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Background Survival rates for ovarian cancer remain poor, and monitoring and prediction of therapeutic response may benefit from additional markers. Ovarian cancers frequently overexpress Folate Receptor alpha (FRα) and the soluble receptor (sFRα) is measurable in blood. Here we investigated sFRα as a potential biomarker. Methods We evaluated sFRα longitudinally, before and during neo-adjuvant, adjuvant and palliative therapies, and tumour FRα expression status by immunohistrochemistry. The impact of free FRα on the efficacy of anti-FRα treatments was evaluated by an antibody-dependent cellular cytotoxicity assay. Results Membrane and/or cytoplasmic FRα staining were observed in 52.7% tumours from 316 ovarian cancer patients with diverse histotypes. Circulating sFRα levels were significantly higher in patients, compared to healthy volunteers, specifically in patients sampled prior to neoadjuvant and palliative treatments. sFRα was associated with FRα cell membrane expression in the tumour. sFRα levels decreased alongside concurrent tumour burden in patients receiving standard therapies. High concentrations of sFRα partly reduced anti-FRα antibody tumour cell killing, an effect overcome by increased antibody doses. Conclusions sFRα may present a non-invasive marker for tumour FRα expression, with the potential for monitoring patient response to treatment. Larger, prospective studies should evaluate FRα for assessing disease burden and response to systemic treatments.

Evaluation of FRalpha expression by tumor cell lines

Table of Patient Characteristics

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.

Description of additional experiments and procedures carried out in this study

IgE contributes to host-protective functions in parasitic and bacterial infections, often by monocyte and macrophage recruitment. We previously reported that monocytes contribute to tumour antigen-specific IgE-mediated tumour growth restriction in rodent models. Here, we investigate the impact of IgE stimulation on monocyte response, cellular signalling, secretory and tumour killing functions. IgE cross-linking on human monocytes with polyclonal antibodies to mimic formation of immune complexes induced upregulation of co-stimulatory (CD40, CD80, CD86), and reduced expression of regulatory (CD163, CD206, MerTK) monocyte markers. Cross-linking and tumour antigen-specific IgE antibody-dependent cellular cytotoxicity (ADCC) of cancer cells by cancer patient-derived monocytes triggered release of pro-inflammatory mediators (TNF&alpha, MCP-1, IL-10, CXCL-10, IL-1&beta, IL-6, IL-23). High intratumoural gene expression of these mediators was associated with favourable five-year overall survival in ovarian cancer. IgE cross-linking of trimeric Fc&epsilon, RI on monocytes stimulated the phosphorylation of intracellular protein kinases widely reported to be downstream of mast cell and basophil tetrameric Fc&epsilon, RI signalling. These included recently-identified Fc&epsilon, RI pathway kinases Fgr, STAT5, Yes and Lck, which we now associate with monocytes. Overall, anti-tumour IgE can potentiate pro-inflammatory signals, and prime tumour cell killing by human monocytes. These findings will inform the development of IgE monoclonal antibody therapies for cancer.

Cytotoxic T-lymphocyte associated protein-4 (CTLA-4) and the Programmed Death Receptor 1 (PD-1) are immune checkpoint molecules that are well-established targets of antibody immunotherapies for the management of malignant melanoma. The monoclonal antibodies, Ipilimumab, Pembrolizumab, and Nivolumab, designed to interfere with T cell inhibitory signals to activate immune responses against tumors, were originally approved as monotherapy. Treatment with a combination of immune checkpoint inhibitors may improve outcomes compared to monotherapy in certain patient groups and these clinical benefits may be derived from unique immune mechanisms of action. However, treatment with checkpoint inhibitor combinations also present significant clinical challenges and increased rates of immune-related adverse events. In this review, we discuss the potential mechanisms attributed to single and combined checkpoint inhibitor immunotherapies and clinical experience with their use.

