Efficacy Of Checkpoint Blockade Research Articles

Improved prediction of immune checkpoint blockade efficacy across multiple cancer types.

Only a fraction of patients with cancer respond to immune checkpoint blockade (ICB) treatment, but current decision-making procedures have limited accuracy. In this study, we developed a machine learning model to predict ICB response by integrating genomic, molecular, demographic and clinical data from a comprehensively curated cohort (MSK-IMPACT) with 1,479 patients treated with ICB across 16 different cancer types. In a retrospective analysis, the model achieved high sensitivity and specificity in predicting clinical response to immunotherapy and predicted both overall survival and progression-free survival in the test data across different cancer types. Our model significantly outperformed predictions based on tumor mutational burden, which was recently approved by the U.S. Food and Drug Administration for this purpose1. Additionally, the model provides quantitative assessments of the model features that are most salient for the predictions. We anticipate that this approach will substantially improve clinical decision-making in immunotherapy and inform future interventions.

CONFIRMing single-drug immune checkpoint blockade efficacy in mesothelioma

Mesothelioma is an uncommon cancer, but, despite a reduction in asbestos use in high-income countries in the past few decades, we are still facing a global epidemic of this disease. Immune checkpoint blockade had an inauspicious start in mesothelioma, when the anti-CTLA-4 antibody, tremelimumab, was shown to not improve overall survival compared with placebo in the second-line setting. 1 Maio M Scherpereel A Calabro L et al. Tremelimumab as second-line or third-line treatment in relapsed malignant mesothelioma (DETERMINE): a multicentre, international, randomised, double-blind, placebo-controlled phase 2b trial. Lancet Oncol. 2017; 18: 1261-1273 Summary Full Text Full Text PDF PubMed Scopus (271) Google Scholar However, outcomes from several subsequent single-arm clinical trials suggested anti-tumour activity of PD-1 or PD-L1 blockade in patients with mesothelioma. 2 Alley EW Lopez J Santoro A et al. Clinical safety and activity of pembrolizumab in patients with malignant pleural mesothelioma (KEYNOTE-028): preliminary results from a non-randomised, open-label, phase 1b trial. Lancet Oncol. 2017; 18: 623-630 Summary Full Text Full Text PDF PubMed Scopus (335) Google Scholar , 3 Quispel-Janssen J van der Noort V de Vries JF et al. Programmed death 1 blockade with nivolumab in patients with recurrent malignant pleural mesothelioma. J Thorac Oncol. 2018; 13: 1569-1576 Summary Full Text Full Text PDF PubMed Scopus (150) Google Scholar , 4 Okada M Kijima T Aoe K et al. Clinical efficacy and safety of nivolumab: results of a multicenter, open-label, single-arm, Japanese phase II study in malignant pleural mesothelioma (MERIT). Clin Cancer Res. 2019; 25: 5485-5492 Crossref PubMed Scopus (138) Google Scholar Objective tumour response rates ranged from 20–30%. Together with promising progression-free survival and overall survival outcomes, these trials triggered inclusion of single drug PD-1 inhibition as an option for subsequent-line therapy in some—but not all—clinical practice guidelines from as early as 2017. Nivolumab versus placebo in patients with relapsed malignant mesothelioma (CONFIRM): a multicentre, double-blind, randomised, phase 3 trialNivolumab represents a treatment that might be beneficial to patients with malignant mesothelioma who have progressed on first-line therapy. Full-Text PDF Open Access

Potential Biomarkers for the Efficacy of PD-1-PD-L Blockade in Cancer.

A decade ago, immune checkpoint blockade emerged as a major breakthrough in oncology, proposing a novel approach by which immune brakes could be released to enhance antitumor responses. Despite apparently modest improvement of the median duration of response, a spectacular doubling of long-term responses as compared to the available standard of care was seen, for instance, in metastatic melanoma. It soon became obvious that the percentage of patients responding to these novel approaches is relatively small, and the importance of an accurate prediction of responders became more and more clear. Strong predictive markers would allow for the administration of immune checkpoint blocker therapy to the patients most likely to benefit from it, and sparing the potential non-responders of a treatment which is far from innocuous, being associated with significant side-effects and, not least, an important price tag. A number of potential response predictors have already been investigated and partly validated, but they do not cover the major unmet need encountered in the current clinical setting. Here, we review biomarkers for immune checkpoint blockade efficacy, either clinically validated and currently in use, or which have been proposed as candidates and are currently under investigation.

