Immunotherapies In Preclinical Models Research Articles

Abstract ND03: Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor

Abstract RASG12D mutant cancers represent a significant unmet medical need with 55,000 new diagnoses annually in the US. The RASG12D mutation increases the abundance of the active, GTP-bound state of RAS (RASG12D(ON)) and occurs commonly in multiple tumor histotypes, including about 20%, 29% and 17% of RAS mutant colorectal, pancreatic, and non-small cell lung cancers, respectively. RMC-6236, an investigational RASMULTI(ON) inhibitor currently in clinical testing, selectively targets the active, GTP-bound state of both mutant and wild-type RAS variants, including RASG12D(ON) and has shown clinical anti-tumor activity against tumors harboring KRASG12D. Efforts to find a mutant-selective RASG12D(ON) inhibitor led to the discovery of RMC-9805.The investigational agent RMC-9805 is a first-in-class, orally bioavailable, mutant selective covalent inhibitor of RASG12D(ON) that forms a tri-complex between the abundant intracellular chaperone cyclophilin A (CypA) and the “ON” state of RASG12D, enabling selective covalent engagement of Asp-12 and disrupting downstream RAS signaling by steric occlusion of effector binding. In contrast to other examples in the literature of covalent inhibitors of KRASG12D that rely on highly reactive, low stability warheads to overcome the low intrinsic reactivity of aspartic acid, RMC-9805 employs a warhead with low intrinsic reactivity and high stability under biological conditions. Rapid, selective, covalent modification of RASG12D by RMC-9805 is enabled by our tri-complex technology. We used structure-guided design to position the warhead in the CypA-RAS interface in an optimal orientation that enhanced crosslinking of Asp-12 allowing us to use a warhead that showed no intrinsic reactivity to a model aspartic acid system and was stable to enable oral dosing. RMC-9805 drove selective and persistent covalent modification of KRASG12D in human cancer cell lines in vitro, leading to deep and durable suppression of RAS pathway activity, inhibition of cell proliferation, and apoptosis. RMC-9805 monotherapy induced tumor regressions at well-tolerated doses in a majority of preclinical PDAC and NSCLC models harboring KRASG12D. Though a more heterogeneous response was observed in KRASG12D CRC models, combinations of RMC-9805 with either RMC-6236 (RASMULTI(ON) inhibitor) or an anti-EGFR antibody improved the depth of response and delayed the onset of resistance in vivo. In addition, RMC-9805 promoted cancer-associated neoantigen recognition and synergized with immunotherapy in preclinical models. RMC-9805 is also CNS penetrant and drove regressions in intracranial xenograft models of human KRASG12D PDAC. RMC-9805 is a first-in-class orally bioavailable, mutant selective and covalent RASG12D inhibitor currently in Phase 1 clinical trial (NCT06040541). Citation Format: John E. Knox, G. Leslie Burnett, Caroline Weller, Lingyan Jiang, Dongyu Zhang, Nicole Vita, Abby Marquez, Kyle J. Seamon, Andrea Gould, Marie Menard, Elsa Quintana, Zhe Chen, Zhican Wang, Zhengping Wang, Elena S. Koltun, Malika Singh, Jingjing Jiang, David Wildes, Jacqueline A.M. Smith, Adrian L. Gill. Discovery of RMC-9805, an oral, covalent tri-complex KRASG12D(ON) inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND03.

Abstract 2611: Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 immunotherapy

Abstract Background: Antibody-drug conjugates (ADCs) play a key role in targeted therapy of solid tumors. However, few studies have examined the impact of ADCs on the tumor microenvironment (TME), particularly on innate immune cells. To elucidate this, this study focuses on investigating the comprehensive impacts of ICAM1-DXd on the tumor microenvironment, especially on tumor-associated neutrophils (TAN) and its synergistic effect with PD-1 immunotherapy. Methods: Firstly, the efficacy and prognosis of ICAM1-DXd, a potent ADC for cervical cancer, were determined in pre-clinical tumor models, providing crucial transcriptomic and biological function information by using RNA-seq. Then, pathological studies were performed to evaluate the abundance and location of TAN (Ly6G+/MPO+) in TME post-ADC treatment. The interaction between TAN and ICAM1-DXd and subsequent impacts on adaptive immune cells (e.g., CD8+ cells) was examined by flow cytometry (FACS), confocal fluorescent imaging, and western blot techniques (WB). Eventually, the synergistic effect of ICAM1-DXd and PD-1 monoclonal antibody were investigated in an immunocompetent animal model. Results: ICAM1-DXd potently inhibits cervical tumor growth in preclinical models, and RNA-seq analysis revealed that ICAM1-DXd treatment induces a strong down-regulation of neutrophil activation, especially for Ly6G+/MPO+ TANs, a subtype with pro-tumor function. IHC and FACS results confirmed that pro-tumor TANs were efficiently eliminated in TME by ICAM1-DXd treatment via non-specific phagocytosis in comparison with peripheral blood neutrophils. ICAM1-DXd-phagocytised TANs also elicit a strong bystander killing effect by cleaving the quadra peptide linker of ICAM1-DXD by cathepsin B. This ADC-mediated TAN elimination effectively ameliorates immunosuppressive TME and works in synergy with PD-1 immunotherapy in preclinical models. Conclusion: In preclinical models of cervical cancer, ICAM1-DXd not only directly ablates cancer cells via antigen recognition but also non-specifically kills tumor-promoting TANs, resulting in an improved tumor immune microenvironment, providing a shred of mechanistic evidence supporting ADC and PD-1 combination therapy. Citation Format: Shili Yao, Lu Sun, Xiu Zhu, Hanfei Xie, Tong Yang, Peng Guo, Huarong Tang, Tao Zhu. Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 immunotherapy [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 2611.

