Immune-based Cancer Therapies Research Articles

Abstract 936: Regulation of CD8+ T-cell function and antitumor activity by DGKα and DGKζ

Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that metabolize the lipid diacylglycerol (DAG) to form phosphatidic acid. In T cells, two isoforms of DGK, DGKα and DGKζ, serve an important role in dampening signal transduction downstream of the T cell receptor (TCR), where they phosphorylate DAG generated by PLCγ1 after activation of proximal signaling molecules. DAG generated downstream of TCR binds to Protein Kinase C θ (PKCθ) and Ras Guanyl Releasing Protein 1 (RasGRP1), facilitating T cell activation. Elimination of either DGK isoform leads to increased generation of DAG and subsequent strengthened signaling downstream of RasGRP1 and PKCθ. Functionally, this results in formation of CD8+ T cells with enhanced cytokine production and proliferation after TCR stimulation. While mice or CD8+ T cells deficient in DGKα or DGKζ are both known to demonstrate enhanced immune responses to tumor, there has not been, to date, a direct comparison of the anti-tumor activity between mice lacking the two isoforms. We sought to determine which isoform of DGK more intensely attenuated anti-tumor immunity in order to identify which would be a better target for immune-based cancer therapies. We inoculated C57BL/6 mice with a variety of syngeneic tumor cell lines - MC38 colon carcinoma, B16F1 melanoma, and C1498 leukemia - and directly compared survival and tumor growth between mice deficient in DGKα or DGKζ in the presence or absence of anti-PD1 treatment. We found that DGKζ-/- mice more efficiently suppressed growth of subcutaneously implanted tumor cells when compared with DGKα-/- mice across the three model systems and further observed that the combination of DGKζ-deficiency and anti-PD1 were additive in tumor control. Additionally, relative to wildtype CD8+ T cells, we found that DGKζ-/- CD8+ T cells demonstrated qualitatively enhanced mRNA changes as assessed by RNA-seq analysis of cells after TCR stimulation. Pathway analyses from RNA-seq similarly indicated enhanced quantitative changes in DGKζ-/- T cells relative to DGKα-/- T cells, findings confirmed with in vitro biochemical and functional assays. Lastly, using CRISPR-based approaches to eliminate DGKα or DGKζ, we observed similar biochemical changes in human T cells when compared with T cells from gene-deficient mice. Our data, employing genetic approaches to modulate DGKα or DGKζ in both human and mouse models, suggests that DGKζ is a dominant regulator of TCR driven T cell activation and is consistent with the observed superior anti-tumor immune response. Citation Format: Susan Wee, Junchen Gu, Cindy Wang, Carolyn Cao, Sandra Holzhauer, Heshani Desilva, Erin Wesley, Susan Tsai, Douglas Evans, Matthew Riese. Regulation of CD8+ T-cell function and antitumor activity by DGKα and DGKζ [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 936.

Regulation of regulatory T cells in cancer

SummaryThe inflammatory response to transformed cells forms the cornerstone of natural or therapeutically induced protective immunity to cancer. Regulatory T (Treg) cells are known for their critical role in suppressing inflammation, and therefore can antagonize effective anti‐cancer immune responses. As such, Treg cells can play detrimental roles in tumour progression and in the response to both conventional and immune‐based cancer therapies. Recent advances in our understanding of Treg cells reveal complex niche‐specific regulatory programmes and functions, which are likely to extrapolate to cancer. The regulation of Treg cells is reliant on upstream cues from haematopoietic and non‐immune cells, which dictates their genetic, epigenetic and downstream functional programmes. In this review we will discuss how Treg cells are themselves regulated in normal and transformed tissues, and the implications of this cross talk on tumour growth.

An IL-2 proaerolysin fusion toxin that selectively eliminates regulatory t cells to enhance antitumor immune response.

