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Harnessing Therapeutic IgE Antibodies to Re-educate Macrophages against Cancer
TRMOME 1552 No. of Pages 12
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Review
Harnessing Therapeutic IgE Antibodies to Re-educate Macrophages against Cancer Giulia Pellizzari,1 Heather J. Bax,1,2 Debra H. Josephs,1,2 Jelena Gotovina,3,4 Erika Jensen-Jarolim,3,4 James F. Spicer,2,* and Sophia N. Karagiannis1,* Currently, IgG is the only class of antibodies employed for cancer therapy. However, harnessing the unique biological properties of a different class ( e.g., IgE) could engender potent effector cell activation, and unleash previously untapped immune mechanisms against cancer. IgE antibodies are best known for pathogenic roles in allergic diseases and for protective effector functions against parasitic infestation, often mediated by IgE Fc receptor-expressing macrophages. Notably, IgE possess a very high affinity for cognate Fc receptors expressed by tumor-associated macrophages (TAMs). This paper reviews pre-clinical studies, which indicate control of cancer growth by tumor antigen-specific IgE that recruit and re-educate TAMs towards activated profiles. The clinical development harnessing the antitumor potential of recombinant IgE antibodies in cancer patients is also discussed.
Highlights IgE antibodies engineered to recognize cancer antigens can activate host immunity and promote tumor killing. Different in vivo models of cancer demonstrate potential for therapeutic superiority of IgE compared to IgG1. In the presence of IgE engagement and immune complex formation, classically activated macrophages (M1) can retain their inflammatory and antigenpresenting profile, while alternatively activated macrophages (M2) are re-activated to express inflammatory markers.
Leveraging IgE for Cancer Immunotherapy The contributions of IgE antibodies in the pathogenesis of allergic diseases and their protective targeting parasitic infestation by activating tissue-resident immune responses are well established [1–3]. Emerging evidence supports the design of IgE antibodies recognizing cancer-associated antigens for the treatment of solid tumors (see Glossary), aiming to take advantage of unique tissue-resident effector mechanisms of this class to be directed against cancer [4]. Even though antibody therapeutics represent a well-established platform to combat disease, only IgG (most often IgG1) is used in cancer immunotherapy at present. Since different antibody classes function through unique Fc-receptors and induce specific immune responses at different tissues, there may be potential benefit to design new therapeutics that exploit antibody isotypes other than IgG, depending on the tumor type to be targeted. IgE-based therapy against solid tumors may offer multiple advantages over those conferred by IgG. Significantly higher affinities (two to five orders of magnitude) for cognate Fc receptors, compared with those of IgG for Fc gamma receptors [5], means that IgE is the only antibody class strongly retained by effector cells without antigen engagement and immune complex formation [6]. This natural retention by immune cells may lead to a higher bioavailability in tissues, where solid tumor antigens on cancer cells are likely to be encountered. Moreover, solid tumors are usually associated with inflammatory responses and with Th2biased conditions in situ. Th2 conditions represent a fertile ambient environment for IgE antibodies to exert immunological functions, such as in allergic disease settings and in solid tissues such as the gut where IgE effector cells such as mast cells and macrophages can be activated by IgE to clear parasites [7–9]. Since it is known that IgE antibodies are immunologically active in Th2 conditions, the binding of IgE to its Fc receptors on inflammatory cells such as tumor-associated macrophages (TAMs) (Box 1) may trigger potent immune cell activation directly in the tumor milieu. Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
Macrophage activity in solid tumors treated with IgE is more similar to IgEmediated physiological protective functions in antiparasitic immunity than to an allergic immune response. The first monoclonal IgE antibody engineered to target a tumor-associated antigen is now under clinical development in cancer patients.
1 St. John’s Institute of Dermatology, School of Basic and Medical Biosciences, Guy’s Hospital, King’s College London, London, UK 2 School of Cancer and Pharmaceutical Sciences, King’s College London, Guy’s Hospital, London, UK 3 Institute of Pathophysiology and Allergy Research, Medical University Vienna, Vienna, Austria 4 The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria
*Correspondence: [email protected] (J.F. Spicer) and [email protected] (S.N. Karagiannis).
https://doi.org/10.1016/j.molmed.2020.03.002 © 2020 Elsevier Ltd. All rights reserved.
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Box 1. Manipulation of TAM for Cancer Therapy Macrophages represent a major component of the TME [49,50]. In response to cancer-associated inflammatory responses, cells of the monocyte-macrophage lineage are either recruited to the TME, or already tissue-resident macrophages undergo further differentiation [51–53]. Depending on the environmental signals these cells receive, they are differentiated broadly between M1 macrophages with proinflammatory functions and M2 phenotypes with immunomodulating or immunosuppressive properties. Nevertheless, TAM transcriptional profiles are reported to differ from those of the physiological M1 and M2 macrophage subsets [54]: TAMs are reported to express high levels of CCL2, CCL5, IL-10 and CD68, CD81, CD163 and CD206, Arginase-1 (Arg-1), nitric oxide synthase 2 (NOS2), and MHC-II [55]. TAMs also transcend the typical functional categorization of M1/M2 and depending on context such as anatomical location, tumor type and stage, location within tumors, and chemotherapy used, they feature a dual prognostic significance [56–59]. M2 macrophages are thought to be favored in the TME and can influence cancer-associated inflammation and cancer progression [60]. Increased infiltration by TAMs may be associated with tumor progression, invasion, neoangiogenesis, and suppression of antitumor immunity in breast, prostate, gastric, and head and neck cancers [61–63]. By contrast, the presence of TAMs in colorectal cancer has been linked with improved overall survival [64,65]. Therapeutic strategies targeting TAMs to reduce tumor growth are emerging [66] and include approaches to block their immunosuppressive functions, limit their influx to the tumor tissue, or repolarize them towards a proinflammatory and antitumor M1-like phenotype [25,67–70]. For instance, an in vivo model of breast cancer demonstrated that inhibition of TAM recruitment to the tumor tissue significantly increased the efficacy of cytotoxic therapies in a CD8+ T cell-dependent fashion [71]. In addition, repolarization of TAMs towards an M1 phenotype was shown to facilitate the depletion of cancer cells when animals were administered with a CD40 agonist antibody in combination with gemtabicine chemotherapy [72]. Similarly, targeting of CSF-1R (colony stimulating factor 1 receptor) with the tyrosine kinase inhibitor PLX3397 was shown to reduce macrophage recruitment to tumors, and to enhance the efficacy of adoptive cell transfer immunotherapy in an in vivo model of melanoma [73]. Influencing monocyte and macrophage lineage cells with therapeutic interventions may therefore hold merit for cancer treatment.