Abstract 4026: The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice

Abstract CTX-009 is a bispecific antibody targeting VEGF-A and DLL4 which is undergoing clinical investigation in patients with multiple tumor types, including biliary tract and colon cancers. In biliary tract cancers, checkpoint inhibitor therapy has been shown to improve outcomes when combined with chemotherapy. In an effort to increase the breadth and duration of response of CTX-009, we set out to investigate the combined anti-tumor efficacy of this bispecific antibody and immunotherapy in preclinical models. Here we report the evaluation of the combination of the mouse surrogates of CTX-009 and CTX-471, an agonistic anti-mouse CD137 antibody, or monoclonal antibodies that disrupt the PD-1/PD-L1 signaling axis. Coupling VEGF-A/DLL4 targeting with either CD137 agonism or PD-1/PD-L1 blockade markedly increased the anti-tumor efficacy of the individual treatments alone in multiple syngeneic mouse tumor models. Tumor growth inhibition was accompanied by pharmacodynamic evidence consistent with enhancement of anti-tumor immunity. Notably, the combination of CTX-009 and CTX-471 retained potent anti-tumor activity even in a tumor line (MC38) which was made MHC I deficient through the deletion of the beta 2 microglobulin gene. This system may model the clinically relevant unmet need characterized by tumor escape through the loss of antigen presentation machinery such as HLA LOH. Interestingly, the model cell lines CT26 and MC38 failed to thrive in the B2M-deficient setting and depletion of NK cells markedly increased their ability to grow. This implies that NK cells might be involved in controlling tumor growth in this setting, and that the combined action of CTX-471 and CTX-009 in the tumor stroma enhances NK cell activity. The findings presented here suggest that CTX-009 may provide enhanced clinical benefit when combined with immunomodulatory agents, such as agonistic anti-CD137 antibodies or PD-1/PD-L1 blockers in tumors where immune checkpoint inhibitors have failed.  Citation Format: Diana I. Albu, Rachael Duffy, Amy Daniel Ulumben, Jason Kong, Jessica Zolotarevsky, Melissa Brundin, Mei Su, Ruturaj Jadhav, Kris F. Sachsenmeier, Bing Gong, Thomas J. Schuetz, Alberto Visintin. The combination of DLL4/VEGF-a blockade and immunomodulation can eliminate MHC class I negative tumors in mice [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 4026.

Inhibition of EZH2 Improves CART19 Immunotherapy By Reprogramming Lymphoma Tumor Cells and Enhancing T-Cell Functionality