Recentsuccess with immune-checkpoint inhibitors in some tumor types has highlighted the power of the immune system to control and eradicate human cancer cells. However, these therapies have demonstrated a limited activity in prostate cancer, which has a more immunosuppressive microenvironment that can be because of the presence of a variety of inhibitory cell types, such as myeloid-derived suppressor cells, mesenchymal stem cells, and regulatory T cells (Tregs). One strategy to improve the efficacy of immune-based therapies for prostate cancer is to selectively eliminate these immunosuppressive cells within the tumor microenvironment. We developed and characterized a chimeric protein consisting of the cytokine IL-2 fused to binding mutant of the highly toxic bacterial toxin proaerolysin (ie IL2-R336A). The IL2-R336A fusion protein selectively kills immunosuppressive Tregs that express the IL-2 receptor while having little to no effect on cells negative for this target. IL2-R336A depleted Tregs in both tumor bearing and nontumor bearing mice. Tumor bearing mice vaccinated with a GMCSF-expressing CT-26 GVAX vaccine had reduced tumor growth when given IL2-R336A before vaccination. IL2-R336A also enhanced immune response to a model hemagglutinin antigen (HA) in HA-tolerized mice. These results suggest that this IL2-R336A toxin may be a useful in improving the therapeutic efficacy of antitumor vaccines by enhancing the immune response against target tumor antigens.

Culturing the Human Natural Killer Cell Line NK-92 in Interleukin-2 and Interleukin-15 - Implications for Clinical Trials.

The human natural killer cell line NK-92 is increasingly being used in adoptive cell immunotherapies, either in vitro or in animal models transduced with different chimeric antigen receptor (CAR) constructs. Herein, NK-92 cells were analyzed with respect to their proliferation and cytotoxicity, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15). A time-resolved fluorometric assay (TDA-labeled K562 target cells) was used for measuring the cytotoxic activity of NK-92 cells treated with IL-2, IL-4, IL-7 and/or IL-15. Their proliferation, in the presence of these common cytokine receptor γ chain (γc)-dependent cytokines, was measured by traditional tritiated thymidine ([3H]-TdR) incorporation. IL-2 and IL-15, but not IL-4 or IL-7, were able to induce a dose-dependent proliferation of NK-92 cells. IL-15 was, depending on the dose and culture time, up to 10 times more potent compared to corresponding concentrations of IL-2, whereas their combination could potentiate the NK-activity almost equally well. No synergistic effects could be noticed with respect to the cytotoxicity and the proliferation of these cells. Data presented here indicate that of the common gamma chain receptor-dependent cytokines tested here, IL-15 alone is able to cultivate and trigger NK-92 cells to such an extent so that they can be used for immune-based cancer therapies. Implications with respect to CAR-transduced NK-92 cells are also discussed.

The BCL-2-Selective Inhibitor Venetoclax Spares Activated T-Cells during Anti-Tumor Immunity