IgE antibodies exert pathogenic roles in allergic diseases, but also have protective effector functions against parasitic infestation often mediated by macrophages (Box 2). Several studies have also demonstrated that therapeutic harnessing of the unique biological properties of IgE antibodies could unleash previously-untapped immune mechanisms against cancer cells [10–12] and have potentiated the emergence of the AllergoOncology field concerned with the interactions between human Th2 immune responses, IgE, allergic responses, and cancer [13–17]. Among these, a study demonstrated that IgE antibodies induce a tumor-targeting immune response with the engagement of, and antigen presentation via, dendritic cells (DCs) [15]. IgE-mediated uptake of tumor antigens via the high affinity IgE receptor (FcεRI) on DCs led to antigen cross-presentation to naïve CD8+ T-cells. Comparing CD8+ T-cell proliferation in Box 2. IgE in Allergy and Parasite Infection IgE antibodies play an important role in eliciting and maintaining allergic inflammation in response to allergens. The allergic response takes place with allergen exposure, where allergen sensitization represents the triggering event, followed by the production of new antigen-specific IgE antibodies by B cells [74]. When re-exposure to the same antigen takes place, mast cells and basophils undergo a rapid degranulation, followed by release of mediators such as histamine and leukotrienes, and extravasation of inflammatory cells to the site of allergen challenge. This leads to a cascade of events to shape the early and late phase inflammatory reactions. However, even though IgE is now the main focus of investigation in allergic reactions, IgE antibodies are thought to have originally evolved from an arm of the adaptive immune response designed to protect against helminthic parasites. For this reason the IgE-related inflammation is thought to be an evolution from the original role of antibodies of the ε class: the rapid recognition and neutralization of invading parasites [75–77]. Evidence that IgEs were an essential component of the immune-system against parasitic infections was first demonstrated in animal models, studying immunological reactions to Schistosoma mansoni and Leishmania major [78,79]. Studies in Leishmania major also contributed to dissect the pivotal role that macrophages play in defending the host from parasitic infections upon activation via IgE antibodies [80]. The invasion of tissues by helminths triggers the rapid release of type 2 cytokines such as IL-4 and IL-13, which polarize tissue macrophages towards an M2-phenotype and increase the expression levels of CD23 [81]. Upon engagement via IgEbound immune complexes, the CD23 triggers intracellular signaling promoting engulfment processes of the pathogens, thus eliciting the killing activity of this immune cell subset [45].
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Glossary AllergoOncology: research at the interface between allergic responses, Th2 immunity, and cancer. Aims are to improve understanding of the immunological functions of Th2 antibodies, including IgE, of innate immune effector cells, to unfold their potential antitumor immune surveillance functions, and to develop more effective treatment options against malignant diseases. Alternatively activated macrophages (M2): cells of the myeloid lineage induced by M-CSF, IL-4, IL-13, IL-10, and TGF-β. They play a role in immune responses to parasitic infections, tissue remodeling, tumor promotion, and immune regulation. They are characterized as IL-10highIL-12lowIL-1Rhigh with a high expression of the mannose receptor CD206. In the TME, M2 macrophages can contribute to cancer inflammation and tumor progression. Antibody-dependent cell-mediated cytotoxicity (ADCC): a mechanism of cell death of antibody-coated target cells by a cytotoxic effector cell (e.g., NK cells, monocytes, and macrophages) triggered by Fc receptor signaling and a subsequent release of proteolytic enzymes or by expression of cell death-inducing molecule. Antibody-dependent cellular phagocytosis (ADCP): another potent mechanism for the elimination of antibody-coated foreign cells through engagement of FcR on, for example, macrophages, which triggers a signaling cascade leading to engulfment of antibody-opsonized particle/target cell. Classically activated macrophages (M1): cells of the myeloid lineage generated by LPS, IFN-γ, and GM-CSF stimuli and that represent prototypical effector cells with enhanced microbicidal and tumoricidal capacity. They are characterized by the expression of MHC II, CD68, and co-stimulatory CD80 and CD86 surface molecules, while at the same time producing high levels of proinflammatory cytokines, including IL-1b, TNFα, and IL-12, which drive antigen-specific Th1 immune responses. MOv18 IgE: a mouse/human chimeric IgE monoclonal antibody against folate receptor alpha (FRα), a tumor-associated marker of ovarian, and other solid tumors. MOv18 IgE is a first in class chimeric IgE antibody that is currently being tested in a Phase I clinical trial in patients with advanced solid tumors.
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response to priming by IgE/FcεRI-dependent and -independent antigen uptake, tumor cell-killing was triggered only in the presence of IgE antibodies [15]. Furthermore, in vivo investigations showed that epithelial damage promoted stress surveillance by tissue-resident γδT cells, which triggered isotype class switching to IgE in skin B cells. This endogenous IgE production provided protection from epithelial tumor development [18], alongside several epidemiological studies supporting protective effects of IgE on cancer incidence [19–23]. Together, the findings support that IgE may confer physiological protective functions against malignant transformation and allude to the potential for IgE antibodies to trigger effector functions, to promote immuneactivating signals, and to initiate potent innate and adaptive immunity against cancer. Following pre-clinical evaluations, a mouse/human chimeric antibody (MOv18 IgE) specific for the tumorassociated antigen folate receptor alpha (FRα) has been translated to a first-in-class, first-inman trial for the treatment of solid tumors (https://clinicaltrials.gov/ct2/show/NCT02546921). The development of monoclonal antibody therapies has traditionally focused on targeting the tumor cell itself; however, attention has more recently shifted towards targeting the tumor microenvironment (TME), and its cellular components. Here we argue that TAMs represent an attractive target because they express both classes of cell surface IgE Fc receptor.
A First-in-Class Monoclonal IgE Antibody: From Bench to Bedside Several pre-clinical studies reported the antitumor efficacy of engineered IgE antibodies recognizing cancer antigens [13,24–26]. These culminated in the first-in-human, first-in-class, IgE clinical trial in cancer patients, which opened in 2015 (ClinicalTrials.gov NCT02546921). The original MOv18 antibody, murine MOv18 IgG1, was generated by immunization of mice with a surgical specimen of ovarian carcinoma. Subsequently, IgG1 and IgE antibodies with human Fc domains were cloned and expressed. The in vivo efficacy of MOv18 IgE and IgG1 antibodies were compared first in a subcutaneous model, and subsequently in an orthotopic patient-derived human FRα-expressing ovarian carcinoma xenograft grown in immunodeficient mice [27–29]. In both models, MOv18 IgE showed superior efficacy, assessed by reduction of tumor growth and prolonged survival compared to the IgG1 counterpart.
Solid tumors: cancers different to cancers of the blood (e.g., leukemias) that do not contain liquid areas or cysts. Examples include sarcomas and carcinomas. Tumor-associated macrophages (TAMs): they represent the largest population of infiltrating inflammatory cells in malignant tumors that can either promote tumor development by fostering their growth and chemo resistance or can enhance antitumor responses (e.g., in colorectal cancer). Depending on the type of environmental signals and stage or localization within a tumor, these cells can exhibit characteristics of either M1 or M2 macrophage phenotype. Tumor microenvironment (TME): a specific niche surrounding a tumor mass that consists of cellular components such as fibroblasts, neuroendocrine cells, adipose cells, immune-inflammatory cells, as well as non-cellular components including blood and lymphatic vessels, secreted factors, and extracellular matrix (ECM). The TME can shape therapeutic responses, and the myriad of cellular interactions in the TME can ultimately determine tumor progression outcomes.