Introduction: Anti-CD19 chimeric antigen receptor T cell (CART19) immunotherapy provides a high response rate in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) patients, but only ~30-40% of patients maintain a durable remission. The factors contributing to lymphoma resistance to CART19 are numerous, multifaceted, and coincidental, such as tumor intrinsic properties, CART dysfunction, the immunosuppressive tumor microenvironment (TME), and host factors. However, most strategies currently in development to increase CART efficacy only address one resistance mechanism at a time; therefore, it is unlikely that these approaches will be consistently effective in a group of heterogeneous patients. In this study, we sought to develop a strategy to overcome multiple resistance mechanisms simultaneously. Epigenetic modulation offers a promising approach to this goal, as it can reshape the tumor landscape, as well as immune cells. DLBCL/FL cells depend for their survival on the methyltransferase EZH2, which represses gene expression by generating histone 3 lysine 27 trimethylation. Interestingly, somatic gain-of-function mutations of EZH2 are frequently found in DLBCL/FL and drive lymphomagenesis and impair tumor B-cell to T-cell interactions, leading to an immune evasive phenotype. Therefore, we hypothesized that EZH2 inhibition could enhance CART anti-tumor effect, remodeling the entire tumor ecosystem by: a. inhibiting lymphoma growth; b. increasing lymphoma immunogenicity; and c. enhancing T cell function ( Fig. 1A). Methods and Results: Through short-term killing assays, we compared CART19 alone to CART19 combined with the EZH2 inhibitor tazemetostat (taz), and demonstrated that taz enhances CART19 tumor killing of DLBCL cell lines [OCI-Ly18 and Toledo (with wild-type EZH2) and SU-DHL-4 (with mutant EZH2)]. Interestingly, the highest effect was observed when tumor cells were pre-incubated with taz and taz was kept in the coculture with CART19. This finding underscores the importance of tumor priming to improve CART19 recognition and activation. Since EZH2 is also expressed by T cells, we tested if EZH2 inhibition impacted CART cell function. Co-treatment of lymphoma cell lines with taz and CART19 was associated with higher cytokine secretion over time (INFy and TNF) by CART cells and CART cell expansion. Most importantly, using the sub-cutaneous OCI-Ly18 xenograft lymphoma model in immunodeficient NSG mice, we demonstrated that taz administration improved tumor control ( Fig. 1B) and CART19 expansion and infiltration in vivo. Mechanistically, we studied the molecular changes in lymphoma cells (OCI-Ly18 and SU-DHL-4) induced by exposure to taz/CART19 or CART19 alone by performing RNA sequencing. We observed upregulation of genes involved in inflammatory response, B-cell activation/differentiation (e.g., BATCH2), adhesion (OX40L, CD80), and motility (CAECAM1, NRCAM1, EPHB2, CXCL16, CXCL10), sustaining the hypothesis that EZH2 inhibition changed lymphoma immunogenicity and the tumor:CART cell interaction. Indeed, functionally, we observed that taz exposure increased tumor:CART cell interaction in vitro as demonstrated by increased avidity (zMovi assay). Further, single-cell RNA sequencing performed on circulating CART19 cells from taz-treated-mice revealed enrichment in naïve and less exhausted CART cells. Lastly, we assessed whether increased activation of EZH2 in tumor cells reduces responses to CART19 in the clinic. We retrospectively analyzed a cohort of large B-cell lymphoma patients treated with commercial CART19 (tisagenlecleucel and axicabtagene ciloleucel) at the University of Pennsylvania between January 2018 and March 2023 for response (Lugano criteria) according to their EZH2 mutational status. Patients harboring EZH2 activating mutations (14/69) had a reduced complete response rate to CART19 as compared to wild-type patients (15.4% vs. 40.0%). Conclusions: Our data demonstrate that modulation of EZH2 enhances CART19 immunotherapy in preclinical models of lymphoma. EZH2 inhibition rewires lymphoma immunogenicity, making tumor cells more susceptible to CART19 killing and reducing CART19 terminal differentiation. By understanding and modulating the interplay between tumor and CART cells, we can unlock the full potential of CART19 immunotherapy in DLBCL/FL patients, aiming at long-lasting treatment outcomes.

Abstract 3475: RMC-9805, a first-in-class, mutant-selective, covalent and orally bioavailable KRASG12D(ON) inhibitor, promotes cancer-associated neoantigen recognition and synergizes with immunotherapy in preclinical models

Abstract Major epithelial tumors harboring the cancer driver KRASG12D, the most common oncogenic KRAS mutation1, remain an outstanding unmet medical need. While oncogenic RAS primarily promotes tumor progression through sustained cellular proliferation, there is also increasing evidence that it adversely shapes the tumor microenvironment (TME) through the induction of immuno-suppressive cytokines2, downregulation of MHCI, and modulation of checkpoint proteins3. KRASG12D oncoproteins can be selectively targeted with the covalent tri-complex RAS(ON) inhibitor, RMC-9805, which has been shown to inhibit RAS signaling in KRASG12D mutant cancer cells and induce apoptosis. RMC-9805 also decreased the secretion of immunosuppressive cytokines by KRASG12D tumor cells, while promoting the secretion of lymphocyte-recruiting factors. Oral administration of RMC-9805 drove durable complete responses and synergized with anti-PD-1 in an immune-infiltrated colorectal KRASG12D mutant model. Re-challenge experiments further demonstrated that RMC-9805 induced immunological memory. The activity of RMC-9805 was also assessed in an orthotopic, immune-evasive, GEMM-derived preclinical model for PDAC, where it induced a profound and sustained inhibition of RAS signaling, driving initial tumor regressions and significantly prolonging survival. Tumors treated with RMC-9805 in vivo showed significant transformation of the TME in favor of anti-tumor immunity, with an increase in T cell infiltration and decrease in immunosuppressive myeloid cells, including M2-like macrophages and MDSCs. RMC-9805 also modulated expression of cancer cell surface proteins, notably decreasing expression of immune checkpoint molecules while significantly increasing MHC-I expression. TCR sequencing of T cells from tumors and blood harvested from treated mice revealed a significant increase in T cell diversity, as well as an increase in the number of shared TCR clones among all KRASG12D(ON) inhibitor-treated samples, suggesting cancer-associated antigen recognition. Overall, in these preclinical experiments, RMC-9805 exhibited direct anti-tumor effects and indirectly transformed the TME through inhibition of cancer cell-intrinsic KRASG12D oncogenic signaling. The increased antigen presentation, recognition, and T cell infiltration induced by inhibition of mutant KRAS may permit a more favorable environment for immune-directed ‘companion’ therapies, such as checkpoint inhibitors, cellular therapies, and/or vaccines. [1] Timar, J. et al, 2020 [2] Hamarsheh, S. et al, 2020 [3] Sumimoto, H. et al, 2016 Citation Format: Marie J. Menard, Christopher Chow, Kevin Chen, Cristina Blaj, Nataliya Tovbis Shifrin, Haley Courtney, Nicole M. Nasholm, Anna Pham, Alice Kumamoto, Swetha Ganesh, James W. Evans, Lindsey Lawrence, Brian Vonmelchert, Amy C. Kwok-Parkhill, Jingjing Jiang, John E. Knox, Jacqueline Smith, Elsa Quintana. RMC-9805, a first-in-class, mutant-selective, covalent and orally bioavailable KRASG12D(ON) inhibitor, promotes cancer-associated neoantigen recognition and synergizes with immunotherapy in preclinical models [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 3475.