Introduction and objectives:During an adaptive immune response antigen-specific T cells rapidly proliferate and differentiate into cytotoxic T lymphocytes. Most of these cells undergo apoptosis but some develop into high-affinity memory CD8+ T cells. The BCL-2 family of proteins regulates apoptosis and has a critical role in development and maintenance of the immune system. Venetoclax (Venclexta™, ABT-199) is a selective BCL-2 inhibitor that increases tumor cell apoptosis, and is approved by the FDA for patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL), with or without 17p deletion, who have received at least one prior therapy. Given the critical role of BCL-2 in the regulation of the immune system, we hypothesized that venetoclax may affect the anti-tumor activity of immune checkpoint inhibitors.Results:To interrogate the effects of venetoclax on T cells we initially performed a series of in vitro studies using human lymphocytes treated with clinically relevant doses of the drug. As previously reported (Khaw et al., Leukemia; 28(6):1207-1215, 2014), human peripheral blood mononuclear cells (PBMCs) treated with venetoclax exhibited a dose-dependent decrease in the number of B-cells and T-cells (CD4+ and CD8+ T-cells). Upon further characterization of the surviving T cells, we found that while the proportion of naïve T-cells decreased with increasing venetoclax concentrations, the proportion of memory T-cells increased, specifically CD8+ and CD4+ T effector memory cells (Figure 1).We next examined the effects of venetoclax on T-cell function in vitro in response to immune stimulation with or without immune checkpoint blockade. To address this we performed a mixed lymphocyte reaction (MLR) assay, in which primary monocyte-derived dendritic cells from one donor were cultured with CD4+ T-cells from another donor. In the MLR reaction we observed that venetoclax reduced CD4+ T-cell viability in a dose-dependent manner, but it did not limit T-cell proliferation of surviving cells. Venetoclax did not affect IFNγ secretion within these surviving cells and, more importantly, did not reduce the effects of the checkpoint inhibitor nivolumab (Figure 2).To test the effects of venetoclax on antigen-specific T cells, we performed a cytomegalovirus (CMV) recall assay where PBMCs from CMV-positive human subjects were incubated with CMV antigen and the activity of T cells was measured by IFNg secretion. Although venetoclax treatment reduced the total number of cells, IFNg production from antigen-specific CMV+ T cells remained comparable to DMSO control and combining venetoclax with nivolumab did not affect the anti-PD-1 response (Figure 3).Finally, to investigate the effects of venetoclax in combination with anti-PD-1 therapy in vivo we used the murine syngeneic tumor model MC38. Venetoclax did not impair the efficacy of anti-PD-1, and in some studies increased efficacy relative to either anti-PD-1 or venetoclax monotherapy alone. To determine whether the efficacy of the venetoclax-anti-PD-1 combination is immune-mediated, we transplanted immunodeficient mice with MC38 cells and repeated the same treatment regimens. The lack of efficacy in any of the treatment arms indicates that the contribution of venetoclax to efficacy in this solid tumor model is immune-mediated (Figure 4).Conclusions:These data suggest that venetoclax treatment results in loss of naïve but not memory T cells. Venetoclax did not affect the viability, the induction or frequency of memory T cells. In human in vitro experiments and in an in vivo syngeneic tumor model venetoclax did not antagonize the therapeutic effect of anti-PD-1. Contrary to our initial hypothesis, we find that modulation of the immune system by venetoclax may support its potential use for immune-based cancer therapy, as memory T-cells can rapidly acquire effector and cytotoxic function to eliminate cancer cells. Taken together, we provide evidence that venetoclax in combination with immune checkpoint inhibitors should be further explored as a therapy for cancer patients.All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie and Genentech participated in the interpretation of data, review, and approval of the publication. [Display omitted] DisclosuresMathew:AbbVie Inc.: Employment. Haribhai:AbbVie Inc.: Employment. Kohlhapp:AbbVie Inc.: Employment. Duggan:AbbVie Inc.: Employment. Ellis:AbbVie Inc.: Employment. Riehm:AbbVie Inc.: Employment. Robinson:AbbVie Inc.: Employment. Shi:AbbVie Inc.: Employment. Bhathena:AbbVie Inc.: Employment. Leverson:AbbVie Inc: Employment, Equity Ownership, Patents & Royalties. Pappano:AbbVie Inc.: Employment. Donawho:AbbVie Inc.: Employment. Uziel:AbbVie Inc.: Employment.

The role of proteomics in the age of immunotherapies.

The antigenic landscape of the adaptive immune response is determined by the peptides presented by immune cells. In recent years, a number of immune-based cancer therapies have been shown to induce remarkable clinical responses through the activation of the patient's immune system. As a result, there is a need to identify immune biomarkers capable of predicting clinical response. Recent advances in proteomics have led to considerable developments in the more comprehensive profiling of the immune response. "Immunoproteomics" utilises a rapidly increasing collection of technologies in order to identify and quantify antigenic peptides or proteins. This includes gel-based, array-based, mass spectrometry (MS), DNA-based, or computer-based (in silico) approaches. Immunoproteomics is yielding an understanding of disease and disease progression, vaccine candidates, and biomarkers to a depth not before understood. This review gives an overview of the emerging role of proteomics in improving personalisation of immunotherapy treatment.

Abstract 712: Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models

Abstract The success of immune checkpoint therapy with anti-PD-1 and anti-CTLA-4 antibodies has generated broad interest in immuno-based interventions for oncology. Preclinical efficacy assessment of these novel immune-oncology therapies requires a functional immune system and therefore is frequently performed in murine syngeneic tumor models. In addition to tumor growth inhibition, syngeneic tumor models provide a platform to investigate the mechanism of action for a given novel therapeutic target. Characterization and mechanism of action for immune-based cancer therapies in murine syngeneic tumor models is often assessed by flow cytometry; measuring changes in tumor infiltrating leukocyte (TIL) populations following treatment induction. We assessed whether transcriptomic gene expression analysis could identify additional relevant biomarkers for immunotherapy response. Mice bearing subcutaneous syngeneic colon cancer tumors were dosed intraperitoneally with either vehicle alone, anti-CTLA-4, anti-PD-1, or a combination of the two immune checkpoint inhibitors on days 1, 4, and 8. Tumors were harvested on day 9 and half from each group were assessed for TIL population composition by flow cytometry and half by Clariom D mouse transcriptome array analysis. We identified a number of immune cell-related genes with differential expression that correlated to the therapeutic response and were consistent with changes identified in TIL populations by flow cytometry. These results provide an additional metric for assessing immunotherapy response in vivo. In addition, these data suggest that a smaller, defined panel of gene targets may be developed as biomarkers for specific immune system responses to immune checkpoint therapies in murine syngeneic models. Citation Format: Patrick Fadden, Douglas Weitzel, Kelli Davis, Thi Bui, David Hurtado, Hannah Haines, Rhiannon Roark, Ashleigh Derrick, Beverly Godfrey, Ian Belle, Katherine Krontz, Cristina Sokolowski, Aidan Synnott, Edgar Wood. Immune checkpoint inhibitor induced tumor gene expression changes in murine syngeneic colon cancer models [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 712.