These findings were corroborated in an immunocompetent rat model bearing syngeneic lung metastases expressing FRα, which was employed for preclinical efficacy and toxicity studies using a MOv18 IgE surrogate molecule with rodent Fc sequence. The rat was selected as a species because the distribution and functions of IgE Fc receptors in human and rat (but not mouse) are comparable [30]. When tumor-bearing rats were administered up to four weekly intravenous doses of MOv18 IgE or the equivalent IgG2b (reported to be equivalent to mouse IgG2a/b and human IgG1 in ability to efficiently mediate antibody dependent cell-mediated cytotoxicity (ADCC) [31]), superior growth-inhibiting efficacy was observed in lung metastases of IgE treated rats (Figure 1A) [13]. In this immunocompetent animal model, administration of MOv18 IgE did not lead to any clear signs of toxic effects, and no evidence for anaphylactic responses or cytokine storm was seen. These negative findings were informative because IgE Fc receptors are expressed on both rat and human IgE effector cells, including on blood basophils and tissue mast cells. These cells have the capacity to degranulate in response to IgE, and rat FcεRI-bound IgE could potentiate type I hypersensitivity responses that can lead to anaphylaxis in vivo, in rats as well as in humans [32]. This biologically-relevant and surrogate model provided a platform for the study of the immunological effects mediated by MOv18 IgE and IgG in an immunocompetent host (as mentioned later) [24]. These findings helped to pave the way towards a first-in-human Phase I clinical trial of an IgE antibody, to evaluate the safety of MOv18 in patients with solid tumors (see Clinician’s Corner). In the past few years, antibody engineering, cloning, and expression platforms have been developed and optimized for seamless production of IgE antibodies, recognizing several antigens relevant in Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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Figure 1. Efficacy of IgE Engaging Immune Cells against Cancer. (A) CD68 macrophage tumor infiltration in MOv18 IgE-treated immunocompetent rats (left) and in orthotopic PDX of IgE-treated mice reconstituted with human PBMCs (right) correlated with lower tumor burden and increased survival, respectively. (B) Survival of orthotopic PDX-bearing mice treated with human PBMCs and MOv18 IgE was decreased when PBMCs were depleted of monocytes; survival was rescued when depleted PBMCs were reconstituted with monocytes. (C) Monocyte-mediated ADCC and ADCP of tumor cells depend on IgE Fc-Receptors FcεRI and CD23, respectively. Abbreviations: ADCC, antibody dependent cell-mediated cytotoxicity; ADCP, antibody dependent cell-mediated phagocytosis, MOv18 lgE, mouse/human chimeric IgE monoclonal antibody; PBMC, peripheral blood mononuclear cells; PDX, patient-derived xenografts.
cancer [33–36]. These have provided reagents for functional assays and in vivo investigations, offering a new understanding of the biophysical and functional attributes of IgE antibodies [37–39]. These properties, including glycosylation, immune cell engagement, and activating mechanisms, as well as effector cell function, will inform the development of IgE antibodies with high specificity and enhanced biological activity.
Monocytes/Macrophages Are Antitumor IgE Effector Cells Macrophages may play multiple roles as pro- and anti-inflammatory immune cells in IgE-driven responses such as in allergy, hypersensitivity, and in host defense against parasitic infestation [40]. Since tissue macrophages express the high-affinity IgE receptor (FcεRI), and under Th2 conditions also upregulate expression of the low affinity receptor CD23 (FcεRII) [41], these cells may represent immunological payloads with potential for activation by IgE antibodies specific for tumor antigens. The hypothesis of an essential role for monocytes/macrophages in exerting IgE-mediated tumor cell killing was suggested by in vivo studies of human xenograft mouse models. The chimeric anti4
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FRα IgE antibody MOv18 was shown to reduce tumor growth and improve survival of mice bearing human ovarian carcinoma xenografts [27,42]. The cellular immunity of these animals is reconstituted using human effector cells, peripheral blood mononuclear cells (PBMCs) (Figure 1A). Efficacy is correlated with the extent of tumor infiltration by CD68-expressing monocytes/macrophages. The central role of monocytes/macrophages in mediating MOv18 IgE antitumor efficacy was demonstrated in a study in which PBMCs were depleted of monocytes [28]. Following MOv18 IgE treatment, the survival of these mice (21 days) was no better than negative controls. This contrasted with mice receiving MOv18 IgE with intact PBMCs (39 days), or those given MOv18 IgE with PBMCs depleted of, but then reconstituted with, human monocytes (44 days) (Figure 1B). These studies suggest an essential role for cells of the monocytic lineage cells in exerting MOv18 IgE antitumor activity [28]. In the immunocompetent rat model bearing a syngeneic FRα-expressing tumor, in which a FRαspecific rat IgE engendered reduced lung tumor burden, IgE expression and cellular expression of FcεRs are comparable in humans and rats, as are the tissue distributions of monocytes and macrophages [13]. Using this system designed to resemble more closely human IgE-FcεR interactions in the TME, infiltration of CD68+ macrophages into the tumor niche was more prominent in animals treated with FRα-specific rat IgE compared with controls. Both immunohistochemical and flow cytometric analyses of tumors in IgE-treated rats revealed larger areas of macrophage infiltration, and more intratumoral than peripheral CD68-expressing cells, compared with tumors from IgG-treated animals. Supporting these in vivo findings, in vitro results from flow cytometric and lactate dehydrogenase release (LDH) assays show that human monocytes, including those derived from patients with solid tumors, can engage with MOv18 IgE and kill tumor cells in an antigen-specific manner. Human monocytes constitutively express FcεRI, which upon engagement with IgE can trigger cytotoxic killing of cancer cells (ADCC) [13,42]. Furthermore, induction of monocyte expression of the low-affinity receptor for IgE, CD23, detected only upon IL-4 stimulation, increased the number of cancer cells killed by antibody-dependent cellular phagocytosis (ADCP) (Figure 1C). This increased the total tumor cell killing, because monocytes retained the ability to kill via IgE-mediated FcεRI-driven ADCC. Subsequent studies confirmed the molecular mechanisms of these findings: (i) the ADCC function mediated by IgE interaction with FcεRI can be blocked using a recombinant FcεRIα receptor subunit and (ii) ADCP function is lost in the presence of an antibody blocking the interaction of IgE with FcεRII/CD23 [27]. These observations led to the understanding that FcεRI mediates cytotoxicity, while CD23 is responsible for IgE-mediated phagocytosis. Evidence of effector functions are not confined to MOv18. A trastuzumab-equivalent humanized IgE antibody specific for the tumor-associated antigen HER2 was shown to potentiate ADCC of HER2-expressing breast cancer cells in vitro [33,43]; and a mouse/human chimeric IgE (SF-25) recognizing a different cancer-associated antigen engaged monocyte-derived macrophages via Fc domains and triggered cytotoxic effects against melanoma cells expressing SF-25 antigen [16]. Together, these insights suggest that FcεRI-expressing tumor-infiltrating monocytes and macrophages can be directed by tumor antigen-specific IgE to exert tumoricidal functions towards cancer cells.
