBMCs). In peripheral blood from patients treated with RBN-2397, neither plasma nor PBMC CXCL10 increased more than 2-fold over baseline. Expression of 42 ISGs was not consistently induced in a dose-dependent manner in PBMCs. However, in tumor types of interest (e.g. cancers of the upper aerodigestive tract), CXCL10 expression increased 1.5 to 8-fold, with similar effects observed for a subset of ISGs in 5 evaluable paired biopsy samples.Confirming preclinical studies [1], up to 8-fold increases in CD8 T cell infiltration along with induction of granzyme B expression were observed in 4 of 5 paired patient tumor biopsies by immunohistochemistry. Using the MIBI-TOF technology, we observed up to 50-fold increases in intratumoral activated T cells as well as monocytes and M1 macrophages, most strikingly in two NSCLC patients. Conclusions Inhibition of PARP7 with RBN-2397 restores tumor-intrinsic Type I IFN signaling in preclinical models leading to enhanced adaptive immunity, resulting in CD8 T cell-dependent durable tumor regressions. These observations are mirrored in samples from patients treated with RBN-2397 in that pharmacodynamic effects of RBN-2397 were preferentially observed in tumor tissue relative to the periphery, including an increase in immune infiltration into the tumor microenvironment. These data provide evidence for induction of an adaptive immune response and confirm the tumor-intrinsic, immunomodulatory mechanism of action of RBN-2397 in patients. References Gozgit, et al . PARP7 negatively regulates the type I interferon response in cancer cells and its inhibition triggers antitumor immunity. Cancer Cell 2021; In press. Falchook, et al . A first-in-human phase 1 study of a novel PARP7 inhibitor RBN-2397 in patients with advanced solid tumors. ASCO 2021; oral presentation.

Abstract CD38 is an ADP-ribosyl cyclase that converts NAD+ to ADP-ribose (ADPR) or cyclic ADPR (cADPR) and nicotinamide. The enzyme can exist in either an ecto- or endo-catalytic orientation with different sub-cellular localization, and therefore can regulate internal and external NAD+ pools. Both NAD+ and cADPR can impact T cell fitness and effector function, and CD38 has been shown to be increased in settings of chronic T cell activation. CD38 can mediate the non-canonical generation of the immune suppressive adenosine by catabolizing extracellular NAD+ resulting in immunosuppression in the microenvironment. Upon immune checkpoint inhibitor (ICI) therapy, CD38 is upregulated on cancer cells to drive ICI resistance. Therefore CD38, through its catalytic activity, has been implicated in tumor immune suppression and ICI resistance. Genetic knockout of CD38 has been shown to prevent tumor growth and improve T cell fitness. Here, we describe the effects of CD38 inhibition using a small molecule inhibitor on these key metabolites in various cellular and tumor models. RBN013209 is a potent and selective small molecule inhibitor of CD38 catalytic function. We demonstrate that inhibition of CD38 with RBN013209 prevents conversion of extracellular NAD+ to ADPR or cADPR in cancer cell lines and PBMCs. Similarly, RBN013209 inhibited intracellular CD38 activity and elevated intracellular NAD+ levels in cultured human primary T cells. Oral administration of RBN013209 to naïve mice resulted in dose-dependent elevation of NAD+ and reduction of ADPR in various tissues such as spleen and liver. We next assessed the expression of CD38 protein by immunohistochemistry following ICI treatment in various syngeneic cancer models to select a model for efficacy studies. We observed increases in CD38 expression on tumor cells and infiltrating immune cells in MC38 colon cancer and B16-F10 and Cloudman S91 melanoma models. In the MC38 tumor model, we observed single agent antitumor activity with RBN013209 that was associated with changes in NAD+ and ADPR. In B16-F10 tumor-bearing mice, we observed antitumor activity with RBN013209 in combination with anti-PD-L1 therapy. To evaluate CD38 as a biomarker in clinical samples, we assessed and confirmed the tumor expression of CD38 protein from lung, prostate and kidney cancer patients. Here, we show that inhibition of CD38 with a small molecule affects both intra- and extra-cellular CD38 activity and modulates key metabolites playing an important role in immunomodulation. Further, our data indicate that CD38 is increased by ICI treatment and that inhibition of CD38 can lead to antitumor activity. Citation Format: Prashant Shambharkar, Danielle J. Blackwell, Melissa M. Vasbinder, Laurie B. Schenkel, Kaiko Kunii, Jenkins L. Lemera, Kristy G. Kuplast-Barr, Yue Ren, Ellen Bamberg, W. David Church, Christina R. Majer, Luke Utley, Kristen McEachern, Mario Niepel, Tim J. Wigle, Kevin W. Kuntz, Victoria M. Richon, Heike Keilhack, Joseph M. Gozgit. Small molecule inhibitor of CD38 modulates its intra- and extracellular functions leading to antitumor activity [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 1344.