IMMUNOSARC: A collaborative Spanish (GEIS) and Italian (ISG) Sarcoma Groups phase I/II trial of sunitinib plus nivolumab in selected bone and soft tissue sarcoma subtypes—Results of the phase I part.

11515Background: Disruption of angiogenesis substantially enhances the efficacy of immune-based cancer therapies. The combination of an antiangiogenic drug (sunitinib or pazopanib) plus anti- PD1 (...

Pushing the Limits of Cancer Therapy: The Nutrient Game.

The standard cancer treatments include chemotherapy, radiotherapy, or their combination, which are generally associated with a multitude of side effects ranging from discomfort to the development of secondary tumors and severe toxicity to multiple systems including immune system. Mounting evidence has highlighted that the fine-tuning of nutrients may selectively sensitize cancer cells to conventional cancer therapies, while simultaneously protecting normal cells from their side effects. Nutrient modulation through diet also improves cancer immunesurveillance in a way that severe immunosuppression could be avoided or even the immune response or immune-based cancer therapies be potentiated also through patient microbiota remodeling. Here, we review recent advances in cancer therapy focusing on the effects of adjuvant dietary interventions (e.g., ketogenic diets, fasting) on the metabolic pathways within cancer cells and tumor environment (e.g., microbiota, immune system, tumor microenvironment) that are involved in cancer progression and resistance as well as cancer cell death. Finally, based on the overall literature data, we designed a nutritional intervention consisting in a plant-based moderate ketogenic diet that could be exploited for future preclinical research in cancer therapy.

Immune-based therapies for metastatic prostate cancer: an update.

Prostate cancer (PC) is a common malignancy among elderly males and is noncurable once it becomes metastatic. In recent years, a number of antigen-delivery systems have emerged as viable and promising immunotherapeutic agents against PC. The approval of sipuleucel-T by the US FDA for the treatment of males with asymptomatic or minimally symptomatic castrate resistant PC was a landmark in cancer immunotherapy, making this the first approved immunotherapeutic. A number of vaccines are under clinical investigation, each having its own set of advantages and disadvantages. Here, we discuss the basic technologies underlying these different delivery modes, we discuss the completed and current human clinical trials, as well as the use of vaccines in combination with immune checkpoint inhibitors.

Evolving notions on immune response in colorectal cancer and their implications for biomarker development.

Colorectal cancer (CRC) still represents the third most commonly diagnosed type of cancer in men and women worldwide. CRC is acknowledged as a heterogeneous disease that develops through a multi-step sequence of events driven by clonal selections; this observation is sustained by the fact that histologically similar tumors may have completely different outcomes, including a varied response to therapy. In "early" and "intermediate" stage of CRC (stages II and III, respectively) there is a compelling need for new biomarkers fit to assess the metastatic potential of their disease, selecting patients with aggressive disease that might benefit from adjuvant and targeted therapies. Therefore, we review the actual notions on immune response in colorectal cancer and their implications for biomarker development. The recognition of the key role of immune cells in human cancer progression has recently drawn attention on the tumor immune microenvironment, as a source of new indicators of tumor outcome and response to therapy. Thus, beside consolidated histopathological biomarkers, immune endpoints are now emerging as potential biomarkers. The introduction of immune signatures and cellular and molecular components of the immune system as biomarkers is particularly important considering the increasing use of immune-based cancer therapies as therapeutic strategies for cancer patients.