Re-education of Macrophages and the Immune Microenvironment in Response to IgE Monocyte and macrophage lineage cells and the immune microenvironment associated with IgE treatment were investigated in the immunocompetent rat model bearing a syngeneic FRαTrends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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expressing tumor, in which a FRα-specific rat IgE restricted lung tumor growth [13,24]. Alongside immunohistochemical and flow cytometric findings of CD68+ macrophage infiltration into tumor lesions in IgE-treated rats over IgG- and PBS-treated groups, these macrophages from IgEtreated rats expressed higher levels of the co-stimulatory classically activated (M1) molecule CD80 compared with IgG- or PBS-treated animals. TAMs from IgE-treated rats also showed elevated levels of intracellular TNFα and IL-10 expression compared with those from IgGtreated and vehicle control rats. Analyses of the bronchoalveolar lavage (BAL) fluid of IgEtreated rats also revealed higher levels of secreted TNFα, IL-10, and the monocyte/macrophage chemoattractant MCP-1 compared with IgG-treated and vehicle control rats [13,24]. These findings suggest that IgE treatment was associated with upregulation of the M1-marker CD80, alongside augmented production of proinflammatory and chemoattractant factors such as TNFα and MCP-1, thus identifying a possible mechanism for modification of the TME in response to IgE therapy (Figure 2A). Further in vitro studies with human monocytes showed that TNFα upregulation is triggered when Fc-receptor-bound human IgE, but not IgG1, is cross-linked on the surface of effector cells
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Figure 2. IgE Promotes Re-education of Tumor-Associated Macrophages (TAMs) towards Proinflammatory Phenotypes. (A) IgE-treated immunocompetent rats bearing lung metastases had higher locally-secreted IL-10, TNFα and MCP-1 (bronchial alveolar lavage, BAL), and lung-infiltrating CD68+ macrophages with increased CD80, IL-10 and TNFα expression. (B) In vitro cross-linking of IgE-bound FcεRI enhanced CD80 expression by M0 and M2 macrophages and retained CD80 expression by M1 macrophages. Abbreviations: M0, human quiescent; M1, classically activated; M2, alternatively activated.
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[13,16]. Upon cross-linking, production of TNFα also resulted in upregulation of MCP-1 by human monocytes and a range of cell types in the TME. Release of MCP-1 could trigger further monocyte chemotaxis and tumor infiltration, consistent with the observations in IgE-treated rodent models (Figure 3A). When the cytokine profile of IgE-mediated ADCC was evaluated in vitro,
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Figure 3. Mechanisms of Macrophage-Recruitment and Re-polarization via IgE Antibodies. (A) In vivo and in vitro crosslinking of IgE-bound FcεRI on monocytes and macrophages triggered TNFα upregulation (i). TNFα potentiated increased secretion of the monocyte chemoattractant, MCP-1, by monocytes and tumor cells (ii). MCP-1 then led to further macrophage recruitment to tumors (iii). (B) Cross-linking of IgE-bound FcεRI on M0 and M2 macrophages triggered newly-polarized phenotypes with enhanced secretion of proinflammatory mediators, while M1 macrophage proinflammatory features were retained. (C) FcεRI cross-linking by IgE in vitro altered macrophage signaling. Abbreviations: M0, human quiescent; M1, classically activated; M2, alternatively activated.
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upregulation of the TNFα/MCP-1/IL-10 cytokine signature similar to that found in vivo was seen [13]. In addition, MCP-1 secretion was significantly reduced by TNFα receptor–blocking antibodies, suggesting that MCP-1 production may be dependent on TNFα secretion. Interrogating publicly-available ovarian cancer gene expression datasets, higher expression levels of these mediators is associated with superior 5-year overall patient survival, pointing to a putative clinical relevance of enhancing the TNFα/MCP-1 axis (Figure 4, left) [13,44]. Together, these findings suggested that a TNFα/MCP-1 cascade may play an important role in IgE-mediated functions against tumors (Figure 3A) [13]. Upregulation of TNFα, MCP-1, nitric oxide, and IL-10 have all been detected during parasiticidal activities of macrophages [45] and, similarly, TNFα and MCP-1 appear to be upregulated in tumors following IgE therapy. This suggested that macrophage activity in solid tumors treated with IgE antibodies may mimic physiological antiparasitic activation, rather than an allergic profile (which is normally characterized by upregulation of IL-4 and IL-13).
IgE Stimulation Primes Activatory Cytokine Profile and Gene Expression Signatures by Alternatively Activated Macrophages Studies in rodent models, and in human monocytes suggested that cancer antigen-specific IgE influenced the spatial distribution, maturation, and activation of monocyte lineage cells. It was therefore explored whether IgE engagement may promote differential activation of specific macrophage subsets. Studies of the human macrophage-associated IgE-Fc receptor (FcεR) axis revealed that proportions of human quiescent (M0), alternatively activated (M2), and classically activated (M1) macrophage subsets express both the high-affinity IgE receptor FcεRI, and to a lesser extent the low affinity receptor CD23. When cell surface-bound IgE was cross-
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Figure 4. Immune Activatory Mediators Upregulated with Antitumor IgE May Be Associated with Improved Cancer Patient Survival. Interrogation of a publicly-available database (https://kmplot.com/analysis/index.php?p=service&cancer=ovar) showed that higher TNFα and MCP-1 combined expression (left) or higher Lyn expression (right) in tumors of patients with ovarian cancer were associated with better 5-year overall survival. Therefore, possible clinical outcomes of anticancer IgE therapy may include improved patient survival following upregulation of key mediators such as TNFα, MCP-1, and Lyn.
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linked on M2 macrophages, an increase in the co-stimulatory CD80 M1 macrophage marker was detected, consistent with observations of rat CD80+ TAMs in immunocompetent rats following MOv18 IgE treatment (Figure 2B) [13,25]. This points to a contribution of IgE class antibodies to maturation, polarization, and antigen-presenting cell capacity of the otherwise immunomodulatory M2 subset [16]. Interestingly, cross-linking of cell-bound IgE on M0 and M2 macrophages triggered enhanced secretion of several cytokines such as IL-1b, IL-4, IL-6, IL-10, IL-12, IL-13, IFNγ, TNFα, RANTES, CXCL9, and CXCL11, some of which are normally associated with immunoactivatory functions. Furthermore, while IgE cross-linking on M0 and M2 macrophages triggered enhanced levels of the proinflammatory M1 cytokine TNFα, MCP-1 production was upregulated only by M2 macrophages (Figure 3B) [16]. These suggest that IgE has the capacity to stimulate normally anti-inflammatory M2 macrophages to adopt activatory phenotypes [13,24,46]. Consistent with this and with evidence of IgE-mediated macrophage activation and antitumor functions in rodent models, antitumor IgE triggered cytotoxicity of cancer cells more effectively than the corresponding IgG1 mediated by all (M0, M1, and M2) macrophage subsets. These findings collectively support the notion of IgE-mediated activation of macrophages including those subsets more likely found in the TME. Cross-linking IgE bound on Fc receptors on human M1 macrophages retained both their classically-activated cell phenotype and production of proinflammatory cytokines such as IFNγ and IL-12 [16]. This suggested that IgE engagement and cross-linking may at least preserve M1 macrophages in a manner consistent with known proinflammatory and antigen-presenting functions of this subset. These findings were in concordance with signs of classical immune activation seen with IgE treatment in tumor bearing rats: enhanced TNFα expression by TAMs, elevated TNFα, MCP-1, and IL-10 in the TME and upregulation of in situ proinflammatory immune-associated pathways including IL-12 and NK cell immune activation, in the absence of signs of local or systemic allergic response activation [24].
Clinician’s Corner Monoclonal antibodies represent one arm of the cancer immunotherapy armamentarium. All currently approved antibodies are of the IgG class. However, Fc-mediated immune effector cell engagement by therapeutic IgGs may be limited by several factors, including the low affinity of IgG for its FcγRs, competition with native IgGs for binding to FcRs, and inhibitory FcRs expressed on cells within the TME. The potential biological advantages of IgE antibodies and the presence of many key FcεR-expressing immune effector cells (including macrophages) in solid tumors, provide a rationale for the development of tumor antigenspecific therapeutic IgE molecules. A MOv18 IgE recognizing the tumorassociated antigen folate receptor alpha (FRα) entered into clinical development in 2015, with the initiation of a first-in-human first-in-class Phase I clinical trial (ClinicalTrials.gov Identifier: NCT02546921). A potential concern of intravenous administration of therapeutic IgE is type I hypersensitivity. Methods to predict and monitor potential hypersensitivity reactions have been developed.