3000 Background: Targeting cytosolic nucleic acid sensing pathways and the Type I interferon (IFN) response is an emerging therapeutic strategy in oncology. PARP7 is a member of the monoPARP class of enzymes and a newly identified negative regulator of nucleic acid sensing in tumor cells. PARP7 expression is increased by cellular stress and aromatic hydrocarbons, and the PARP7 gene is amplified in multiple cancers. RBN-2397 is a potent, selective inhibitor of PARP7. In preclinical models, RBN-2397 restored Type I IFN signaling in tumors, caused complete tumor regressions, and induced adaptive immunity. Methods: Patients (pts) with advanced solid tumors were treated with RBN-2397 on either a continuous or 14-of-21-day intermittent schedule using a 3+3 dose escalation design. Primary objective: establish MTD and/or RP2D. Secondary obj.: safety, activity, PK of unmicronized/micronized tablets. Exploratory obj.: Pd. Results: As of 4 January 2021, 47 pts were treated: 25 pts in the intermittent schedule (25 to 500 mg BID) and 22 patients in the continuous schedule (100 to 400 mg BID). The most frequent RBN-2397-related AEs (all grades) were dysgeusia (26%), decreased appetite (13%), fatigue (11%), and diarrhea (11%). Gr 3/4 RBN-2397-related AEs all occurred in 7 pts (15%) at doses ≥ 200 mg: diarrhea (2 pts, 4%), increased ALT, AST, and bilirubin (1 pt, 2%), and fatigue, anemia, neutropenia, and thrombocytopenia in 1 pt (2%) each. The 2 DLTs were Gr 3 febrile neutropenia (400 mg continuous schedule) and Gr 4 increase in ALT/AST (500 mg intermittent schedule). Plasma exposures generally increased dose dependently with the majority at or above the projected efficacious range based on animal studies. All evaluable baseline tumor biopsies showed evidence of PARP7 expression as measured by mRNA in situ hybridization (n = 11; Median tumor H score: 128). In 5 evaluable tumor biopsy pairs, increases in interferon-stimulated gene expression were observed post RBN-2397, consistent with activation of Type I IFN. CXCL10 mRNA increased in all evaluable on-treatment biopsies (1.5 to 8-fold). Several on-treatment biopsies showed enrichment for immune response gene sets that was accompanied by an increase in CD8+ T cells and Granzyme B expression, evidence for induction of an adaptive immune response post RBN-2397. This increase in immune response related genes and CD8+ T cells was observed in a pt with metastatic squamous NSCLC who has been on study for 16+ months. 1 pt with HR+, HER2- breast cancer achieved a confirmed PR at 100 mg and 8 pts had SD for ≥18 weeks (RECIST 1.1). Conclusions: To date, RBN-2397 is well tolerated and demonstrates dose dependent increases in plasma exposures, evidence of target inhibition, and preliminary signs of clinical activity. Determination of MTD/RP2D is imminent and study expansion is planned to evaluate safety and efficacy in squamous NSCLC, HNSCC, HR+ breast cancer, and PARP7 amplified tumors. Clinical trial information: NCT04053673.

New immuno-oncology therapies targeting programmed cell death receptor 1 (PD-1) have improved patient outcomes in a broad range of cancers. The objective of this analysis was to evaluate the PK, pharmacodynamics (PDy), and safety of dostarlimab monotherapy in adult patients with previously-treated advanced solid tumors who participated in parts 1 and 2A of the phase 1 GARNET study. Part 1 featured a 3 + 3 weight-based dose–escalation study, in which 21 patients received dostarlimab 1, 3, or 10 mg/kg intravenously every 2 weeks. The 2 fixed-dose nonweight-based dosing regimens of dostarlimab 500 mg every 3 weeks (Q3W) and 1000 mg every 6 weeks (Q6W) were evaluated using a modified 6 + 6 design in part 2A (n = 13). In parts 1 and 2A, treatment with dostarlimab could continue for up to 2 years or until progression, unacceptable toxicity, patient withdrawal, investigator’s decision, or death. The dostarlimab PK profile was dose proportional, and maximal achievable receptor occupancy (RO) was observed at all dose levels in the weight-based and fixed-dose cohorts. Trough dostarlimab concentration after administration of dostarlimab 500 mg Q3W was similar to that after dostarlimab 1000 mg Q6W, the values of which (≈40 µg/mL) projected well above the lowest dostarlimab concentration required for full peripheral RO. No dose-limiting toxicities were observed. Dostarlimab demonstrated consistent and predictable PK and associated PDy. The observed safety profile was acceptable and characteristic of the anti-PD-1 drug class. Trial registration: ClinicalTrials.gov, NCT02715284. Registration date: March 9, 2016.

Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.

Abstract PARP14 is an interferon-stimulated gene that is overexpressed in multiple tumor types and has been shown to promote the pro-tumor M2 polarization of macrophages and support Th2/Th17 signaling in models of allergic airway disease. PARP14 is a large 203 kDa protein that possesses a catalytic domain responsible for the transfer of mono-ADP-ribose to its substrates, three macrodomains that bind mono-ADP-ribose, a WWE domain that serves as a binding module for poly-ADP-ribose, and an RNA recognition motif. We have previously shown that the potent and reversible enzymatic inhibitor, RBN012759 (IC50 < 0.003 μM, 300-fold selective over monoPARPs, > 1,000-fold selective over polyPARPs), links PARP14 catalytic inhibition with suppression of the antitumor immune response in human primary macrophages and human kidney cancer explants. While this catalytic inhibitor of PARP14 was able to suppress IL-4-driven pro-tumor gene expression in macrophages, it is unknown what roles the non-enzymatic biomolecular recognition motifs play in the biological function of PARP14. To further understand this, we describe a heterobifunctional small molecule, RBN012811, based on a catalytic inhibitor of PARP14 that binds in the enzyme's NAD+-binding site and recruits the E3 ligase cereblon to ubiquitinate PARP14 and selectively target it for degradation. RBN012811 has a IC50 of 0.01 μM against PARP14 in a biophysical assay and is at least 200-fold selective over all other PARPs. In KYSE-270 cancer cells, RBN012811 has a half-maximal degradation concentration (DC50) of 0.005 μM and it does not cause degradation of other PARP enzymes. In human primary macrophages PARP14 degradation by RBN012811 led to a dose-dependent decrease of IL-10 release induced by IL-4 stimulation. Our data demonstrates that RBN012811 is a useful tool to enable further exploration of the role of PARP14 in inflammation and cancer. Citation Format: Tim Wigle, Yue Ren, Jennifer Molina, Danielle Blackwell, Laurie Schenkel, Kerren Swinger, Anne Cheug, Ryan Abo, Elena Minissale, Alvin Lu, Christina Majer, William Church, Bryan Dorsey, Mario Niepel, Nicholas Perl, Kristy Kuplast-Barr, Kristen McEachern, Melissa Vasbinder, Heike Keilhack, Kevin Kuntz. Targeted degradation of PARP14 Using a heterobifunctional small molecule [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 1348.

PARP7 (TIPARP) is a monoPARP which catalyzes the transfer of single units of ADP-ribose onto substrates to change their function. Its expression is upregulated during cellular stress, including viral infection or through the activation of the aryl hydrocarbon receptor after exposure to cigarette smoke. We and others have shown that PARP7 activity suppresses the Type I interferon (IFN) response following activation by cytosolic nucleic acid sensing pathways. RBN-2397 is a first-in class PARP7 inhibitor, which induces cancer cell autonomous and immune stimulatory effects in preclinical models through enhanced Type I IFN signaling in cancer cells. Here we describe the presence of PARP7 genomic amplification with corresponding increased mRNA expression across select cancers. Elevated PARP7 expression or amplification may identify cancer patients that could derive benefit from treatment with RBN-2397. In characterizing PARP7 copy number and mRNA expression from The Cancer Genome Atlas (TCGA) database, we found the presence of PARP7 copy number amplification in a subset of tumor types, particularly those of squamous histology, as well as ovarian cancer that corresponded to higher mRNA expression levels. High PARP7 expression correlated with poor survival in squamous cancers, while it had no effect on survival in ovarian cancer. Interestingly, tumor types with high PARP7 expression also expressed higher levels of baseline interferon stimulated genes (ISGs). This parallels our findings that cancer cell lines with higher ISGs at baseline are more responsive to PARP7 inhibition. To explore PARP7 copy number variations (CNVs) in advanced cancer patients, we queried the FoundationCore® (Foundation Medicine, Inc) database. Similar to TCGA, squamous cancers as well as ovarian, breast, and pancreatic ductal adenocarcinoma (PDAC) were among the tumor types with PARP7 amplifications. Moreover, PARP7 was found to be amplified both on the background of chromosome 3q arm-level amplifications as well as focally. Congruent to our analysis of PARP7 amplifications, we evaluated PARP7 mRNA expression in both squamous and non-squamous non-small cell lung carcinoma (NSCLC), breast cancer, and PDAC primary tumor samples. Using a validated RNAscope ISH probe set, we analyzed over 1,000 patient samples and found that PARP7 was more highly expressed in tumor cells relative to corresponding normal tissues. Overall, there were varying levels of PARP7 expression across samples with higher expression levels of PARP7 in tumor cells, compared to stroma, across all cancers examined. Our analyses describing the presence of PARP7 amplifications as well as high expression levels in several tumor types including NSCLC, breast, and PDAC provides evidence for the therapeutic relevance of PARP7 inhibition and highlights potential patient selection strategies to identify those patients more likely to benefit from RBN-2397 treatment. Citation Format: Jodie Wong, Kristy Kuplast-Barr, Ryan P. Abo, Anupriya S. Kulkarni, Linda T. Nieman, Azfar Neyaz, Katherine H. Xu, Radwa Sharaf, Lee A. Albacker, David T. Ting, Heike Keilhack, Kristen A. McEachern. Elevated PARP7 expression in select cancers identifies a target population for RBN-2397 therapy [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 381.