Using a Machine-Learning Approach to Predict Discontinuous Antibody-Specific B-Cell Epitopes

Background: Predicting B-cell epitopes is important for understanding disease pathogenesis, identifying potential autoantigens, and designing vaccines and immune-based cancer therapies. The experimental approaches used for detecting B-cell epitopes are often laborious and resource-intensive. Thus, several computational methods have been developed for predicting the epitopes of a given antigen. However, most of these methods are coarse binary classifications of antigen regions within epitopes or non-epitopes and do not specify antibodies. Therefore, we aim to solve this antibodyspecified epitope prediction problem using a developed structure-based computational machine-learning method, Epitopia, to reflect this biological reality accurately. Result: We selected 60 non-redundant antibody-antigen protein complexes to train and applied the leave-one-out cross-validation method to test the accuracy of our proposed methods; we compared the results with the Epitopia, Discotope 2.0,PEASE and the state-of-the-art tool SEPPA 2.0. We considered the role of both complementarity determining region residues and antigen surface residues in antigenantibody interactions and assigned a score for each antigen surface residue. If we considered a prediction to be successful if the average score of “epitope residues” exceeded the average score over all surface residues. Then the success rates of our proposed methods were all higher than Epitopia and the best one was 83.3%, which is 7% higher than the rate obtained with Epitopia (76%). The results show that antibody-specific methods are competitive with, and sometimes even better than, Epitopia, which only considers the antigen residues. Conclusion: Our antibody-specific methods provide sufficient accuracy in locating epitopes because whether an antigen surface residue is an epitope depends on whether the antibody recognizes it. This approach is a new method for identifying B-cell epitopes. Based on our results, we believe that the prediction of antibody-specific B cell epitopes is more meaningful and efficient than those which are only considering antigen residues. Keywords: Antibody-specific B-cell epitope, paratope-epitope residue pair, machine-learning, bioinformatics, antigen, epitopia.

Abstract 2935: Sex disparity in the response of the immune microenvironment to colon cancer liver metastases

Abstract Background Metastasis remains the most life-threatening aspect of malignant disease and a major clinical challenge. Myeloid-derived suppressor cells (MDSC) and regulatory T lymphocytes (Tregs), which are frequently found in the tumor microenvironment, are known to impair immune surveillance and promote a state of immune tolerance that leads to metastatic expansion. Previously, we have shown that the incidence of colon carcinoma liver metastases was reduced in TNF receptor 2 (TNFR2) deficient female mice, as a consequence of reduced recruitment of MDSCs and Tregs into the liver. Intriguingly however, no reduction in liver metastasis or MDSC/Treg accumulation was observed in TNFR2 deficient male mice, suggesting that their recruitment and survival were distinctly regulated. The objective of this study was, therefore, to elucidate the mechanisms underlying the sex disparity observed in the response of the immune microenvironment to liver metastases, in particular the role of sex hormones in immune response regulation in this context. Methods Hormone-ablated female mice were generated using surgical ovariectomy (OVX). Estrogen-deprived mice, sham-operated controls and hormone reconstituted OVX mice were injected with syngeneic murine colon carcinoma MC-38 cells via the intrasplenic/portal route and experimental liver metastases enumerated 2 weeks later. MDSCs and Tregs recruited into the livers of tumor-bearing mice were analyzed 7-10 days post-tumor injection using flow cytometry, as well as IHC performed on liver cryostat sections. Results We found that the number of experimental liver metastases was markedly reduced in ovariectomized female mice, as compared to sham-operated controls but was restored in estradiol supplemented OVX mice. Reduced incidence of hepatic metastases in OVX mice correlated with reduced accumulations of CD11b+Gr-1+ myeloid cells (MDSCs) and CD4+Foxp3+ cells (Tregs) at metastatic sites. IHC also revealed an increased CD8+ : CD4+Foxp3+ (i.e. cytotoxic T cell : Treg) ratio around hepatic metastases in OVX mice as compared to sham-operated controls. Estradiol reconstitution in OVX mice restored MDSC and Treg cell accumulation to the levels observed in control mice. Conclusion Together, our data identify female sex hormones as major players in the regulation of the tumor immune microenvironment and a potential contributing factor in the patients’ response to immune-based cancer therapy. Supported by the Canadian Institute for Health Research, MDEIE and FRSQ. Citation Format: Simon Milette, Ni Wang, Stéphanie Perrino, Pnina Brodt. Sex disparity in the response of the immune microenvironment to colon cancer liver metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2935. doi:10.1158/1538-7445.AM2017-2935

Immunotherapy of cancer.