Further investigations revealed that IgE can also influence macrophage signaling. IgE crosslinking on the surface of M2 macrophages enhanced phosphorylation of FcεRI-dependent intracellular pathway molecules Lyn, ERK1/2, Lck, Fyn, and p38 (Figure 3C) [16]. Upregulated Lyn gene expression was detected by M0, M1, and M2 macrophages (Figure 3C). These findings may have clinical significance: elevated Lyn gene expression is associated with improved 5-year overall survival in gastric, lung, and ovarian cancers (Figure 4, right) [16]; transgenic mouse models lacking Lyn have defective macrophage populations and in vivo studies suggest that a loss-offunction of Lyn may predispose to carcinogenesis [47]. Furthermore, the MAPK, PI3K/AKT, Rho GTPase, and other signaling pathways were differentially-activated in M2 versus M1 macrophages with IgE stimulation [16], and IgE cross-linking downregulated expression of the checkpoint molecule T cell, or transmembrane immunoglobulin and mucin domain protein 3 (TIM-3), a tyrosine kinase activated downstream of FcεRI, thought to participate in multiple immune suppressing pathways [48]. Overall, retention and upregulation of Lyn, downregulation of TIM-3, alongside activation of multiple immune mediators and signaling pathways point to significant functions for the FcεR axis in re-educating M2 macrophages towards enhanced immune activating profiles.
Concluding Remarks Findings from disparate rodent models of cancer alongside in vitro and ex vivo functional studies suggest that IgE engagement and Fc receptor cross-linking on TAMs could reactivate these cells against cancer. Antitumor IgE can prime human macrophages of different polarization states to elicit effector function responses and restrict tumor cell growth. Importantly, emerging evidence Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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also supports a more prominent role for IgE than for IgG in re-educating M2 macrophages towards activated states, to feature higher expression of M1 macrophage markers such as CD80 and TNFα. These observations suggest that delivery of a tumor-specific IgE may alter the cellular and immunological dynamics in the TME, ultimately leading to tumor regression [13]. In vitro and in vivo functional studies of MOv18 IgE targeting FRα formed the basis for clinical investigation in a first-in-class, first-in-human clinical trial in patients with advanced solid cancers. Further dissection of the molecular interactions between macrophages and IgE in cancer may provide a promising avenue for the development of a new therapeutic strategy for solid tumors (see Outstanding Questions). These observations strengthen the case for the therapeutic potential of antitumor IgE, which seems able not only to mediate killing of cancer cells by ADCC, but also to play a role in re-education of TAMs from an anti-inflammatory profile to an M1-like phenotype. Observations of the TNFα/MCP-1 axis as an IgE-specific cytokine profile in a human context await the clinical development of IgE therapy in patients with cancer. Collectively, despite the frequent tumor-promoting properties of TAMs, new avenues are emerging for their engagement and activation by IgE to induce effective immune surveillance against cancer. Acknowledgments The authors acknowledge support by Cancer Research UK, United Kingdom (C30122/A11527; C30122/A15774); the Medical Research Council, United Kingdom (MR/L023091/1); the Academy of Medical Sciences, United Kingdom; The Inman Charity, United Kingdom; Breast Cancer Now, United Kingdom (147), working in partnership with Walk the Walk; CR UK//NIHR in England/DoH for Scotland, Wales, and Northern Ireland Experimental Cancer Medicine Centre, United Kingdom (C10355/A15587); and the Cancer Research UK King’s Health Partners Centre at King’s College London. J.G. was supported by a grant of the Austrian Science Fund, Austria, CCHD, grant number W1205-B09 (to E.J.J.). The authors also acknowledge support by the European Academy of Allergy and Clinical Immunology (EAACI) (AllergoOncology Task Force). The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) based at Guy's and St Thomas' NHS Foundation Trust and King's College London, United Kingdom (IS-BRC1215-20006). The authors are solely responsible for study design, data collection, analysis, decision to publish, and preparation of the manuscript. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.
Disclaimer Statement S.N.K. and J.F.S. are founders and shareholders of IGEM Therapeutics Ltd. S.N.K. holds a patent on antitumor IgE antibodies. H.J.B. is funded through a grant by IGEM Therapeutics Ltd. The other authors declare no conflicts of interest.
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Outstanding Questions What are the molecular pathways involved in the re-polarization of TAMs by IgE antibodies? What effects are triggered by combination treatment of cancer cells with both tumor-specific IgE and IgG antibodies? In the context of selective macrophage depletion in vivo, would therapeutic IgE antibodies still deliver anticancer functions? If yes, how? What is the clinical safety of therapeutic IgE antibodies, and how can this be best monitored? Would combination therapy with targeted agents or biologics be a possibility for cancer patients administered with IgE immunotherapy?
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76. Burton, O.T. and Oettgen, H.C. (2011) Beyond immediate hypersensitivity: evolving roles for IgE antibodies in immune homeostasis and allergic diseases. Immunol. Rev. 242, 128–143 77. Rosenwasser, L.J. (2011) Mechanisms of IgE inflammation. Curr Allergy Asthma Rep 11, 178–183 78. Capron, M. and Capron, A. (1994) Immunoglobulin E and effector cells in schistosomiasis. Science 264, 1876–1877 79. Verwaerde, C. et al. (1987) Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni. J. Immunol. 138, 4441–4446 80. Soulat, D. and Bogdan, C. (2017) Function of macrophage and parasite phosphatases in leishmaniasis. Front. Immunol. 8, 1838 81. Gordon, S. and Martinez, F.O. Alternative activation of macrophages: mechanism and functions. Immunity 32, 593–604.
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Review
Harnessing Therapeutic IgE Antibodies to Re-educate Macrophages against Cancer Giulia Pellizzari,1 Heather J. Bax,1,2 Debra H. Josephs,1,2 Jelena Gotovina,3,4 Erika Jensen-Jarolim,3,4 James F. Spicer,2,* and Sophia N. Karagiannis1,* Currently, IgG is the only class of antibodies employed for cancer therapy. However, harnessing the unique biological properties of a different class ( e.g., IgE) could engender potent effector cell activation, and unleash previously untapped immune mechanisms against cancer. IgE antibodies are best known for pathogenic roles in allergic diseases and for protective effector functions against parasitic infestation, often mediated by IgE Fc receptor-expressing macrophages. Notably, IgE possess a very high affinity for cognate Fc receptors expressed by tumor-associated macrophages (TAMs). This paper reviews pre-clinical studies, which indicate control of cancer growth by tumor antigen-specific IgE that recruit and re-educate TAMs towards activated profiles. The clinical development harnessing the antitumor potential of recombinant IgE antibodies in cancer patients is also discussed.
Highlights IgE antibodies engineered to recognize cancer antigens can activate host immunity and promote tumor killing. Different in vivo models of cancer demonstrate potential for therapeutic superiority of IgE compared to IgG1. In the presence of IgE engagement and immune complex formation, classically activated macrophages (M1) can retain their inflammatory and antigenpresenting profile, while alternatively activated macrophages (M2) are re-activated to express inflammatory markers.
