Andrew J.S. Furness

Anti-programmed cell death-1 therapy and insulin-dependent diabetes: a case report

Abstract The anti programmed cell death-1 (PD-1) antibodies pembrolizumab and nivolumab have been recently licensed by the Food and Drug Administration for the treatment of advanced melanoma. Immune checkpoint inhibitors such as these can induce endocrine adverse events but autoimmune diabetes has not been described to date. However, there is a strong preclinical rationale that supports this autoimmune toxicity. We describe for the first time the case of an adult patient who developed autoimmune diabetes likely as a consequence of PD-1 inhibition with pembrolizumab. The presence of high serum titres of anti-glutamic acid decarboxylase antibodies together with a suggestive clinical presentation, age of the patient and preclinical data strongly support an autoimmune aetiology of the diabetes. Moreover, the patient was found to have a well-known high-risk human leucocyte antigen type for the development of type 1 diabetes in children, so the PD-1 inhibition is very likely to have triggered the autoimmune phenomenon. Our case suggests that insulin-dependent diabetes might be a rare but important anti-PD-1 immune-related adverse event.

Impact of tumour microenvironment and Fc receptors on the activity of immunomodulatory antibodies.

Immunomodulatory antibodies influence the direction and magnitude of immune responses against cancer. Significant efficacy has been demonstrated across multiple solid tumour types within clinical trials. Recent preclinical studies indicate that successful outcome relies upon mechanistic activity extending beyond simple receptor stimulation or blockade. In addition to blocking co-inhibitory signals in secondary lymphoid organs, cytotoxic T-lymphocyte antigen (CTLA)-4 antibodies mediate depletion of tumour-infiltrating regulatory T cells by antibody-dependent cellular cytotoxicity (ADCC). This mechanism appears to be common to other immunomodulatory antibodies including those targeting OX40 and glucocorticoid-induced TNFR-related protein (GITR). If verified in the human setting, these findings have significant implications for antibody design, biomarker discovery, and the development of synergistic combinatorial therapies.

Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade

Intravenous infusion of oncolytic reovirus in patients leads to infection of brain tumors, infiltration by cytotoxic T cells, and up-regulation of PD-L1. Viruses team up with cancer immunotherapy Immune checkpoint inhibitors have shown great promise for cancer therapy, but they do not treat all cancers, and neither breast nor brain tumors are usually treatable with these drugs. However, Bourgeois-Daigneault et al. discovered a way to address this for breast cancer, and Samson et al. discovered a way to address this for brain tumors. In both cases, the authors found that oncolytic virus treatment given early, before surgical resection, alters the antitumor immune response and potentiates the effects of subsequent treatment with immune checkpoint inhibitors. Although these studies differ in the details of their methods and the immune effects induced by the oncolytic viruses, they indicate the potential of such viruses for enhancing the potential of checkpoint therapy and expanding it to new types of cancer. Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are reshaping cancer therapeutic strategies. Evidence suggests, however, that tumor response and patient survival are determined by tumor programmed death ligand 1 (PD-L1) expression. We hypothesized that preconditioning of the tumor immune microenvironment using targeted, virus-mediated interferon (IFN) stimulation would up-regulate tumor PD-L1 protein expression and increase cytotoxic T cell infiltration, improving the efficacy of subsequent checkpoint blockade. Oncolytic viruses (OVs) represent a promising form of cancer immunotherapy. For brain tumors, almost all studies to date have used direct intralesional injection of OV, because of the largely untested belief that intravenous administration will not deliver virus to this site. We show, in a window-of-opportunity clinical study, that intravenous infusion of oncolytic human Orthoreovirus (referred to herein as reovirus) leads to infection of tumor cells subsequently resected as part of standard clinical care, both in high-grade glioma and in brain metastases, and increases cytotoxic T cell tumor infiltration relative to patients not treated with virus. We further show that reovirus up-regulates IFN-regulated gene expression, as well as the PD-1/PD-L1 axis in tumors, via an IFN-mediated mechanism. Finally, we show that addition of PD-1 blockade to reovirus enhances systemic therapy in a preclinical glioma model. These results support the development of combined systemic immunovirotherapy strategies for the treatment of both primary and secondary tumors in the brain.

Intratumoural evolutionary landscape of high-risk prostate cancer: The PROGENY study of genomic and immune parameters

Abstract Background Intratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies. Patients and methods Forty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification. Results We demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of β-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of β-catenin. Analysis of all patients with activating Wnt/β-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion. Conclusions The PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/β-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies. Clinical Trials.gov Identifier NCT02022371

Neoantigen heterogeneity: a key driver of immune response and sensitivity to immune checkpoint blockade?

Modulation of co-inhibitory and co-stimulatory immune checkpoint pathway activity with antibody-based therapies has emerged as a promising anticancer strategy. Although responses to such agents are limited to a modest fraction of treated patients, those deriving benefit have the potential for durable remissions and possibly even cure [1-8]. The identification of biomarkers predictive of response and resistance to such therapies therefore remains an area of high scientific priority.