The immune system employs several checkpoint pathways to regulate responses, maintain homeostasis and prevent self-reactivity and autoimmunity. Tumor cells can hijack these protective mechanisms to enable immune escape, cancer survival and proliferation. Blocking antibodies, designed to interfere with checkpoint molecules CTLA-4 and PD-1/PD-L1 and counteract these immune suppressive mechanisms, have shown significant success in promoting immune responses against cancer and can result in tumor regression in many patients. While inhibitors to CTLA-4 and the PD-1/PD-L1 axis are well-established for the clinical management of melanoma, many patients do not respond or develop resistance to these interventions. Concerted efforts have focused on combinations of approved therapies aiming to further augment positive outcomes and survival. While CTLA-4 and PD-1 are the most-extensively researched targets, results from pre-clinical studies and clinical trials indicate that novel agents, specific for checkpoints such as A2AR, LAG-3, IDO and others, may further contribute to the improvement of patient outcomes, most likely in combinations with anti-CTLA-4 or anti-PD-1 blockade. This review discusses the rationale for, and results to date of, the development of inhibitory immune checkpoint blockade combination therapies in melanoma. The clinical potential of new pipeline therapeutics, and possible future therapy design and directions that hold promise to significantly improve clinical prognosis compared with monotherapy, are discussed.

Immunoglobulin E (IgE) antibodies are well known for their role in allergic diseases and for contributions to antiparasitic immune responses. Properties of this antibody class that mediate powerful effector functions may be redirected for the treatment of solid tumours. This has led to the rise of a new class of therapeutic antibodies to complement the armamentarium of approved tumour targeting antibodies, which to date are all IgG class. The perceived risk of type I hypersensitivity reactions following administration of IgE has necessitated particular consideration in the development of these therapeutic agents. Here, we bring together the properties of IgE antibodies pivotal to the hypothesis for superior antitumour activity compared to IgG, observations of in vitro and in vivo efficacy and mechanisms of action, and a focus on the safety considerations for this novel class of therapeutic agent. These include in vitro studies of potential hypersensitivity, selection of and observations from appropriate in vivo animal models and possible implications of the high degree of glycosylation of IgE. We also discuss the use of ex vivo predictive and monitoring clinical tools, as well as the risk mitigation steps employed in, and the preliminary outcomes from, the first-in-human clinical trial of a candidate anticancer IgE therapeutic.