Leveraging IgE for Cancer Immunotherapy The contributions of IgE antibodies in the pathogenesis of allergic diseases and their protective targeting parasitic infestation by activating tissue-resident immune responses are well established [1–3]. Emerging evidence supports the design of IgE antibodies recognizing cancer-associated antigens for the treatment of solid tumors (see Glossary), aiming to take advantage of unique tissue-resident effector mechanisms of this class to be directed against cancer [4]. Even though antibody therapeutics represent a well-established platform to combat disease, only IgG (most often IgG1) is used in cancer immunotherapy at present. Since different antibody classes function through unique Fc-receptors and induce specific immune responses at different tissues, there may be potential benefit to design new therapeutics that exploit antibody isotypes other than IgG, depending on the tumor type to be targeted. IgE-based therapy against solid tumors may offer multiple advantages over those conferred by IgG. Significantly higher affinities (two to five orders of magnitude) for cognate Fc receptors, compared with those of IgG for Fc gamma receptors [5], means that IgE is the only antibody class strongly retained by effector cells without antigen engagement and immune complex formation [6]. This natural retention by immune cells may lead to a higher bioavailability in tissues, where solid tumor antigens on cancer cells are likely to be encountered. Moreover, solid tumors are usually associated with inflammatory responses and with Th2biased conditions in situ. Th2 conditions represent a fertile ambient environment for IgE antibodies to exert immunological functions, such as in allergic disease settings and in solid tissues such as the gut where IgE effector cells such as mast cells and macrophages can be activated by IgE to clear parasites [7–9]. Since it is known that IgE antibodies are immunologically active in Th2 conditions, the binding of IgE to its Fc receptors on inflammatory cells such as tumor-associated macrophages (TAMs) (Box 1) may trigger potent immune cell activation directly in the tumor milieu. Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
Macrophage activity in solid tumors treated with IgE is more similar to IgEmediated physiological protective functions in antiparasitic immunity than to an allergic immune response. The first monoclonal IgE antibody engineered to target a tumor-associated antigen is now under clinical development in cancer patients.
1 St. John’s Institute of Dermatology, School of Basic and Medical Biosciences, Guy’s Hospital, King’s College London, London, UK 2 School of Cancer and Pharmaceutical Sciences, King’s College London, Guy’s Hospital, London, UK 3 Institute of Pathophysiology and Allergy Research, Medical University Vienna, Vienna, Austria 4 The Interuniversity Messerli Research Institute of the University of Veterinary Medicine Vienna, Medical University Vienna and University Vienna, Vienna, Austria
*Correspondence: [email protected] (J.F. Spicer) and [email protected] (S.N. Karagiannis).
https://doi.org/10.1016/j.molmed.2020.03.002 © 2020 Elsevier Ltd. All rights reserved.
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Box 1. Manipulation of TAM for Cancer Therapy Macrophages represent a major component of the TME [49,50]. In response to cancer-associated inflammatory responses, cells of the monocyte-macrophage lineage are either recruited to the TME, or already tissue-resident macrophages undergo further differentiation [51–53]. Depending on the environmental signals these cells receive, they are differentiated broadly between M1 macrophages with proinflammatory functions and M2 phenotypes with immunomodulating or immunosuppressive properties. Nevertheless, TAM transcriptional profiles are reported to differ from those of the physiological M1 and M2 macrophage subsets [54]: TAMs are reported to express high levels of CCL2, CCL5, IL-10 and CD68, CD81, CD163 and CD206, Arginase-1 (Arg-1), nitric oxide synthase 2 (NOS2), and MHC-II [55]. TAMs also transcend the typical functional categorization of M1/M2 and depending on context such as anatomical location, tumor type and stage, location within tumors, and chemotherapy used, they feature a dual prognostic significance [56–59]. M2 macrophages are thought to be favored in the TME and can influence cancer-associated inflammation and cancer progression [60]. Increased infiltration by TAMs may be associated with tumor progression, invasion, neoangiogenesis, and suppression of antitumor immunity in breast, prostate, gastric, and head and neck cancers [61–63]. By contrast, the presence of TAMs in colorectal cancer has been linked with improved overall survival [64,65]. Therapeutic strategies targeting TAMs to reduce tumor growth are emerging [66] and include approaches to block their immunosuppressive functions, limit their influx to the tumor tissue, or repolarize them towards a proinflammatory and antitumor M1-like phenotype [25,67–70]. For instance, an in vivo model of breast cancer demonstrated that inhibition of TAM recruitment to the tumor tissue significantly increased the efficacy of cytotoxic therapies in a CD8+ T cell-dependent fashion [71]. In addition, repolarization of TAMs towards an M1 phenotype was shown to facilitate the depletion of cancer cells when animals were administered with a CD40 agonist antibody in combination with gemtabicine chemotherapy [72]. Similarly, targeting of CSF-1R (colony stimulating factor 1 receptor) with the tyrosine kinase inhibitor PLX3397 was shown to reduce macrophage recruitment to tumors, and to enhance the efficacy of adoptive cell transfer immunotherapy in an in vivo model of melanoma [73]. Influencing monocyte and macrophage lineage cells with therapeutic interventions may therefore hold merit for cancer treatment.
IgE antibodies exert pathogenic roles in allergic diseases, but also have protective effector functions against parasitic infestation often mediated by macrophages (Box 2). Several studies have also demonstrated that therapeutic harnessing of the unique biological properties of IgE antibodies could unleash previously-untapped immune mechanisms against cancer cells [10–12] and have potentiated the emergence of the AllergoOncology field concerned with the interactions between human Th2 immune responses, IgE, allergic responses, and cancer [13–17]. Among these, a study demonstrated that IgE antibodies induce a tumor-targeting immune response with the engagement of, and antigen presentation via, dendritic cells (DCs) [15]. IgE-mediated uptake of tumor antigens via the high affinity IgE receptor (FcεRI) on DCs led to antigen cross-presentation to naïve CD8+ T-cells. Comparing CD8+ T-cell proliferation in Box 2. IgE in Allergy and Parasite Infection IgE antibodies play an important role in eliciting and maintaining allergic inflammation in response to allergens. The allergic response takes place with allergen exposure, where allergen sensitization represents the triggering event, followed by the production of new antigen-specific IgE antibodies by B cells [74]. When re-exposure to the same antigen takes place, mast cells and basophils undergo a rapid degranulation, followed by release of mediators such as histamine and leukotrienes, and extravasation of inflammatory cells to the site of allergen challenge. This leads to a cascade of events to shape the early and late phase inflammatory reactions. However, even though IgE is now the main focus of investigation in allergic reactions, IgE antibodies are thought to have originally evolved from an arm of the adaptive immune response designed to protect against helminthic parasites. For this reason the IgE-related inflammation is thought to be an evolution from the original role of antibodies of the ε class: the rapid recognition and neutralization of invading parasites [75–77]. Evidence that IgEs were an essential component of the immune-system against parasitic infections was first demonstrated in animal models, studying immunological reactions to Schistosoma mansoni and Leishmania major [78,79]. Studies in Leishmania major also contributed to dissect the pivotal role that macrophages play in defending the host from parasitic infections upon activation via IgE antibodies [80]. The invasion of tissues by helminths triggers the rapid release of type 2 cytokines such as IL-4 and IL-13, which polarize tissue macrophages towards an M2-phenotype and increase the expression levels of CD23 [81]. Upon engagement via IgEbound immune complexes, the CD23 triggers intracellular signaling promoting engulfment processes of the pathogens, thus eliciting the killing activity of this immune cell subset [45].