Pan-cancer deconvolution of cellular composition identifies molecular correlates of antitumour immunity and checkpoint blockade response

The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a novel DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: ‘immune hot’ and ‘immune cold’, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity, and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to hot tumours, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations and define a catalogue of novel and known mediators of active antitumour immunity, deriving biomarkers and potential targets for precision immunotherapy.

Pan-cancer deconvolution of tumour composition using DNA methylation

The nature and extent of immune cell infiltration into solid tumours are key determinants of therapeutic response. Here, using a DNA methylation-based approach to tumour cell fraction deconvolution, we report the integrated analysis of tumour composition and genomics across a wide spectrum of solid cancers. Initially studying head and neck squamous cell carcinoma, we identify two distinct tumour subgroups: ‘immune hot’ and ‘immune cold’, which display differing prognosis, mutation burden, cytokine signalling, cytolytic activity and oncogenic driver events. We demonstrate the existence of such tumour subgroups pan-cancer, link clonal-neoantigen burden to cytotoxic T-lymphocyte infiltration, and show that transcriptional signatures of hot tumours are selectively engaged in immunotherapy responders. We also find that treatment-naive hot tumours are markedly enriched for known immune-resistance genomic alterations, potentially explaining the heterogeneity of immunotherapy response and prognosis seen within this group. Finally, we define a catalogue of mediators of active antitumour immunity, deriving candidate biomarkers and potential targets for precision immunotherapy.Determining the extent of immune cell infiltration into solid tumours is essential for adequate therapeutic response. Here the authors develop a DNA methylation-based approach for tumour cell fraction deconvolution and analyse tumour composition and genomics across a wide spectrum of solid cancers.

Urine-derived lymphocytes as a non-invasive measure of the bladder tumor immune microenvironment

Despite the advances in cancer immunotherapy, only a fraction of patients with bladder cancer exhibit responses to checkpoint blockade, highlighting a need to better understand drug resistance and identify rational immunotherapy combinations. However, accessibility to the tumor prior and during therapy is a major limitation in understanding the immune tumor microenvironment (TME). Herein, we identified urine-derived lymphocytes (UDLs) as a readily accessible source of T cells in 32 patients with muscle invasive bladder cancer (MIBC). We observed that effector CD8+ and CD4+ cells and regulatory T cells within the urine accurately map the immune checkpoint landscape and T cell receptor repertoire of the TME. Finally, an increased UDL count, specifically high expression of PD-1 (PD-1hi) on CD8+ at the time of cystectomy, was associated with a shorter recurrence-free survival. UDL analysis represents a dynamic liquid biopsy that is representative of the bladder immune TME that may be used to identify actionable immuno-oncology (IO) targets with potential prognostic value in MIBC.

Abstract B179: Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers

Different components of the tumor microenvironment hamper the immune response against cancer cells and are a critical impediment for the success of cancer immunotherapy. Among these, the accumulation of regulatory T cells (Treg) and the reduction of the effector T cells (Teff)/Treg ratio favors tumor progression and is associated with worse prognosis in several human cancers. Treg elimination in the tumor microenvironment can be achieved through antibodies that target molecules expressed preferentially in Treg and that induce antibody-mediated cell cytotoxicity (ADCC). CTLA-4 is highly expressed on tumor-infiltrating Treg and therapy with anti-CTLA-4 has been effective for cancer treatment in murine models and in humans. In mice, we and others have shown that anti-CTLA-4 depletes Treg in the tumor but not in peripheral lymphoid organs. This site specificity in mice is given by the expression of the Fcγ-receptor IV (FcγRIV) in myeloid cells present in the tumor microenvironment but not in the periphery. However, the role of FcγRs in the effectiveness of anti-CTLA-4 therapy or therapies with other immunomodulatory antibodies in humans is not known. We therefore investigated the density of targets of clinically relevant immunomodulatory antibodies in different subpopulations of tumor-infiltrating immune cells as well as the expression of FcγR subtypes in murine tumor models. Because of the inter-species differences in the repertoire and the cellular distribution of FcγRs, we also studied this in humanized-FcγR (huFcγR) mice, in which there is no expression of murine FcγRs but of their human homologues. We compared this with samples of human melanoma and observed surprisingly similar expression profiles of both FcγRs and targets of immunomodulatory antibodies, including CTLA-4. We then asked whether anti-CTLA-4 would also lead to selective Treg depletion in the tumor microenvironment in huFcγR mice. Using an antibody targeting mCTLA-4 with a human IgG1 isotype, we observed that was the case. Furthermore, mutation of the Fc region of the antibody to enhance its affinity to activating huFcγRs resulted in more effective selective Treg depletion in the tumor. These findings are not only relevant for CTLA-4 but also for other targets expressed preferentially on Treg instead of Teff and must be taken into account when designing immunomodulatory antibodies for clinical use. Citation Format: Frederick Arce Vargas, Andrew J. Furness, Marta H. Lesko, Martin Pule, Jeffrey V. Ravetch, Karl S. Peggs, Sergio A. Quezada. Enhancing intratumoural Treg depletion through antibody engineering and analysis of the checkpoint landscape of mouse and human cancers. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B179.