Local immunotherapy with the RNA-based immune stimulator CV8102 induces substantial anti-tumor responses and enhances checkpoint inhibitor activity

Immunotherapy has revolutionized cancer treatment in recent years. Although currently approved checkpoint inhibitors (CPIs) yield remarkable anti-tumoral responses in several cancer types, a substantial proportion of patients do not benefit from such therapies. Local activation of innate immune signaling pathways is a promising approach to overcome the immunosuppressive tumor microenvironment, induce anti-tumor immunity, and improve the efficacy of CPI therapies. Here, we assessed the mode of action and efficacy of the RNA-based innate immune stimulator CV8102 for local immunotherapy in preclinical models. Intratumoral (i.t.) administration of CV8102 activated innate immune responses in the tumor microenvironment and draining lymph nodes, resulting in a dose-dependent anti-tumoral response. Combining i.t. CV8102 with systemic anti-programmed death protein 1 (PD-1) treatment further enhanced anti-tumoral responses, inducing tumor infiltration and activation of CD8+ T cells. The resulting memory response prevented tumor growth in rechallenged animals and impaired the growth of non-injected distal tumors. Therefore, i.t. CV8102 delivery is a promising approach for local cancer immunotherapy, especially in combination with CPIs. Clinical testing of CV8102 is ongoing (NCT03291002).

Latest Advances in the Use of Therapeutic Focused Ultrasound in the Treatment of Pancreatic Cancer.

Simple SummaryPancreatic cancer is a devastating disease with the worst survival rates of all cancers. Advances in immunotherapy have revolutionized the treatment of cancer, providing cures to patients with a previously lethal disease. Sadly, clinical trials of currently established immunotherapies have not provided significant therapeutic benefits to pancreatic cancer patients. For this reason, alternative treatment approaches that could sensitise the pancreatic tumours to immunotherapy are needed. Therapeutic focused ultrasound is a non-invasive physical modality that can kill cells by raising the temperature, and by disrupting and destroying tissue mechanically. It can also sensitise tumours to the effects of radiotherapy. This review article describes the use of therapeutic focused ultrasound in the treatment of pancreatic cancer. Attention is given to the immunological effects induced by therapeutic focused ultrasound, and how these can be used to sensitise the pancreatic tumours to immunotherapy.Traditional oncological interventions have failed to improve survival for pancreatic cancer patients significantly. Novel treatment modalities able to release cancer-specific antigens, render immunologically “cold” pancreatic tumours “hot” and disrupt or reprogram the pancreatic tumour microenvironment are thus urgently needed. Therapeutic focused ultrasound exerts thermal and mechanical effects on tissue, killing cancer cells and inducing an anti-cancer immune response. The most important advances in therapeutic focused ultrasound use for initiation and augmentation of the cancer immunity cycle against pancreatic cancer are described. We provide a comprehensive review of the use of therapeutic focused ultrasound for the treatment of pancreatic cancer patients and describe recent studies that have shown an ultrasound-induced anti-cancer immune response in several tumour models. Published studies that have investigated the immunological effects of therapeutic focused ultrasound in pancreatic cancer are described. This article shows that therapeutic focused ultrasound has been deemed to be a safe technique for treating pancreatic cancer patients, providing pain relief and improving survival rates in pancreatic cancer patients. Promotion of an immune response in the clinic and sensitisation of tumours to the effects of immunotherapy in preclinical models of pancreatic cancer is shown, making it a promising candidate for use in the clinic.

Senescence induction dictates response to chemo- and immunotherapy in preclinical models of ovarian cancer

High-grade serous ovarian carcinoma (HGSOC) is a cancer with dismal prognosis due to the limited effectiveness of existing chemo- and immunotherapies. To elucidate mechanisms mediating sensitivity or resistance to these therapies, we developed a fast and flexible autochthonous mouse model based on somatic introduction of HGSOC-associated genetic alterations into the ovary of immunocompetent mice using tissue electroporation. Tumors arising in these mice recapitulate the metastatic patterns and histological, molecular, and treatment response features of the human disease. By leveraging these models, we show that the ability to undergo senescence underlies the clinically observed increase in sensitivity of homologous recombination (HR)-deficient HGSOC tumors to platinum-based chemotherapy. Further, cGas/STING-mediated activation of a restricted senescence-associated secretory phenotype (SASP) was sufficient to induce immune infiltration and sensitize HR-deficient tumors to immune checkpoint blockade. In sum, our study identifies senescence propensity as a predictor of therapy response and defines a limited SASP profile that appears sufficient to confer added vulnerability to concurrent immunotherapy and, more broadly, provides a blueprint for the implementation of electroporation-based mouse models to reveal mechanisms of oncogenesis and therapy response in HGSOC.