Abstract Purpose: To determine the tumor tissue/cell distribution, functional associations, and clinical significance of PD-1, LAG-3, and TIM-3 protein expression in human non–small cell lung cancer (NSCLC). Experimental Design: Using multiplexed quantitative immunofluorescence, we performed localized measurements of CD3, PD-1, LAG-3, and TIM-3 protein in >800 clinically annotated NSCLCs from three independent cohorts represented in tissue microarrays. Associations between the marker's expression and major genomic alterations were studied in The Cancer Genome Atlas NSCLC dataset. Using mass cytometry (CyTOF) analysis of leukocytes collected from 20 resected NSCLCs, we determined the levels, coexpression, and functional profile of PD-1, LAG-3, and TIM-3 expressing immune cells. Finally, we measured the markers in baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers and known response to treatment. Results: PD-1, LAG-3, and TIM-3 were detected in tumor-infiltrating lymphocytes (TIL) from 55%, 41.5%, and 25.3% of NSCLC cases, respectively. These markers showed a prominent association with each other and limited association with major clinicopathologic variables and survival in patients not receiving immunotherapy. Expression of the markers was lower in EGFR-mutated adenocarcinomas and displayed limited association with tumor mutational burden. In single-cell CyTOF analysis, PD-1 and LAG-3 were predominantly localized on T-cell subsets/NKT cells, whereas TIM-3 expression was higher in NK cells and macrophages. Coexpression of PD-1, LAG-3, and TIM-3 was associated with prominent T-cell activation (CD69/CD137), effector function (Granzyme-B), and proliferation (Ki-67), but also with elevated levels of proapoptotic markers (FAS/BIM). LAG-3 and TIM-3 were present in TIL subsets lacking PD-1 expression and showed a distinct functional profile. In baseline samples from 90 patients with advanced NSCLC treated with PD-1 axis blockers, elevated LAG-3 was significantly associated with shorter progression-free survival. Conclusions: PD-1, LAG-3, and TIM-3 have distinct tissue/cell distribution, functional implications, and genomic correlates in human NSCLC. Expression of these immune inhibitory receptors in TILs is associated with prominent activation, but also with a proapoptotic T-cell phenotype. Elevated LAG-3 expression is associated with insensitivity to PD-1 axis blockade, suggesting independence of these immune evasion pathways.