Background For 50 years, cancer physicians have relied on just three primary treatment modalities: surgery, radiation therapy and chemotherapy. Over that time, enormous progress has been made in understanding cancer biology, targeted anti-cancer drugs have emerged, and thousands of clinical trials have taught us how best to craft treatment combinations that improve clinical outcomes. Only five years ago, a fourth and radically different form of therapy finally emerged: immune‑based cancer therapies. Objective This review briefly outlines the history and theoretical framework underpinning cancer immunotherapy, and recent progress on several immunotherapeutic approaches. Discussion Immune-based cancer therapies are already revolutionising the management of several types of hitherto intractable cancer, while offering immense hope that the burden of personal suffering and community cost due to cancer will diminish appreciably over the coming decades. At least two immunotherapeutic approaches, checkpoint inhibition and cellular therapy with autologous ('self') chimeric antigen receptor T cells (CAR T cells), now show indisputable evidence of efficacy in several cancer types, and promise yet more rapid progress as they are refined and we learn to combine them with existing conventional therapies and each other.

Immune-based Therapies for Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide. Treatment of non-small cell lung cancer has evolved tremendously over the past decade. Specifically, immune checkpoint inhibitors have become an increasingly interesting target of pharmacological blockade. These immune inhibitors have shown promising results in front-line therapy and after failure of multiple lines, as well as in monotherapy and combination with other therapies. Vaccination in non-small cell lung cancer is also an emerging field of research that holds promising results for the future of immunotherapy in non-small cell lung cancer. This review presents a concise update on the most recent data regarding the role of checkpoint inhibitors as well as vaccination in non-small cell lung cancer.

The functional interplay between systemic cancer and the hematopoietic stem cell niche.

Hematopoietic cells are increasingly recognized as playing key roles in tumor growth and metastatic progression. Although many studies have focused on the functional interaction of hematopoietic cells with tumor cells, few have examined the regulation of hematopoiesis by the hematopoietic stem cell (HSC) niche in the setting of cancer. Hematopoiesis occurs primarily in the bone marrow, and processes including expansion, mobilization, and differentiation of hematopoietic progenitors are tightly regulated by the specialized stem cell niche. Loss of niche components or the ability of stem cells to localize to the stem cell niche relieves HSCs of the restrictions imposed under normal homeostasis. In this review, we discuss how tumor-derived factors and therapeutic interventions disrupt structural and regulatory properties of the stem cell niche, resulting in niche invasion by hematopoietic malignancies, extramedullary hematopoiesis, myeloid skewing by peripheral tissue microenvironments, and lymphopenia. The key regulatory roles played by the bone marrow niche in hematopoiesis has implications for therapy-related toxicity and the successful development of immune-based therapies for cancer.

Abstract 4010: Identification of novel targeted and immunotherapy combinations by a high throughput assay of T cell-mediated cytotoxicity

Abstract Melanoma is the major cause of skin cancer-related deaths. However, the recent emergence of immune checkpoint inhibitors such as Ipilimumab (anti-CTLA-4) and Nivolumab (anti-PD-1), and adoptive cell therapy, using tumor infiltrating lymphocytes (TILs), has improved clinical outcomes and produced response rates of approximately 50%. Despite the progress made in melanoma immunotherapy, there is a large cohort of patients for which these treatments are currently not applicable. They are either inherently resistant to or acquire resistance to immunotherapy. As such, understanding mechanisms of resistance to immune response and ascertaining efficacious therapy combinations is important in overcoming immunoresistance and developing novel treatment regimens. As proof of principle, preliminary work using a unique protocol for an in vitro T-cell mediated cytotoxicity screen was established. In this assay, intracellular staining of active caspase-3 in tumor cells, a marker of apoptosis, is measured by flow cytometry in a 96-well format. Using patient-derived tumor and TIL pairs, a screen of 850 compounds was performed to find candidate drugs that could modulate the sensitivity of tumor cells to T- cell mediated killing. Initial screens identified classes of compounds that inhibit Heat shock protein 90 (Hsp90), Aurora Kinase and Topoisomerase I, and studies to understand their mechanisms of action are currently underway. However, as this screening process is very time-intensive, we are currently increasing the efficiency and feasibility of this screen by miniaturizing this workflow. This work presents the development of a screening method that is performed in a 384-well format, with a greater drug concentration range, and utilizing robotics to streamline and increase output. With this, we can specifically identify novel targeted and immunotherapy combinations that may be used in the clinic to treat melanoma. Additionally, by understanding the signaling pathways and molecular factors that regulate tumor response to T cell mediated killing, we can translate these findings into making immune-based cancer therapies applicable to other tumor types. This project aims to discover novel compounds that work synergistically with T-cell mediated tumor cell cytotoxicity, understand their mechanism of action and how they contribute to resistance and as a result identify innovative immune-based therapies that can be translated into clinical use. Citation Format: Leila Williams, Shruti Malu, Jodi McKenzie, Rina Mbofung, Marie-Andree Forget, Chantale Bernatchez, Patrick Hwu. Identification of novel targeted and immunotherapy combinations by a high throughput assay of T cell-mediated cytotoxicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4010.