CYCLON and NPM1 Cooperate within an Oncogenic Network Predictive of R-CHOP Response in DLBCL.

Simple SummaryCYCLON is a nuclear protein, which has been associated with disease progression and treatment resistance in DLBCL, the most common form of aggressive B-cell lymphoma, but also represents a predictive factor of refractory disease and relapse for immuno-chemotherapy-treated DLBCL patients. The molecular mechanisms related to this unstructured protein remain largely uncharacterized. Here, we performed a mass-spectrometry-based identification of the CYCLON protein interactome that suggested it could exert nucleolar functions related to cell proliferation. Among the CYCLON oncogenic network, we performed an immunohistochemical evaluation of the multi-functional nucleolar protein NPM1 in a DLBCL cohort and showed that CYCLON/NPM1 concomitant expression delineates a poor prognosis subgroup of patients. Multivariate survival analyses demonstrated that specific sub-cellular localizations of CYCLON and NPM1 represent independent novel predictors specifically associated with refractory DLBCL.R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30–40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

Off-tumor IDO1 target engagements determine the cancer-immune set point and predict the immunotherapeutic efficacy

BackgroundIndoleamine-2,3-dioxygenase 1 (IDO1) has been intensively pursued as a therapeutic target to reverse the immunosuppressive cancer-immune milieu and promote tumor elimination. However, recent failures of phase III clinical trials with...

Immunomodulation by HDAC inhibition: Results from a phase I study with entinostat in combination with atezolizumab and bevacizumab in metastatic renal cell carcinoma patients.

5064 Background: Immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) limit the efficacy of immunotherapies. We have previously reported that the HDAC inhibitor entinostat has synergistic antitumor effect in combination with immunotherapies in preclinical models by inhibiting Tregs and MDSCs function. The combination of atezolizumab (PD-L1 inhibitor) and bevacizumab (VEGF inhibitor) is active in renal cell carcinoma (RCC). Thus, we have conducted a Phase I study with entinostat, atezolizumab and bevacizumab in patients (pts) with metastatic RCC. Methods: The primary objective was to evaluate safety and tolerability. The phase I portion included 3 dose levels of entinostat (1 mg, 3 mg or 5 mg, PO weekly) and fixed, standard doses of atezolizumab (1200 mg IV every 21 days) and bevacizumab (15 mg/Kg IV every 21 days). Pts with any histological type and prior therapies were included. Results: Dose levels were completed with up to 1 DLT/dose level. 5 mg was the Phase II recommended dose for entinostat. DLTs included hypertension, encephalopathy, hyponatremia and pruritus. The most common resolved grade 3/4 toxicities were hypophosphatemia (33%), hypertension (17%), and pneumonitis (11%). We have enrolled 18 pts (17 evaluable for ORR by RECIST). 5 pts continue on treatment. 3 pts discontinued treatment because of adverse events, 9 pts for disease progression, and one pt for physician decision. Good risk and intermediate risk pts were 61% and 39%, respectively. Overall ORR was 47.1% (95% CI 23.0-72.2) and median PFS was 7.6 months (95% CI 1.6-16.3). In pts with no prior therapies (12) the ORR was 58.3% (95% CI 27.7-84.8) and median PFS was 13.4 months (95% CI 1.5-28.9). One additional PR was observed by ir-RC but was not confirmed within the data cut-off date of 11/11/19. In pts with prior immune checkpoint inhibitors (ICIs) (5) ORR was 20% (95% CI 0.5-71.6) and median PFS was 7.6 months (95% CI 1.3-NR). Preliminary data show a statistically significant lower % of circulating monocytic MDSC (HLADR−1CD11b+CD33highCD14+CD15−) and exhausted T cells (CD45+CD3+CD8+TIM3+) following treatment in pts (4) with objective responses as compared to pts (4) with progressive disease. Conclusions: The results from this phase I suggest that the combination of entinostat, atezolizumab and bevacizumab is relatively well tolerated and is active in renal cell carcinoma patients, in both ICIs naïve and resistant disease. A phase II portion of this study is currently accruing patients. Clinical trial information: NCT03024437 .

A personalised approach for anti-GITR-based immunotherapy in pre-clinical models of pancreatic ductal adenocarcinoma