Abstract Introduction: T-cells recognizing tumor antigens can express diverse immune inhibitory receptors such as PD-1, LAG-3 and TIM-3. Blockade of PD-1 induces prominent clinical benefit in patients with melanoma, lung, bladder and gastric carcinomas. However, the relative expression and significance of immune inhibitory receptors across different tumors is unknown. Methods: Using multiplexed quantitative immunofluorescence (QIF) we measured the levels of CD3 (rabbit polyclonal, Dako), PD-1 (EH33, CST), LAG-3 (17B4, Abcam) and TIM-3 (D5D5R, CST) in >1,300 human malignancies including 119 triple negative breast carcinomas (TNBC), 225 ovarian carcinomas, 382 melanomas, 259 colorectal tumors, 229 bladder urothelial carcinomas and 130 gastric malignancies. The cohorts included FFPE tumor tissues and were studied using tissue microarrays (TMAs). The targets were measured in all cells of the preparation using fluorescence co-localization with DAPI and specifically in CD3-positive T-lymphocytes. Associations between the markers across tumors and with survival were studied. Results: PD-1, LAG-3, and TIM-3 were expressed predominantly in T-cells and showed a positive association with each other. The highest levels were detected in gastric adenocarcinomas followed by urothelial bladder tumors, ovarian malignancies, melanoma, TNBC and colorectal carcinomas. Gastric and bladder tumors showed comparably high levels of all three markers and were significantly higher than the other tumor types (P<0.001). TNBC and colorectal carcinomas showed comparably lower expression. The levels of PD-1, LAG-3 and TIM-3 were positively associated with CD3+ T-cell infiltration in all tumors except in gastric cancer. Melanoma showed the highest CD3 levels and gastric tumors had the lowest. Elevated T-cell PD-1 was significantly associated with longer overall survival in melanoma and colorectal cancer; and elevated LAG-3 was associated with better survival only in melanoma. The levels of TIM-3 were not associated with survival. Conclusion: PD-1, LAG-3 and TIM-3 are differentially expressed in human solid tumors and show limited prognostic value. Gastric and bladder carcinomas show the highest levels of the targets, while TNBC and colorectal cancer show the lowest. Despite having the highest T-cell infiltration, melanomas show intermediate levels of the immune inhibitory receptors. These data demonstrate the differences in the immune composition of human solid tumors and could be used for optimal design and interpretation of clinical trials. Citation Format: Micaela Morgado, Ila Datar, Jun Wang, Miguel F. Sanmamed, Kristen McEachern, David Jenkins, Lieping Chen, Daniel Carvajal-Hausdorf, David L. Rimm, Roy S. Herbst, Kurt A. Schalper. Simultaneous measurement and significance of PD-1, LAG-3 and TIM-3 expression in human solid tumors [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 1681.

Introduction:TSR-042 is a humanized monoclonal antibody targeting programmed death (PD)-1,effectively blocking interaction with its ligands PD-L1 and PD-L2. TSR-042 is being evaluated in patients (pts) with advanced solid tumors in the ongoing phase 1 GARNET trial (NCT02715284). Weight based dose escalation (Part 1) and fixed-dose safety studies (Part 2A) have been completed,1 and the study is currently enrolling pts with specific tumor types into expansion cohorts. Here, we present safety and efficacy data from the microsatellite instability-high (MSI-H) endometrial cancer (EC) and non-small cell lung cancer (NSCLC) cohorts, as well as pharmacokinetic (PK) and receptor occupancy (RO) characterization at the recommended phase 2 dose (RP2D). Methods/Procedures: 30 NSCLC pts and 19 MSI-H EC pts with previously treated recurrent or advanced disease were enrolled; median number of prior lines of therapy for metastatic disease was 1.0 for both cohorts. MSI status for EC pts was determined centrally using a next generation sequencing-based assay. Key exclusion criteria included prior therapy with agents targeting anti-PD-1, PD-L1, or PD-L2, and uncontrolled CNS metastases. Pts were treated at the R2PD of TSR-042: 500 mg Q3W for the first 4 cycles and 1000 mg Q6W thereafter. Serum and PBMCs were collected for PK and RO analyses, respectively. Antitumor activity was assessed by investigators using immune-related Response Evaluation Criteria in Solid Tumors (irRECIST). RO was assessed using a CD3+ binding assay (direct receptor occupancy).1 Results: Among the 30 NSCLC and 19 MSI-H EC pts, 42 (85.7%) pts reported ≥1 adverse event (AE), with grade ≥3 AEs reported in 13/49 (26.5%) pts. The most common AEs were diarrhea and nausea (11 pts each), arthralgia and fatigue (9 pts each), and cough, decreased appetite, and dyspnea (7 pts each). 25/49 (51.0%) pts reported treatment-related AEs; 11/49 (22.4%) pts had serious AEs; 1 case of grade 3 fatigue and 1 case of grade 3 leukopenia and grade 3 neutropenia were deemed treatment-related. 21 NSCLC and 11 MSI-H EC pts had at least 1 tumor assessment. Among NSCLC pts, 7/21 (33.3%) had a partial response (irPR; confirmed and unconfirmed), and 6/21 (28.6%) had stable disease (irSD); among MSI-H EC pts, 4/11 (36.4%) had irPR, and 2/11 (18.2%) had irSD. TSR-042 demonstrated dose-proportional PK. The maximal achievable RO was observed in pts treated at the RP2D, consistent with the results reported for Parts 1 and 2A.1 Conclusions: These results indicate clinical benefit of TSR-042 in previously treated NSCLC and MSI-H EC patients, with a safety profile similar to other PD-1 inhibitors. PK results were consistent across all patients evaluated and show that maximal achievable receptor occupancy was attained at the RP2D. 1. Sachdev JC et al. Ann Oncol. 2017(suppl 5):28:420;1185P Citation Format: Victor Moreno, Maria-Pilar Barretina-Ginesta, Wei Guo, Sharon Lu, David Jenkins, Kristen McEachern, Vienna Reichert, Steven Dunlap, Ellie Im, Lucy Gilbert, Ana Oaknin, Charles Leath, III, Janakiraman Subramanian. Preliminary safety, efficacy, and PK/PD characterization from GARNET, a phase 1 clinical trial of the anti-PD-1 monoclonal antibody, TSR-042, in patients with recurrent or advanced NSCLC and MSI-H endometrial cancer [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 CT053.