Background: All antibodies approved for the treatment of cancer are monoclonal IgGs, and no IgE therapy has yet been tested in humans. The biology of IgE, compared with IgG, offers potential for enhanced immune surveillance and superior effector cell potency against tumor cells. IgE antibodies in preclinical cancer models are not associated with allergic toxicity even in immunocompetent animals, and in vivo efficacy compares favorably with equivalent IgGs. Methods: We conducted a first-in-human first-in-class trial of MOv18, a chimeric monoclonal IgE, in patients with solid tumors expressing folate receptor-alpha, the antigen recognized by this antibody. Antigen expression was deemed positive in the presence of >5% membrane staining of any intensity using the mouse clone BN3.2. Intravenous treatment was administered weekly for 6 weeks, then two-weekly. The trial incorporated pre-treatment skin prick testing with MOv18 IgE, and an ex vivo basophil activation test (BAT) using patient whole blood, with the aim of predicting systemic allergic toxicity and excluding patients at potential risk. Safety, efficacy, markers of immune response, and pharmacokinetics have been evaluated in 24 patients to date, at total doses ranging from 0.07 to 3.0mg. Results: Treatment was well tolerated in almost all patients. The most common toxicity was readily manageable urticaria, without systemic symptoms, signs or tryptase elevation. One patient treated at the 0.5mg dose experienced anaphylaxis, with tryptase elevation, despite a negative pre-dose skin prick test. This was the only patient in the trial with baseline circulating basophils that could be activated by ex vivo exposure to MOv18 IgE. This BAT assay was subsequently used to ensure no further patient with reactive basophils was exposed. Maximum tolerated dose has not yet been reached. Dose-dependent increases in Cmax were observed, and plasma concentrations of 70-100ng/mL achieved at the 1.5mg dose are comparable to typical levels of endogenous IgE. No consistent anti-drug antibody response has been detected. Preliminary evidence of anti-tumor activity was seen in a patient with ovarian cancer at a total MOv18 IgE dose of 0.7mg. Shrinkage of peritoneal metastases was accompanied by a tumor marker reduction meeting Gynecologic Cancer InterGroup criteria for response. Conclusions: These results support for the first time the safety of IgE as a treatment for cancer, and provide preliminary evidence for anti-tumor efficacy of this new therapeutic class. The mechanism of cutaneous toxicity is being investigated. Clinical testing of class-switched IgE versions of approved IgG-based therapeutic antibodies is warranted. Citation Format: James Spicer, Bristi Basu, Ana Montes, Udai Banerji, Rebecca Kristeleit, Gareth J. Veal, Christopher Corrigan, Stephen Till, George Nintos, Timothy Brier, Ionut G. Funingana, Joo Ern Ang, Kam Zaki, Annie Griffin, Claire Barton, Paul Jones, Sarah Mellor, Susan Brook, Katie Stoddart, Christopher Selkirk, Simon Carroll, Heike Lentfer, Natalie Woodman, Amy Pope, Giulia Pellizzari, Mano Nakamura, Kristina M. Ilieva, Atousa Khiabany, Chara Stavraka, Hannah Gould, Jitesh Chauhan, Heather Bax, Sarah Pinder, Debra Josephs, Sophia Karagiannis. Phase 1 trial of MOv18, a first-in-class IgE antibody therapy for cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT141.

Basophils are involved in manifestations of hypersensitivity, however, the current understanding of their propensity for activation and their prognostic value in cancer patients remains unclear. As in healthy and atopic individuals, basophil populations were identified in blood from ovarian cancer patients (n = 53) with diverse tumor histologies and treatment histories. Ex vivo basophil activation was measured by CD63 expression using the basophil activation test (BAT). Irrespective of prior treatment, basophils could be activated by stimulation with IgE- (anti-Fc&epsilon, RI and anti-IgE) and non-IgE (fMLP) mediated triggers. Basophil activation was detected by ex vivo exposure to paclitaxel, but not to other anti-cancer therapies, in agreement with a clinical history of systemic hypersensitivity reactions to paclitaxel. Protein and gene expression analyses support the presence of basophils (CCR3, CD123, Fc&epsilon, RI) and activated basophils (CD63, CD203c, tryptase) in ovarian tumors. Greater numbers of circulating basophils, cells with greater capacity for ex vivo stimulation (n = 35), and gene signatures indicating the presence of activated basophils in tumors (n = 439) were each associated with improved survival in ovarian cancer. Circulating basophils in cancer patients respond to IgE- and non-IgE-mediated signals and could help identify hypersensitivity to therapeutic agents. Activated circulating and tumor-infiltrating basophils may be potential biomarkers in oncology.

Identification of mutations in the gene encoding the serine/threonine-protein kinase, BRAF, and constitutive activation of the mitogen-activated protein kinase (MAPK) pathway in around 50% of malignant melanomas have led to the development and regulatory approval of targeted pathway inhibitor drugs. A proportion of patients are intrinsically resistant to BRAF inhibitors, and most patients who initially respond, acquire resistance within months. In this review, we discuss pathway inhibitors and their mechanisms of resistance, and we focus on numerous efforts to improve clinical benefits through combining agents with disparate modes of action, including combinations with checkpoint inhibitor antibodies. We discuss the merits of combination strategies based on enhancing immune responses or overcoming tumor-associated immune escape mechanisms. Emerging insights into mechanisms of action, resistance pathways and their impact on host-tumor relationships will inform the design of optimal combinations therapies to improve outcomes for patients who currently do not benefit from recent treatment breakthroughs.

IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcϵ receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.