Abstract Background Patient outcomes with pancreatic ductal adenocarcinoma (PDA) remain dismal, despite the introduction of combination chemotherapies. Response to therapy is hampered by tumour stroma, which is highly heterogeneous but poorly characterised. The clinical success of immunotherapy in certain cancer types justifies investigation in PDA using a personalised approach of selecting pre-clinical models. Methods Patient PDA samples (n = 70; International Cancer Genome Consortium) were subjected to hierarchical clustering using a gene set representing CD8 and regulatory T cells to identify sub-groups. Syngeneic mouse models recapitulating these sub-groups (cross-species analysis) were developed using cell lines from genetically engineered mouse (GEM) models. Models bearing subcutaneous tumours were treated with anti-GITR agonist (DTA-1; 10mg/kg) or isotype (IgG2b) and tissues were collected for 12-color fluorescence activated cell sorting (FACS), NanoString and immunohistochemistry (IHC). Results Using clustering analysis, we identified four distinct patient PDA immune sub-groups with differential expression of T cell genes. These sub-groups showed differential expression of glucocorticoid-induced TNF receptor (GITR) and its associated genes. Hence, we hypothesised that these groups may respond differently to anti-GITR therapy in vivo. As expected, the three syngeneic models showed differential response to anti-GITR treatment. Model with increased GITR expression (>2-fold; NanoString and immunoblot) showed a significant (p = 0.02) survival benefit and a modest decrease in tumour burden upon anti-GITR treatment (n = 10) compared to the isotype control (n = 7) and the other two models. We observed anti-GITR mediated increase in GITR+ CD8+ cells and no change in GITR+ FOXP3+ cells (FACS, NanoString and IHC) along with increased gene expression of Pdcd1, Lag3, Spn and Itk. Conclusion Our models recapitulate the immunological gene expression profiles observed in patient PDA samples making them a highly useful resource to study personalized immunotherapies. This integrated study highlights the importance of patient selection to maximize therapy benefit and spare immune related adverse events. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure D. Cunningham: Research grant / Funding (self): Amgen; Research grant / Funding (self): Sanofi; Research grant / Funding (self): Merrimack; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Celgene; Research grant / Funding (self): MedImmune; Research grant / Funding (self): Bayer; Research grant / Funding (self): 4SC; Research grant / Funding (self): Clovis; Research grant / Funding (self): Eli Lilly; Research grant / Funding (self): Janssen; Research grant / Funding (self): Merck. N. Starling: Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Pfizer; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Bristol-Myers Squibb; Travel / Accommodation / Expenses: Eli Lilly; Travel / Accommodation / Expenses: Merck; Travel / Accommodation / Expenses: Roche; Honoraria (self): Eli Lilly; Honoraria (self): Merck Serono; Honoraria (self): MSD Oncology; Honoraria (self): Pierre Fabre; Advisory / Consultancy: Pfizer; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Servier. A. Sadanandam: Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Merck KGaa; Research grant / Funding (self): Pierre Fabre. All other authors have declared no conflicts of interest.

MEK inhibition suppresses B regulatory cells and augments anti-tumor immunity.

Mitogen-activated protein kinase (MAPK) kinase (MEK) is an integral component of the RAS pathway and a therapeutic target in RAS-driven cancers. Although tumor responses to MEK inhibition are rarely durable, MEK inhibitors have shown substantial activity and durable tumor regressions when combined with systemic immunotherapies in preclinical models of RAS-driven tumors. MEK inhibitors have been shown to potentiate anti-tumor T cell immunity, but little is known about the effects of MEK inhibition on other immune subsets, including B cells. We show here that treatment with a MEK inhibitor reduces B regulatory cells (Bregs) in vitro, and reduces the number of Bregs in tumor draining lymph nodes in a colorectal cancer model in vivo. MEK inhibition does not impede anti-tumor humoral immunity, and B cells contribute meaningfully to anti-tumor immunity in the context of MEK inhibitor therapy. Treatment with a MEK inhibitor is associated with improved T cell infiltration and an enhanced response to anti-PD1 immunotherapy. Together these data indicate that MEK inhibition may reduce Bregs while sparing anti-tumor B cell function, resulting in enhanced anti-tumor immunity.

Targeting interferon signaling and CTLA-4 enhance the therapeutic efficacy of anti-PD-1 immunotherapy in preclinical model of HPV+ oral cancer

BackgroundThe US is experiencing an epidemic of HPV+ oropharyngeal cancers (OPC), the rates and burden of which now exceed that for cervical cancer. Immunotherapy targeting programmed death 1 (PD-1) on tumor-infiltrating lymphocytes and/or its ligand PD-L1 on tumor cells, which was effective in several cancers has however, showed efficacy in only less than 15% of patients.MethodsWe used a preclinical HPV+ oral tumor model, mEER, consisting of mouse tonsil derived epithelial cells expressing HPV-16 E6 and E7 genes, along with the H-ras oncogene to test strategies for enhancing the efficacy of anti-PD-1 therapy.ResultsMonotherapy with PD-1 blocking antibody was ineffective against flank-implanted tumors, but induced regression in 54% of mice bearing orthotopic tongue tumors that correlated with higher CD8 T cell responses. Since the CD8+ T cells derived from tongue tumors also showed high levels of the immune checkpoint inhibitory receptor CTLA-4, we tested combination immunotherapy targeting both CTLA-4 and PD-1 together and observed 93.3% survival of mice bearing tumors in the tongue for the duration of our 100-day study. Protective immunity correlated with a significant decrease in immunosuppressive lymphoid and myeloid populations within the tumor microenvironment. Consistent with the reported capacity of interferon-driven PD-L1/PD-1 pathway induction to serve as a biomarker of response to PD-1 blockade, we observed elevated interferon signaling and significantly higher levels of PD-1/PD-L1 in tongue-implanted mEER tumors compared to those growing on the flank correlating with their preferential responsiveness to PD-1 blockade. More importantly, in a pseudometastasic mouse model bearing both flank and tongue tumors to represent metastatic disease, delivery of Stimulator of Interferon Induced Genes (STING) agonist into the flank tumors combined with systemic treatment with α-PD-1 and α-CTLA-4 antibodies resulted in sustained tumor regression in 71% of mice. In this case, productive abscopal anti-tumor immunity was associated with robust increases in the ratios of cytotoxic CD8+ T cells (CTL) versus regulatory T cells (Treg) and versus functional myeloid-derived suppressor cells (MDSC).ConclusionsThese results support combining α-PD-1 therapy with induction of IFN-α/β signaling via provision of STING agonist and/or through CTLA-4 blockade as potential treatment option for HNSCC patients, especially, those not responding to α-PD-1 monotherapy.