Proviral integration Moloney virus (PIM) kinases (PIM1, 2 and 3) are overexpressed in several tumour types and contribute to oncogenesis. AZD1208 is a potent ATP-competitive PIM kinase inhibitor investigated in patients with recurrent or refractory acute myeloid leukaemia (AML) or advanced solid tumours. Two dose-escalation studies were performed to evaluate the safety and tolerability, and to define the maximum tolerated dose (MTD), of AZD1208 in AML and solid tumours. Secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics (PD) and preliminary efficacy of AZD1208. Sixty-seven patients received treatment: 32 in the AML study over a 120–900 mg dose range, and 25 in the solid tumour study over a 120–800 mg dose range. Nearly all patients (98.5%) in both studies experienced adverse events, mostly gastrointestinal (92.5%). Dose-limiting toxicities included rash, fatigue and vomiting. AZD1208 was not tolerated at 900 mg, and the protocol-defined MTD was not confirmed. AZD1208 increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance. There were no clinical responses; PD analysis showed biological activity of AZD1208. Despite the lack of single-agent clinical efficacy with AZD1208, PIM kinase inhibition may hold potential as an anticancer treatment, perhaps in combination with other agents.

While immunotherapies directed against PD-1 and PD-L1 have proven effective across multiple indications, there is still a large unmet medical need for therapies for patients who do not respond or who develop acquired resistance during the course of treatment. There are several emerging hypotheses to explain the lack of response, one of which is the upregulated expression of additional checkpoint receptors, such as TIM-3 and LAG-3, that limit the ability of PD-1 pathway blockade to re-establish effective anti-tumor immunity. To investigate the potential role of TIM-3 and LAG-3 relative to PD-1, we set out to evaluate the expression and function of these receptors in several systems. Firstly, we used a flow cytometry approach to enumerate immune cell populations in a panel of human tumor samples, including non-small cell lung cancer (NSCLC). In these samples, PD-1, TIM-3 and LAG-3 expression was observed in both T-cell and non-T-cell populations. PD-1 was found to be co-expressed with TIM-3 and LAG-3 in CD8+ T-cells and we also found expression of TIM-3 and LAG-3 in other cell types, including regulatory T-cells and myeloid cells. Building on the expression data, we explored the functional effects of targeting these receptors, evaluating the effect of single agent and combination effects of anti-PD-1, anti-TIM-3 and anti-LAG-3 antibodies in mouse models. In these studies, we found not only that dual-blockade of PD-1 with either anti-TIM-3 or anti-LAG-3 resulted in improvements in efficacy over monotherapy, but also that the triple combination of all 3 inhibitors was associated with further anti-tumor activity. In a humanized mouse model of NSCLC, the triple combination was associated with pharmacodynamic effects not only in T-cells, but also reductions in Tregs and macrophages. Taken together, these studies provide further evidence to suggest that in addition to PD-1, LAG-3 and TIM-3 are important emerging immunotherapy targets and provide rationale for not only doublet but also triplet combinations as an approach to cancer therapy. Citation Format: Srimoyee Ghosh, Jon Travers, Kristen McEachern, Sujatha Kumar, Sridhar Ramaswamy, David Jenkins. Investigation of the expression profile and functional role of PD-1, TIM-3 and LAG-3 in human tumors [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 2722.