Recent advances in immuno-oncology and its application to urological cancers

Recent advances in immuno-oncology have the potential to transform the practice of medical oncology. Antibodies directed against negative regulators of T-cell function (checkpoint inhibitors), engineered cell therapies and innate immune stimulators, such as oncolytic viruses, are effective in a wide range of cancers. Immune'based therapies have had a clinically meaningful impact on the treatment of advanced melanoma, and the lessons regarding use of single agents and combinations in melanoma may be applicable to the treatment of urological cancers. Checkpoint inhibitors, cytokine therapy and therapeutic vaccines are already showing promise in urothelial bladder cancer, renal cell carcinoma and prostate cancer. Critical areas of future immuno-oncology research include the prospective identification of patients who will respond to current immune-based cancer therapies and the identification of new therapeutic agents that promote immune priming in tumours, and increase the rate of durable clinical responses.

Myeloid-Derived Suppressor Cells Express Bruton's Tyrosine Kinase and Can Be Depleted in Tumor-Bearing Hosts by Ibrutinib Treatment.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of immature myeloid cells that expand in tumor-bearing hosts in response to soluble factors produced by tumor and stromal cells. MDSC expansion has been linked to loss of immune effector cell function and reduced efficacy of immune-based cancer therapies, highlighting the MDSC population as an attractive therapeutic target. Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and IL2-inducible T-cell kinase (ITK), is in clinical use for the treatment of B-cell malignancies. Here, we report that BTK is expressed by murine and human MDSCs, and that ibrutinib is able to inhibit BTK phosphorylation in these cells. Treatment of MDSCs with ibrutinib significantly impaired nitric oxide production and cell migration. In addition, ibrutinib inhibited in vitro generation of human MDSCs and reduced mRNA expression of indolamine 2,3-dioxygenase, an immunosuppressive factor. Treatment of mice bearing EMT6 mammary tumors with ibrutinib resulted in reduced frequency of MDSCs in both the spleen and tumor. Ibrutinib treatment also resulted in a significant reduction of MDSCs in wild-type mice bearing B16F10 melanoma tumors, but not in X-linked immunodeficiency mice (XID) harboring a BTK mutation, suggesting that BTK inhibition plays an important role in the observed reduction of MDSCs in vivo Finally, ibrutinib significantly enhanced the efficacy of anti-PD-L1 (CD274) therapy in a murine breast cancer model. Together, these results demonstrate that ibrutinib modulates MDSC function and generation, revealing a potential strategy for enhancing immune-based therapies in solid malignancies. Cancer Res; 76(8); 2125-36. ©2016 AACR.

Targeting the programmed cell death-1 pathway in breast and ovarian cancer.

Immune checkpoint blockade is changing cancer therapy. Targeting the programmed cell death-1 (PD-1) pathway releases T cells from inhibitory signals within the tumor microenvironment, thereby activating a latent antitumor immune response. Here, we review the biology underlying the activity of PD-1/programmed cell death-ligand 1 (PD-L1) antagonists, and data describing their clinical activity in breast and ovarian cancer. Several antagonists of PD-1 and PD-L1 have been tested in breast and ovarian cancer. These drugs are generally well tolerated, with some immune-related adverse events that are typically easily managed. Objective response rates generally range from about 10 to 20% in both breast cancer and ovarian cancer, with durable responses noted in multiple trials. Selecting patients with PD-L1 expression by cells within the tumor microenvironment appears to enrich for responses. These agents are under accelerated development based on these promising early data. Monoclonal antibody-based blockade of the PD-1 pathway results in objective and durable clinical responses in a subset of patients with breast or ovarian cancers, particularly those with PD-L1-positive cells within the tumor microenvironment. Current priorities are to refine biomarkers of therapeutic response, and to develop combination immunotherapy strategies that integrate PD-1/PD-L1 antagonists with both standard and immune-based cancer therapies to increase efficacy.