Biomaterials-Based Cancer Immunotherapy in Preclinical Models of Treatment-Resistant Head and Neck Cancer

Biomaterials-Based Cancer Immunotherapy in Preclinical Models of Treatment-Resistant Head and Neck Cancer

Abstract 2245: Improving the effectiveness of immunotherapy in breast cancer by targeting the tumor microenvironment

Abstract Large cohorts of recent clinical studies have firmly established that increased levels of tumor-infiltrating lymphocytes (TILs) in TNBC and HER2+ subtypes predicted better clinical outcome compared to the luminal subtype. These observations led to the hypothesis that women with TNBC or HER2+ subtypes may respond to a checkpoint blockade. However, early results from these trials using check point inhibitors alone or in combination with chemotherapy have shown very little promise in breast cancer patients, despite the remarkable long-lasting responses in other hard to treat malignancies such as non-small cell lung and melanoma. Although the outcome falls short of the expectation, it has suggested that the combinations of check point blockade with therapeutics that target immunosuppression may potentiate its efficacy. TNFα exhibits paradoxical roles; it may fuel tumor cell growth, invasion and metastasis in some tumor types, while in others it induces cytotoxic cell death. We recently demonstrated that TNFα distinctly induces A20 in TNBC subtype and protects these cells from TNFα-induced cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models. Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, we proposed that targeting HSP70 may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors do respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody ed with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (>10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility. Citation Format: Hasan Korkaya, Eunmi Lee, Raziye Piranioglu, Maria Ouzounova, Ahmet Korkaya, Jason Gestwicki, Max S. Wicha, Esteban Celis. Improving the effectiveness of immunotherapy in breast cancer by targeting 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 2245.

A Novel Form of 4-1BBL Prevents Cancer Development via Nonspecific Activation of CD4+ T and Natural Killer Cells.

Costimulation through 4-1BB (CD137) receptor generates robust CD8+ T-effector and memory responses. The only known ligand, 4-1BBL, is a trimeric transmembrane protein that has no costimulatory activity as a soluble molecule. Thus, agonistic antibodies to the receptor have been used for cancer immunotherapy in preclinical models and are currently being evaluated in the clinic. Here, we report that treatment with an oligomeric form of the ligand, SA-4-1BBL, as a single agent is able to protect mice against subsequent tumor challenge irrespective of the tumor type. Protection was long-lasting (>8 weeks) and a bona fide property of SA-4-1BBL, as treatment with an agonistic antibody to the 4-1BB receptor was ineffective in generating immune protection against tumor challenge. Mechanistically, SA-4-1BBL significantly expanded IFNγ-expressing, preexisting memory-like CD44+CD4+ T cells and NK cells in naïve mice as compared with the agonistic antibody. In vivo blockade of IFNγ or depletion of CD4+ T or NK cells, but not CD8+ T or B cells, abrogated the immunopreventive effects of SA-4-1BBL against cancer. SA-4-1BBL as a single agent also exhibited robust efficacy in controlling postsurgical recurrences. This work highlights unexpected features of SA-4-1BBL as a novel immunomodulator with implications for cancer immunoprevention and therapy. SIGNIFICANCE: This study demonstrates the unique and unexpected immunomodulatory features of SA-4-1BBL that bridge innate and adaptive immune responses with both preventive and therapeutic efficacy against cancer.

Biomaterials-Based Cancer Immunotherapy in Preclinical Models of Treatment-Resistant Head and Neck Cancer

Biomaterials-Based Cancer Immunotherapy in Preclinical Models of Treatment-Resistant Head and Neck Cancer

Abstract CT141: Safety and efficacy results from a phase I dose-escalation trial of Nintedanib in combination with Pembrolizumab in patients with advanced solid tumors (PEMBIB trial)