Identification of an Autophagy-Related Risk Signature Correlates With Immunophenotype and Predicts Immune Checkpoint Blockade Efficacy of Neuroblastoma.

Neuroblastoma is one of the malignant solid tumors with the highest mortality in childhood. Targeted immunotherapy still cannot achieve satisfactory results due to heterogeneity and tolerance. Exploring markers related to prognosis and evaluating the immune microenvironment remain the major obstacles. Herein, we constructed an autophagy-related gene (ATG) risk model by multivariate Cox regression and least absolute shrinkage and selection operator regression, and identified four prognostic ATGs (BIRC5, GRID2, HK2, and RNASEL) in the training cohort, then verified the signature in the internal and external validation cohorts. BIRC5 and HK2 showed higher expression in MYCN amplified cell lines and tumor tissues consistently, whereas GRID2 and RNASEL showed the opposite trends. The correlation between the signature and clinicopathological parameters was further analyzed and showing consistency. A prognostic nomogram using risk score, International Neuroblastoma Staging System stage, age, and MYCN status was built subsequently, and the area under curves, net reclassification improvement, and integrated discrimination improvement showed more satisfactory prognostic predicting performance. The ATG prognostic signature itself can significantly divide patients with neuroblastoma into high- and low-risk groups; differentially expressed genes between the two groups were enriched in autophagy-related behaviors and immune cell reactions in gene set enrichment analysis (false discovery rate q -value < 0.05). Furthermore, we evaluated the relationship of the signature risk score with immune cell infiltration and the cancer-immunity cycle. The low-risk group was characterized by more abundant expression of chemokines and higher immune checkpoints (PDL1, PD1, CTLA-4, and IDO1). The risk score was significantly correlated with the proportions of CD8+ T cells, CD4+ memory resting T cells, follicular helper T cells, memory B cells, plasma cells, and M2 macrophages in tumor tissues. In conclusion, we developed and validated an autophagy-related signature that can accurately predict the prognosis, which might be meaningful to understand the immune microenvironment and guide immune checkpoint blockade.

Next generation of tumor-activating type I IFN enhances anti-tumor immune responses to overcome therapy resistance

Type I interferon is promising in treating different kinds of tumors, but has been limited by its toxicity, lack of tumor targeting, and very short half-life. To target tumors, reduce systemic toxicity, and increase half-life, here we engineer a masked type I IFN-Fc (ProIFN) with its natural receptor connected by a cleavable linker that can be targeted by tumor-associated proteases. ProIFN has a prolonged serum half-life and shows an improved tumor-targeting effect. Interestingly, ProIFN-treated mice show enhanced DC cross-priming and significant increased CD8+ infiltration and effector function in the tumor microenvironment. ProIFN is able to improve checkpoint blockade efficacy in established tumors, as well as radiation efficacy for both primary and metastatic tumors. ProIFN exhibits superior long-term pharmacokinetics with minimal toxicity in monkeys. Therefore, this study demonstrates an effective tumor-activating IFN that can increase targeted immunity against primary tumor or metastasis and reduce periphery toxicity to the host.

Tumor organoid-originated biomarkers predict immune response to PD-1 blockade.

Despite the demonstrated efficacy and broad applicability of checkpoint blockade, the mechanisms by which it exerts its antitumor effects are incompletely understood. A recent article in Nature Medicine describes an exvivo platform for assessing early responses to checkpoint blockade and the properties of tumor immune contexture in correlation to clinical responses.

The Prognosis and Immune Checkpoint Blockade Efficacy Prediction of Tumor-Infiltrating Immune Cells in Lung Cancer.