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Glossary AllergoOncology: research at the interface between allergic responses, Th2 immunity, and cancer. Aims are to improve understanding of the immunological functions of Th2 antibodies, including IgE, of innate immune effector cells, to unfold their potential antitumor immune surveillance functions, and to develop more effective treatment options against malignant diseases. Alternatively activated macrophages (M2): cells of the myeloid lineage induced by M-CSF, IL-4, IL-13, IL-10, and TGF-β. They play a role in immune responses to parasitic infections, tissue remodeling, tumor promotion, and immune regulation. They are characterized as IL-10highIL-12lowIL-1Rhigh with a high expression of the mannose receptor CD206. In the TME, M2 macrophages can contribute to cancer inflammation and tumor progression. Antibody-dependent cell-mediated cytotoxicity (ADCC): a mechanism of cell death of antibody-coated target cells by a cytotoxic effector cell (e.g., NK cells, monocytes, and macrophages) triggered by Fc receptor signaling and a subsequent release of proteolytic enzymes or by expression of cell death-inducing molecule. Antibody-dependent cellular phagocytosis (ADCP): another potent mechanism for the elimination of antibody-coated foreign cells through engagement of FcR on, for example, macrophages, which triggers a signaling cascade leading to engulfment of antibody-opsonized particle/target cell. Classically activated macrophages (M1): cells of the myeloid lineage generated by LPS, IFN-γ, and GM-CSF stimuli and that represent prototypical effector cells with enhanced microbicidal and tumoricidal capacity. They are characterized by the expression of MHC II, CD68, and co-stimulatory CD80 and CD86 surface molecules, while at the same time producing high levels of proinflammatory cytokines, including IL-1b, TNFα, and IL-12, which drive antigen-specific Th1 immune responses. MOv18 IgE: a mouse/human chimeric IgE monoclonal antibody against folate receptor alpha (FRα), a tumor-associated marker of ovarian, and other solid tumors. MOv18 IgE is a first in class chimeric IgE antibody that is currently being tested in a Phase I clinical trial in patients with advanced solid tumors.
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response to priming by IgE/FcεRI-dependent and -independent antigen uptake, tumor cell-killing was triggered only in the presence of IgE antibodies [15]. Furthermore, in vivo investigations showed that epithelial damage promoted stress surveillance by tissue-resident γδT cells, which triggered isotype class switching to IgE in skin B cells. This endogenous IgE production provided protection from epithelial tumor development [18], alongside several epidemiological studies supporting protective effects of IgE on cancer incidence [19–23]. Together, the findings support that IgE may confer physiological protective functions against malignant transformation and allude to the potential for IgE antibodies to trigger effector functions, to promote immuneactivating signals, and to initiate potent innate and adaptive immunity against cancer. Following pre-clinical evaluations, a mouse/human chimeric antibody (MOv18 IgE) specific for the tumorassociated antigen folate receptor alpha (FRα) has been translated to a first-in-class, first-inman trial for the treatment of solid tumors (https://clinicaltrials.gov/ct2/show/NCT02546921). The development of monoclonal antibody therapies has traditionally focused on targeting the tumor cell itself; however, attention has more recently shifted towards targeting the tumor microenvironment (TME), and its cellular components. Here we argue that TAMs represent an attractive target because they express both classes of cell surface IgE Fc receptor.
A First-in-Class Monoclonal IgE Antibody: From Bench to Bedside Several pre-clinical studies reported the antitumor efficacy of engineered IgE antibodies recognizing cancer antigens [13,24–26]. These culminated in the first-in-human, first-in-class, IgE clinical trial in cancer patients, which opened in 2015 (ClinicalTrials.gov NCT02546921). The original MOv18 antibody, murine MOv18 IgG1, was generated by immunization of mice with a surgical specimen of ovarian carcinoma. Subsequently, IgG1 and IgE antibodies with human Fc domains were cloned and expressed. The in vivo efficacy of MOv18 IgE and IgG1 antibodies were compared first in a subcutaneous model, and subsequently in an orthotopic patient-derived human FRα-expressing ovarian carcinoma xenograft grown in immunodeficient mice [27–29]. In both models, MOv18 IgE showed superior efficacy, assessed by reduction of tumor growth and prolonged survival compared to the IgG1 counterpart.
Solid tumors: cancers different to cancers of the blood (e.g., leukemias) that do not contain liquid areas or cysts. Examples include sarcomas and carcinomas. Tumor-associated macrophages (TAMs): they represent the largest population of infiltrating inflammatory cells in malignant tumors that can either promote tumor development by fostering their growth and chemo resistance or can enhance antitumor responses (e.g., in colorectal cancer). Depending on the type of environmental signals and stage or localization within a tumor, these cells can exhibit characteristics of either M1 or M2 macrophage phenotype. Tumor microenvironment (TME): a specific niche surrounding a tumor mass that consists of cellular components such as fibroblasts, neuroendocrine cells, adipose cells, immune-inflammatory cells, as well as non-cellular components including blood and lymphatic vessels, secreted factors, and extracellular matrix (ECM). The TME can shape therapeutic responses, and the myriad of cellular interactions in the TME can ultimately determine tumor progression outcomes.
These findings were corroborated in an immunocompetent rat model bearing syngeneic lung metastases expressing FRα, which was employed for preclinical efficacy and toxicity studies using a MOv18 IgE surrogate molecule with rodent Fc sequence. The rat was selected as a species because the distribution and functions of IgE Fc receptors in human and rat (but not mouse) are comparable [30]. When tumor-bearing rats were administered up to four weekly intravenous doses of MOv18 IgE or the equivalent IgG2b (reported to be equivalent to mouse IgG2a/b and human IgG1 in ability to efficiently mediate antibody dependent cell-mediated cytotoxicity (ADCC) [31]), superior growth-inhibiting efficacy was observed in lung metastases of IgE treated rats (Figure 1A) [13]. In this immunocompetent animal model, administration of MOv18 IgE did not lead to any clear signs of toxic effects, and no evidence for anaphylactic responses or cytokine storm was seen. These negative findings were informative because IgE Fc receptors are expressed on both rat and human IgE effector cells, including on blood basophils and tissue mast cells. These cells have the capacity to degranulate in response to IgE, and rat FcεRI-bound IgE could potentiate type I hypersensitivity responses that can lead to anaphylaxis in vivo, in rats as well as in humans [32]. This biologically-relevant and surrogate model provided a platform for the study of the immunological effects mediated by MOv18 IgE and IgG in an immunocompetent host (as mentioned later) [24]. These findings helped to pave the way towards a first-in-human Phase I clinical trial of an IgE antibody, to evaluate the safety of MOv18 in patients with solid tumors (see Clinician’s Corner). In the past few years, antibody engineering, cloning, and expression platforms have been developed and optimized for seamless production of IgE antibodies, recognizing several antigens relevant in Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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Figure 1. Efficacy of IgE Engaging Immune Cells against Cancer. (A) CD68 macrophage tumor infiltration in MOv18 IgE-treated immunocompetent rats (left) and in orthotopic PDX of IgE-treated mice reconstituted with human PBMCs (right) correlated with lower tumor burden and increased survival, respectively. (B) Survival of orthotopic PDX-bearing mice treated with human PBMCs and MOv18 IgE was decreased when PBMCs were depleted of monocytes; survival was rescued when depleted PBMCs were reconstituted with monocytes. (C) Monocyte-mediated ADCC and ADCP of tumor cells depend on IgE Fc-Receptors FcεRI and CD23, respectively. Abbreviations: ADCC, antibody dependent cell-mediated cytotoxicity; ADCP, antibody dependent cell-mediated phagocytosis, MOv18 lgE, mouse/human chimeric IgE monoclonal antibody; PBMC, peripheral blood mononuclear cells; PDX, patient-derived xenografts.
cancer [33–36]. These have provided reagents for functional assays and in vivo investigations, offering a new understanding of the biophysical and functional attributes of IgE antibodies [37–39]. These properties, including glycosylation, immune cell engagement, and activating mechanisms, as well as effector cell function, will inform the development of IgE antibodies with high specificity and enhanced biological activity.