Abstract Background: Antiangiogenic drugs can synergize with immunotherapy in preclinical models. However some molecules have shown unacceptable levels of toxicity when combined with anti-PD-1. We aimed to determine the safety and activity of the nintedanib+pembrolizumab combination. Nintedanib is an oral angiokinase inhibitor aiming the vascular endothelial growth factor receptors 1,2,3, platelet-derived growth factor receptors α,β, fibroblast growth factor receptors 1,2,3 as well as RET. Pembrolizumab is a highly selective, humanized monoclonal IgG4-kappa isotype antibody against PD-1 designed to block the negative immune regulatory signaling of the PD-1 receptor expressed by T cells. Methods: PEMBIB is a monocentric phase Ib trial which evaluated escalating doses of continuous oral intake of nintedanib (Dose level 1 (DL1) = 150 mg BID; DL2 =200 mg BID) in combination with intravenous flat dose of pembrolizumab at 200 mg every 21 days in patients with advanced solid tumors using the rolling 6 design. A lead-in monotherapy of nintedanib was performed 7 days prior starting the C1D1 of pembrolizumab with the aim of modifying the tumor microenvironment. The primary objective was to establish the MTD of nintedanib in combination with pembrolizumab based on the assessment of DLT occurrence during the first 4 weeks (28 days since C1D1) and to determine the recommended phase II dose (RP2D). Secondary objectives included antitumor activity assessed by both RECIST v1.1 and irRECIST. The clinical activity of the combination is being further evaluated in the expansion part of this trial. Results: As of November 24, 2016, 13 patients (12 evaluable for DLT) have been enrolled in the escalation part with the following pathology: 2 squamous cervical carcinoma, 1 MSI colorectal cancer, 1 triple negative breast cancer, 2 thymic carcinoma, 1 malignant pleural mesothelioma, 1 peritoneal mesothelioma, 1 gastric adenocarcinoma, 1 clear cell renal carcinoma, 1 neuroendocrine tumor, 1 nasopharyngeal cancer. 50% patients were male, ECOG 0 (83%) or 1. There were no grade 4-5 toxicities. The most frequent adverse events reported for more than 2 patients were alanine & aspartate aminotransferase increase, fatigue, decreased appetite, diarrhea, nausea, vomiting, hypothyroidism. Three dose-limiting toxicities of liver enzymes elevation were observed in 200 mg BID nintedanib thus recommending 150 mg BID nintedanib for the phase II part. One nasopharyngeal carcinoma, 1 squamous cervical carcinoma and 1 thymic carcinoma developed an objective RECIST partial response (ORR=25%). Conclusions: Toxicity was consistent with the drugs profile and no unexpected events have been seen with the combination. Nintedanib at 150 mg BID with 200 mg flat dose of Pembrolizumab was well tolerated. Additional data for safety and efficacy is further evaluated in the expansion part. Citation Format: Andreea Varga, Capucine Baldini, Patricia Martin-Romano, Antoine Hollebecque, Anas Gazzah, Rastislav Bahleda, Jessica Menis, Stephane Champiat, Yolla El-Dakdouki, Xavier Paoletti, Vincent Ribrag, Jean-Marie Michot, Saloomeh Rafie, David Planchard, Benjamin Besse, Christophe Massard, Jean-Charles Soria, Aurelien Marabelle. Safety and efficacy results from a phase I dose-escalation trial of Nintedanib in combination with Pembrolizumab in patients with advanced solid tumors (PEMBIB trial) [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 CT141.

PO-413 Immune modulating properties of cyclophosphamide synergise with immunotherapy in preclinical models of neuroblastoma

IntroductionNeuroblastoma (NB) is one of the most common childhood cancers accounting for 15% of paediatric cancer deaths. Current therapies, which include chemotherapy, are highly toxic with significant treatment related mortality. There is little scope for further intensification therefore alternatives strategies such as immunotherapy, are a priority for improving patient outcomes. Classically, chemotherapy is regarded as immunosuppressive but recent work has highlighted that some may illicit an immunogenic form of cell death. This work sets out to identify the immunomodulating effects of cyclophosphamide (CPM) on tumour infiltrating immune cells and whether these properties can synergise with immunomodulatory antibodies in preclinical models of NB.Material and methodsThe effects of CPM and doxorubicin on tumour cells were investigated in vitro by analyses of immunogenic cell death (ICD) markers. Two different in vivo subcutaneous murine NB models (NXS2 +NB9464D), were treated i.p with different doses of CPM. Tissues were harvested and detailed immunophenotyping performed. Combination of CPM and anti-PD-1 therapy was investigated in both models using tumour growth and survival as end points. Combination therapy was also assessed in TH-MYCN mice which develop spontaneous neuroblastoma.Results and discussionsChemotherapy application in vitro led to an increase in expression of the ICD markers, ecto-calreticulin and Hsp-70. CPM was found to have numerous immune modulating activities in vivo, including the reduction of intratumoral Treg cells, even at low doses (20 mg/kg), in both NB models. Combination therapy was shown to increase CD8+ and CD4+ percentages within tumours, along with an increase CD4+ effector and memory cell proportions. Anti-PD-1 therapy synergised with CPM to improve median survival and slow tumour growth. Metronomic dosing of CPM and anti-PD-1 antibody led to tumour regression in TH-MYCN tumour bearing mice.ConclusionThis work supports combining low dose CPM to enhance immunomodulatory antibody therapy, to generate therapeutic anti-neuroblastoma immunity. Ongoing work is focused on elucidating the mechanism of action of the CPM and anti-PD-1 combination therapy, along with identifying the optimal chemotherapy and antibody dosing strategies.