BackgroundsThe high morbidity and mortality of lung cancer are serious public health problems. The prognosis of lung cancer and whether to apply immune checkpoint blockade (ICB) are currently urgent problems to be solved.MethodsUsing R software, we performed Kaplan–Meier (K-M) analysis, Cox regression analysis, functional enrichment analysis, Spearman correlation analysis, and the single-sample gene set enrichment analysis.ResultsOn the Tumor IMmune Estimation Resource (TIMER2.0) website, we calculated the abundance of tumor-infiltrating immune cells (TIICs) of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. B cell and myeloid dendritic cell (DC1) were independent prognostic factors for LUAD and LUSC patients, respectively. Enrichment analysis confirmed that genes highly related to B cell or DC1 were closely related to the immune activation of lung cancer patients. In terms of adaptive immune resistance markers, CD8A, CD8B, immunomodulators (immunostimulants, major histocompatibility complex, receptors, and chemokines), immune-related pathways, tumor microenvironment score, and TIICs, high B cell/DC1 infiltration tissue was inflamed and immune-activated and might benefit more from the ICB. Genes most related to B cell [CD19, toll-like receptor 10 (TLR10), and Fc receptor-like A (FCRLA)] and DC1 (ITGB2, LAPTM5, and SLC7A7) partially clarified the roles of B cell/DC1 in predicting ICB efficacy. Among the 186 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, there were three and four KEGG pathways, which partially explained the molecular mechanisms by which B cell and DC1 simultaneously predicted the prognosis and efficacy of immunotherapy, respectively. Among five immune subtypes, the abundance of B cell/DC1 and expression of six hub genes were higher in immune C2, C3, and C6.ConclusionB cell and DC1 could predict the prognosis and ICB efficacy of LUAD and LUSC patients, respectively. The six hub genes and seven KEGG pathways might be novel immunotherapy targets. Immune C2, C3, and C6 subtypes of lung cancer patients might benefit more from ICB therapy.

O-5 Frequency of minimal residual disease as measured by ctDNA in mismatch repair deficient tumors following curative resection

Mismatch repair deficient (MMRd) tumors are highly sensitive to checkpoint blockade (CPB) in patients with metastatic disease, regardless of tumor type. However, the efficacy of CPB in the adjuvant setting is unknown, especially since MMRd is considered a favorable biomarker for most resected tumor types. Circulating tumor DNA (ctDNA) could be used to screen for patients at high risk for recurrence following surgery or adjuvant chemotherapy, and identify patients (pts) that would most benefit from CPB.

Abstract 2964: Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy

Abstract Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy. These differences in sensitivity can be either innate or acquired. Yet, there has been limited 3D in vitro models to assess tumor immune-reactivity. These systems are ideal for isolating specific molecular mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with cytotoxic T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic response levels in patients. Methods: We created tumor organoids consisting of tumor and immune cells, embedded in extracellular matrix (ECM)-like hydrogels. Organoids were treated with therapeutic equivalent doses of anti-PD-1 and anti-CTLA-4 or single dose of anti-CD-47 and cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry staining were performed to determine the effects of ICB. Physiologic concentrations of metabolites derived from likely effector bacterial species were then evaluated for their effects on genetic expression and immunotherapy efficacy. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR resulted in some elucidation of potential cellular changes due to bacterial metabolites with further quantification ongoing. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We can now continue to change individual microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive or detrimental to successful ICB treatment. Citation Format: Ethan Shelkey, David Soto-Pantoja, Yong Lu, Shay Soker. Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy [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 2964.

In Situ Vaccination Improves Efficacy of PD-1 Blockade in Unresponsive Lymphoma Tumors through Induction of a Highly Efficient Cross-Presenting Dendritic Cell Subset Expressing TLR3

BackgroundLow-grade non-Hodgkin's B-cell lymphomas are generally incurable, with standard therapies inducing only temporary remissions; and preliminary results with anti-PD-1 therapy have yielded low response rates. It has been shown that tumoral DC infiltration correlates with efficacy of checkpoint blockade. Tumor-targeted vaccines represent promising, novel treatment strategies able to induce anti-tumor T cells. Therefore, we tested the combination of in situ vaccination with PD-1 blockade in a pre-clinical mouse model.MethodsA20 lymphoma-bearing mice were treated with a PD-1 blocking antibody with or without an in situ vaccine consisting of intratumoral injections of FMS-like tyrosine kinase-3 ligand (Flt3L), local irradiation (XRT) of the tumor and intratumoral injections of the TLR3 agonist poly-ICLC (pIC).DataUntreated lymphoma tumors contained very low numbers of DC and treatment with anti-PD1 alone did not induce tumor regression or increase survival. After Flt3L treatment, flow cytometry analysis revealed a dramatic increase of IRF8+TLR3+ DC at the tumor site, the tumor-draining lymph node (TdLN) and the spleen. XRT of A20 cells induced activation of Flt3L-treated splenic DC in vitro and local XRT of the tumor in vivo induced expression of CD103 on infiltrating TLR3+ DC, indicating maturation of these cells. Local XRT also increased the amount of intratumoral DC taking up tumor-associated antigen (TAA) , suggesting enhanced uptake of dying tumor cells. Interestingly, tumor antigens were taken up mainly by CD103+ DC and not CD103- subtypes. CD103+ expression distinguishes a subset of migratory DC that are very efficient at cross-presenting exogenous antigens to CD8+ T cells. Accordingly, CD103+ DC isolated from the tumor induced proliferation of tumor-specific CD8+ T cells more efficiently than CD103- subsets. The combination of Flt3L with XRT and pIC induced tumor-reactive, Interferon γ (IFN γ)-producing T cells, but delayed tumor growth and improved survival only in 40% of mice. Importantly, in situ vaccination also increased expression of PD-L1 on tumor cells and tumor infiltrating DC. Consistent with these findings, combination of in situ vaccination with PD-1 blockade led to complete tumor regression and increased long-term survival in the majority of mice. Moreover, PD-1 blockade also increased the number of tumor-reactive T cells and depletion of CD8+ T cells abrogated the anti-tumor effect.ConclusionsIn situ vaccination combining intratumoral Flt3L injection with local XRT and poly-ICLC can improve efficacy of anti-PD-1 in checkpoint-unresponsive lymphoma tumors through induction of a highly efficient cross-presenting DC subset leading to long-term regression of established lymphoma tumors DisclosuresKeler:Celldex Therapeutics: Employment. Davis:Celldex Therapeutics: Employment. Salazar:Oncovir, Inc: Employment. Brody:Janssen Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Research Funding; Synergy pharmaceuticals: Equity Ownership; Immunogen: Equity Ownership; Novavax: Equity Ownership; Pharmacyclics: Other: Travel expenses, Speakers Bureau; Acerta: Research Funding; Gilead Sciences, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses, Speakers Bureau.

Anti-Inflammatory Drugs Remodel the Tumor Immune Environment to Enhance Immune Checkpoint Blockade Efficacy.

Identifying strategies to improve the efficacy of immune checkpoint blockade (ICB) remains a major clinical need. Here, we show that therapeutically targeting the COX2/PGE2/EP2-4 pathway with widely used nonsteroidal and steroidal anti-inflammatory drugs synergized with ICB in mouse cancer models. We exploited a bilateral surgery model to distinguish responders from nonresponders shortly after treatment and identified acute IFNγ-driven transcriptional remodeling in responder mice, which was also associated with patient benefit to ICB. Monotherapy with COX2 inhibitors or EP2-4 PGE2 receptor antagonists rapidly induced this response program and, in combination with ICB, increased the intratumoral accumulation of effector T cells. Treatment of patient-derived tumor fragments from multiple cancer types revealed a similar shift in the tumor inflammatory environment to favor T-cell activation. Our findings establish the COX2/PGE2/EP2-4 axis as an independent immune checkpoint and a readily translatable strategy to rapidly switch the tumor inflammatory profile from cold to hot. SIGNIFICANCE: Through performing in-depth profiling of mice and human tumors, this study identifies mechanisms by which anti-inflammatory drugs rapidly alter the tumor immune landscape to enhance tumor immunogenicity and responses to immune checkpoint inhibitors.See related commentary by Melero et al., p. 2372.This article is highlighted in the In This Issue feature, p. 2355.

Frequency of minimal residual disease as measured by ctDNA in mismatch repair deficient tumors following curative resection.

e14520 Background: Mismatch repair deficient (MMRd) tumors are highly sensitive to checkpoint blockade (CPB) in patients with metastatic disease regardless of tumor type. However, the efficacy of CPB in the adjuvant setting is unknown, especially since MMRd is considered a favorable biomarker for most resected tumor types. Circulating tumor DNA (ctDNA) could be used to screen for patients at high risk for recurrence following surgery or adjuvant chemotherapy and identify patients (pts) that would most benefit from CPB. Methods: To assess the frequency of ctDNA in the resected MMRd population, we prospectively screened pts with MMRd tumors who completed standard perioperative chemotherapy and surgery (NCT03832569). DNA from resected tumors and matched postoperative plasma was sequenced for the presence of somatic mutations. Patients were considered to have minimal residual disease (MRD) when mutations were identified in tumor and found to be identical to those in matched plasma DNA. Somatic tissue mutations were assessed using MSK-IMPACT and ctDNA was assessed using FoundationOne, Guardant360 or MSK-ACCESS. Results: A total of 86 pts were screened for the presence of MRD. These represented 7 tumor types with colorectal (63%), endometrial (16%) and esophagogastric (13%) being the most common. The majority of pts were stage III (49%). MRD was detected in 18% of cases (14 of 79). Among the MRD negative group (n=62), only one pt developed disease recurrence. Three samples failed ctDNA analysis for technical reasons. Conclusions: MRD was identified in 18% of resected MMRd tumors using ctDNA analysis, suggesting this to be a feasible tumor agnostic approach to test the efficacy of CPB in a pts at high-risk for recurrence. Future studies will assess the impact of CPB in MRD positive MMRd tumors.[Table: see text]

Unaltered IL-2 encapsulated in acoustically active lipid microspheres for systemic but targeted microdose delivery to tumor microenvironment.

e14535 Background: In order to take local advantage of exogenous interleukin-2 (IL-2) on effector T-cells within the tumor microenvironment while simultaneously avoiding systemic Treg immunosuppressive effects, microdoses of recombinant human IL-2 (rhIL-2) were encapsulated in gas-filled lipid microspheres (eIL-2), then activated with ultrasound. This novel therapeutic agent (eIL-2) was selectively delivered to the tumor via sonoporation and its anti-tumor effects were determined alone and in combination with PD-1 blockade. Methods: In an MC38 colon cancer model, jugular button catheters (Instech) were surgically implanted in a total of 56 animals (8 animals per arm) prior to tumor inoculation to facilitate repeatable IV injection of both anti-PD-1 (RMP1-14; BioXCell) and microspheres. Imagent perflexane lipid microspheres were incubated with rhIL-2 and the high concentration infranatant was separated, discarded, and replaced with sterile saline to resuspend microspheres to formulate eIL-2 on-site. Tumor-bearing mice were dosed on Days 1, 4, 8, and 12. Ultrasound (US) settings used were 2.2 MHz frequency, and 0.339 mechanical index (MI) during insonation. Once every 10 seconds a microbubble destruct pulse of 1.304 MI lasting 1,100 milliseconds was applied. Total insonation time was 5 minutes using a commercial ultrasound scanner (Mindray TE7) operating within diagnostically safe exposure limits. Study groups included Group 1: no treatment; Group 2: aPD-1, 3 mg/kg; Group 3: aPD-1, 3 mg/kg, Imagent + US; Group 4: Free rhIL-2, 10,000 IU (0.61μg)/animal; Group 5: eIL-2, approximately 0.2 μg encapsulated/animal; Group 6: Free rhIL-2, 10,000 IU (0.61μg)/animal, aPD-1, 3 mg/kg; Group 7: eIL-2, approximately 0.1 μg encapsulated/animal, aPD-1, 3 mg/kg. Results: The anti-PD-1 dose of 3 mg/kg demonstrated an expected tumor growth inhibition over control of -38.2% on Day 6, -35.6% on Day 8 and -30.3% on Day 12. Adding eIL-2 + ultrasound external stimuli approximately doubled the tumor inhibition effect of RMP1-14 to -68.1% on Day 6, -69.2% on Day 8 and -62.0% on Day 12. We also analyzed cytokine levels in the peripheral blood (Day 12) using a multiplex ELISA and observed elevated levels of pro-inflammatory cytokines including IFN-g, IL-12, and IL-1a, which likely serve to enhance anti-tumor immunity. IL-15 was also increased following treatment, which supports effector T cell survival. Conclusions: Using a novel form of recombinant human interleukin-2 encapsulated within a gas-filled lipid microsphere, we demonstrated that ultrasound-activated local delivery via sonoporation plus systemic PD-1 blockade led to improved tumor control and increased expression of pro-inflammatory cytokines. This novel approach enhances the efficacy of checkpoint blockade for the treatment of solid tumors. Further experiments are underway to interrogate mechanisms of action.

EORTC 1707 VESTIGE: Adjuvant immunotherapy in patients with resected gastric cancer following preoperative chemotherapy with high risk for recurrence (ypN+ and/or R1)—An open-label randomized controlled phase II study.

TPS4156 Background: Gastroesophageal adenocarcinoma (GEA) patients with metastatic lymph nodes (ypN+) or a microscopically incomplete surgical resection (R1) following neoadjuvant chemotherapy are at high risk of disease recurrence. Current practice is to continue with the same perioperative chemotherapy used prior to surgery, despite these suboptimal outcomes. Adjuvant immunotherapy with nivolumab has shown efficacy in poor risk GEA patients following chemoradiotherapy and surgery in the CheckMate 577 trial, and nivolumab and ipilimumab have demonstrated activity in advanced GEA. We hypothesise that high risk (ypN+ and/or R1) post resection GEA patients who are treated with nivolumab and ipilimumab will have better disease free survival than patients who continue with standard post-operative chemotherapy. Methods: VESTIGE is an ongoing, international, open label randomized phase II study designed to evaluate the efficacy of adjuvant nivolumab plus ipilimumab versus standard post-operative chemotherapy in high risk (ypN+ and/or R1) post resection GEA patients. Eligible patients (n=240) will be randomised 1:1 to receive post-operative adjuvant chemotherapy (identical regimen as pre-operatively) or nivolumab 3mg/kg IV q2w plus ipilimumab 1mg/kg IV q6w x 1 year. Key inclusion criteria include ypN+ and/or R1 status following neoadjuvant chemotherapy plus surgery and an adequate pre-specified surgical resection. The primary endpoint of the study is disease free survival, with secondary endpoints of overall survival, safety, toxicity and quality of life. Since start of recruitment in August 2019, the trial has recruited 85 of 240 patients at 18 sites in the Czech Republic, France, Germany, Israel, Italy, Norway, Poland, Spain, and United Kingdom. The VESTIGE translational research programme includes collection of pre-treatment biopsies, post-chemotherapy resection specimens and serial liquid biopsy on treatment to explore biomarkers predictive of immune checkpoint blockade efficacy. Clinical trial information: NCT03443856.

Immuno‐Reactive Cancer Organoid Models to Examine Microbiome Metabolite Effects on Immune Checkpoint Blockade Efficacy

Introduction As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, a sizable percentage of patients are non-responders to all types of immunotherapy. Yet, there has been limited number of in vitro models to assess tumor immune-reactivity and enable directed study of the mechanisms of immunotherapy resistance in solid tumors. The goal of this project is develop a model in which we could validate cancer cell killing due to immune cell action in an easily modifiable environment. Towards that, we created 3D tumor tissue constructs (organoids) containing cancer cells paired with cytotoxic T-cells to model immunotherapy efficacy. Our choice of immune checkpoint inhibitor antibodies were a paired course of PD-1 and CTLA-4, which are used extensively in the clinic, or anti-CD47. Upon validation of our organoid model, they could address specific mechanisms behind certain tumor microenvironment elements, such as bacterial metabolites, on immunotherapy efficacy. Methods We created extracellular matrix like 3D structures using collagen/hyaluronic acid-based hydrogels into which tumor cell were mixed, along with T-cells. The cancer cells incorporated were either freshly-isolated CT-26 colon adenocarcinoma cells, 4T1 breast cancer cells, or cultured CT-26 cells. The T-cells were either derived from murine lymph nodes or were isolated from the murine tumor infiltrating mononuclear cell population. Organoids were treated with microbial metabolites and their response to therapeutic equivalent doses of anti- PD-1, CTLA-4 and CD47 antibodies was examined using cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry (IHC) staining. Results The results show that in our immune cell supplemented tumor organoid models the immune checkpoint inhibitor regimens stimulated internally localizing T-cells and inducing T-cell-mediated tumor cell killing. Checkpoint inhibitor treated samples showed proportionally greater loss of viability when compared to untreated controls (p<0.01). The results were corroborated by IHC, showing increased numbers of CD-4+ T-cells and cytotoxic proteins such as granzyme B in the stimulated samples. RT-qPCR results show an increase in key proteins for immune function when both immunotherapy and bacterial metabolites are administered simultaneously. Conclusion: We have created an ex-vivo immune-reactive tumor organoid model for studying immunotherapy. This model will allow us to modulate facets of the tumor cell component in the organoid system including cancer type and grade, tumor cell mutations, biochemical signals, and physical properties of the microenvironment. We can then observe the impacts of these changes on immunotherapy efficacy in order to identify factors that could be targeted to improve immunotherapy efficacy. In this study, we have focused on the effects of bacterial metabolites on immunotherapy efficacy. Preliminary data suggest that there may be a connection between these microbial factors and tumor sensitivity to immunotherapy.

Bromodomain protein BRD4 directs CD8 T cell differentiation during infection and cancer

Abstract In response to infection and malignancy, antigen-specific CD8 T cells differentiate into functionally diverse populations that are critical for resolving disease and providing durable immunity. Here, we utilized an in vivo RNAi screening strategy to identify transcriptional and epigenetic regulators controlling CD8 T cell fate. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we revealed an essential role for the chromatin modifier and BET family member BRD4 in supporting T cell differentiation during infection and cancer. BRD4 was found to bind diverse regulatory regions critical to effector T cell differentiation and was essential for transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion or chemical inhibition of BRD4 resulted in impaired maintenance of a terminal effector phenotype. In preclinical models of cancer, BRD4 was also required for formation of a terminally differentiated CD8 T cell state in the tumor microenvironment, which we show holds implications for immunotherapies. Tailored inhibition of BRD4 enhanced immune checkpoint blockade efficacy; however, complete ablation of BRD4 activity blunted T cell effector function and adoptive cell therapy efficacy. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis-regulatory elements to control CD8 T cell fate and stability of lineage identity, and provide insight for understanding the efficacy and full therapeutic potential of BET inhibition and T cell immunotherapies.

Tumor-Secreted Extracellular Vesicles Regulate T-Cell Costimulation and Can Be Manipulated To Induce Tumor-Specific T-Cell Responses

Colorectal cancer is a major cause of cancer-related deaths worldwide. Immune checkpoint blockade therapies are effective in 30%-60% of the microsatellite instable-high subtype. Unfortunately, most patients with colorectal cancer (>85%) have microsatellite stable tumors that do not respond. In this study, we aimed to decipher the underlying tumor-intrinsic mechanisms critical for improving immunotherapy in colorectal cancer. We used human and mouse tumor samples, cell lines, human colorectal cancer organoids, and various syngeneic orthotopic mouse models of late-stage colorectal cancer to define the effects of tumor cell-secreted extracellular vesicles (EVs) on antitumor immune response. Our analyses of human colorectal cancer immune profiles and tumor-immune cell interactions showed that tumor-secreted EVs containing microRNA miR-424 suppressed the CD28-CD80/86 costimulatory pathway in tumor-infiltrating T cells and dendritic cells, leading to immune checkpoint blockade resistance. Modified tumor-secreted EVs with miR-424 knocked down enhanced T-cell-mediated antitumor immune response in colorectal cancer tumor models and increased the immune checkpoint blockade response. Intravenous injections of modified tumor-secreted EVs induced tumor antigen-specific immune responses and boosted the immune checkpoint blockade efficacy in colorectal cancer models that mimic aggressively progressing, late-stage disease. Collectively, we show a critical role for tumor-secreted EVs in antitumor immune regulation and immunotherapy response, which could be developed as a novel treatment for immune checkpoint blockade-resistant colorectal cancer.

Novel Combinatorial Approaches to Tackle the Immunosuppressive Microenvironment of Prostate Cancer.

Simple SummaryThe mainstay of treatment for advanced prostate cancer (PCa) is androgen deprivation therapy (ADT), and although patients initially respond, almost all will eventually develop progressive and metastatic disease. Metastatic, hormone-resistant prostate cancer is essentially incurable. A key problem with ADT is that it is not effective in the long term, partly due to the fact that prostate cancers can switch from requiring androgen to grow to surviving without it and these new tumor cells are more sensitive to stress hormones released by the brain as a consequence of chronic stress/depression. Moreover, both extensive treatment with ADT and chronic stress/depression lead to the recruitment of immature myeloid-derived suppressor cells (MDSCs) which are known to suppress immune responses against tumors. The future of prostate cancer treatments will most certainly include a combination of vaccine with beta-blockers (to interfere with signals from stress hormones) and/or the use of histone deacetylase (HDAC) inhibitors which prevent the function/recruitment of MDSC where timing will be of critical importance.Prostate cancer (PCa) is the second-most common cancer in men worldwide and treatment options for patients with advanced or aggressive prostate cancer or recurrent disease continue to be of limited success and are rarely curative. Despite immune checkpoint blockade (ICB) efficacy in some melanoma, lung, kidney and breast cancers, immunotherapy efforts have been remarkably unsuccessful in PCa. One hypothesis behind this lack of efficacy is the generation of a distinctly immunosuppressive prostate tumor microenvironment (TME) by regulatory T cells, MDSCs, and type 2 macrophages which have been implicated in a variety of pathological conditions including solid cancers. In PCa, Tregs and MDSCs are attracted to TME by low-grade chronic inflammatory signals, while tissue-resident type 2 macrophages are induced by cytokines such as IL4, IL10, IL13, transforming growth factor beta (TGFβ) or prostaglandin E2 (PGE2) produced by Th2 cells. These then drive tumor progression, therapy resistance and the generation of castration resistance, ultimately conferring a poor prognosis. The biology of MDSC and Treg is highly complex and the development, proliferation, maturation or function can each be pharmacologically mediated to counteract the immunosuppressive effects of these cells. Herein, we present a critical review of Treg, MDSC and M2 involvement in PCa progression but also investigate a newly recognized type of immune suppression induced by the chronic stimulation of the sympathetic adrenergic signaling pathway and propose targeted strategies to be used in a combinatorial modality with immunotherapy interventions such as ICB, Sipuleucel-T or antitumor vaccines for an enhanced anti-PCa tumor immune response. We conclude that a strategic sequence of therapeutic interventions in combination with additional holistic measures will be necessary to achieve maximum benefit for PCa patients.

RNA m6A methylation orchestrates cancer growth and metastasis via macrophage reprogramming

N6-methyladenosine (m6A) is a reversible mRNA modification that has been shown to play important roles in various biological processes. However, the roles of m6A modification in macrophages are still unknown. Here, we discover that ablation of Mettl3 in myeloid cells promotes tumour growth and metastasis in vivo. In contrast to wild-type mice, Mettl3-deficient mice show increased M1/M2-like tumour-associated macrophage and regulatory T cell infiltration into tumours. m6A sequencing reveals that loss of METTL3 impairs the YTHDF1-mediated translation of SPRED2, which enhances the activation of NF-kB and STAT3 through the ERK pathway, leading to increased tumour growth and metastasis. Furthermore, the therapeutic efficacy of PD-1 checkpoint blockade is attenuated in Mettl3-deficient mice, identifying METTL3 as a potential therapeutic target for tumour immunotherapy.

48218 Preclinical modeling of BRAF(V600E)/PTEN-/- melanoma leptomeningeal disease (LMD) to assess intrathecal checkpoint blockade

ABSTRACT IMPACT: Melanoma leptomeningeal disease (LMD) is a devastating subtype of central nervous system (CNS) metastatic disease that is associated with limited treatment options and an extremely poor prognosis, thus requiring the development of preclinical models of LMD for therapeutic development. OBJECTIVES/GOALS: 1.Develop an immunocompetent murine model of melanoma LMD with tumors bearing genetic mutations commonly found in patients, specifically BRAF(V600E)/PTEN-/-2.Assess the safety of intrathecal (IT) immunotherapy, specifically anti-PD1 antibody (aPD1)3.Evaluate the therapeutic efficacy of IT aPD1 checkpoint blockade in murine melanoma LMD METHODS/STUDY POPULATION: To develop BRAF(V600E)/PTEN-/- LMD models, we acquired BP, D4M, and D4M-UV2 (irradiated) murine melanoma cell lines and luciferase-tagged them. 1.5x10^4 cells were suspended in 10 uL serum-free media and injected into the cisterna magna of female C57BL/6 mice. Brain and spinal cord were harvested for histologic assessment once mice were moribund. To assess safety of IT aPD1, we injected IT control IgG or IT aPD1 (13 ug, 26 ug, 39 ug) and monitored weights or harvested at days 7 or 14 for IHC staining of inflammation markers. To evaluate therapeutic efficacy of IT aPD1, BP cells were directly injected as above. After 3 days, mice underwent imaging to confirm tumor uptake and randomization to receive 13 ug IT control IgG or aPD1 once + 200 ug systemic (Sys) control IgG or aPD1 (days 0, 3, and 5), and then monitored for survival. RESULTS/ANTICIPATED RESULTS: For LMD development, all mice survived cisternal injection of BP, D4M, and D4M-UV2 cells and median survival was 17, 19, and 30 days, respectively. Presence of leptomeningeal deposits was confirmed for all tumor-bearing mice by IHC for MART1. For safety of IT aPD1, all mice survived the procedure and no mice displayed morbidity or &gt;10% weight loss over 14 days of observation. IHC assessment of brain and spinal cord samples from mice treated with 13 ug aPD1 revealed focal ischemia related to injection site and no other signs of neurological damage or inflammation. IT aPD1 treatment of mice with BP leptomeningeal tumors demonstrated no significant survival advantage, although both IT aPD1 +/- Sys aPD1 had mice live up to days 29 and 26, respectively, compared to both IT control IgG +/- Sys aPD1, for which all mice died by day 22. DISCUSSION/SIGNIFICANCE OF FINDINGS: We demonstrate that cisternal injection of murine BRAF(V600E)/PTEN-/- melanoma cell lines yield LMD with reproducible survival and that treatment with IT aPD1 in this model is feasible and safe. Together these findings establish a new model to facilitate the development of more effective immunotherapy strategies for melanoma patients with LMD.