Monocytes/Macrophages Are Antitumor IgE Effector Cells Macrophages may play multiple roles as pro- and anti-inflammatory immune cells in IgE-driven responses such as in allergy, hypersensitivity, and in host defense against parasitic infestation [40]. Since tissue macrophages express the high-affinity IgE receptor (FcεRI), and under Th2 conditions also upregulate expression of the low affinity receptor CD23 (FcεRII) [41], these cells may represent immunological payloads with potential for activation by IgE antibodies specific for tumor antigens. The hypothesis of an essential role for monocytes/macrophages in exerting IgE-mediated tumor cell killing was suggested by in vivo studies of human xenograft mouse models. The chimeric anti4
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FRα IgE antibody MOv18 was shown to reduce tumor growth and improve survival of mice bearing human ovarian carcinoma xenografts [27,42]. The cellular immunity of these animals is reconstituted using human effector cells, peripheral blood mononuclear cells (PBMCs) (Figure 1A). Efficacy is correlated with the extent of tumor infiltration by CD68-expressing monocytes/macrophages. The central role of monocytes/macrophages in mediating MOv18 IgE antitumor efficacy was demonstrated in a study in which PBMCs were depleted of monocytes [28]. Following MOv18 IgE treatment, the survival of these mice (21 days) was no better than negative controls. This contrasted with mice receiving MOv18 IgE with intact PBMCs (39 days), or those given MOv18 IgE with PBMCs depleted of, but then reconstituted with, human monocytes (44 days) (Figure 1B). These studies suggest an essential role for cells of the monocytic lineage cells in exerting MOv18 IgE antitumor activity [28]. In the immunocompetent rat model bearing a syngeneic FRα-expressing tumor, in which a FRαspecific rat IgE engendered reduced lung tumor burden, IgE expression and cellular expression of FcεRs are comparable in humans and rats, as are the tissue distributions of monocytes and macrophages [13]. Using this system designed to resemble more closely human IgE-FcεR interactions in the TME, infiltration of CD68+ macrophages into the tumor niche was more prominent in animals treated with FRα-specific rat IgE compared with controls. Both immunohistochemical and flow cytometric analyses of tumors in IgE-treated rats revealed larger areas of macrophage infiltration, and more intratumoral than peripheral CD68-expressing cells, compared with tumors from IgG-treated animals. Supporting these in vivo findings, in vitro results from flow cytometric and lactate dehydrogenase release (LDH) assays show that human monocytes, including those derived from patients with solid tumors, can engage with MOv18 IgE and kill tumor cells in an antigen-specific manner. Human monocytes constitutively express FcεRI, which upon engagement with IgE can trigger cytotoxic killing of cancer cells (ADCC) [13,42]. Furthermore, induction of monocyte expression of the low-affinity receptor for IgE, CD23, detected only upon IL-4 stimulation, increased the number of cancer cells killed by antibody-dependent cellular phagocytosis (ADCP) (Figure 1C). This increased the total tumor cell killing, because monocytes retained the ability to kill via IgE-mediated FcεRI-driven ADCC. Subsequent studies confirmed the molecular mechanisms of these findings: (i) the ADCC function mediated by IgE interaction with FcεRI can be blocked using a recombinant FcεRIα receptor subunit and (ii) ADCP function is lost in the presence of an antibody blocking the interaction of IgE with FcεRII/CD23 [27]. These observations led to the understanding that FcεRI mediates cytotoxicity, while CD23 is responsible for IgE-mediated phagocytosis. Evidence of effector functions are not confined to MOv18. A trastuzumab-equivalent humanized IgE antibody specific for the tumor-associated antigen HER2 was shown to potentiate ADCC of HER2-expressing breast cancer cells in vitro [33,43]; and a mouse/human chimeric IgE (SF-25) recognizing a different cancer-associated antigen engaged monocyte-derived macrophages via Fc domains and triggered cytotoxic effects against melanoma cells expressing SF-25 antigen [16]. Together, these insights suggest that FcεRI-expressing tumor-infiltrating monocytes and macrophages can be directed by tumor antigen-specific IgE to exert tumoricidal functions towards cancer cells.
Re-education of Macrophages and the Immune Microenvironment in Response to IgE Monocyte and macrophage lineage cells and the immune microenvironment associated with IgE treatment were investigated in the immunocompetent rat model bearing a syngeneic FRαTrends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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expressing tumor, in which a FRα-specific rat IgE restricted lung tumor growth [13,24]. Alongside immunohistochemical and flow cytometric findings of CD68+ macrophage infiltration into tumor lesions in IgE-treated rats over IgG- and PBS-treated groups, these macrophages from IgEtreated rats expressed higher levels of the co-stimulatory classically activated (M1) molecule CD80 compared with IgG- or PBS-treated animals. TAMs from IgE-treated rats also showed elevated levels of intracellular TNFα and IL-10 expression compared with those from IgGtreated and vehicle control rats. Analyses of the bronchoalveolar lavage (BAL) fluid of IgEtreated rats also revealed higher levels of secreted TNFα, IL-10, and the monocyte/macrophage chemoattractant MCP-1 compared with IgG-treated and vehicle control rats [13,24]. These findings suggest that IgE treatment was associated with upregulation of the M1-marker CD80, alongside augmented production of proinflammatory and chemoattractant factors such as TNFα and MCP-1, thus identifying a possible mechanism for modification of the TME in response to IgE therapy (Figure 2A). Further in vitro studies with human monocytes showed that TNFα upregulation is triggered when Fc-receptor-bound human IgE, but not IgG1, is cross-linked on the surface of effector cells
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Figure 2. IgE Promotes Re-education of Tumor-Associated Macrophages (TAMs) towards Proinflammatory Phenotypes. (A) IgE-treated immunocompetent rats bearing lung metastases had higher locally-secreted IL-10, TNFα and MCP-1 (bronchial alveolar lavage, BAL), and lung-infiltrating CD68+ macrophages with increased CD80, IL-10 and TNFα expression. (B) In vitro cross-linking of IgE-bound FcεRI enhanced CD80 expression by M0 and M2 macrophages and retained CD80 expression by M1 macrophages. Abbreviations: M0, human quiescent; M1, classically activated; M2, alternatively activated.
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[13,16]. Upon cross-linking, production of TNFα also resulted in upregulation of MCP-1 by human monocytes and a range of cell types in the TME. Release of MCP-1 could trigger further monocyte chemotaxis and tumor infiltration, consistent with the observations in IgE-treated rodent models (Figure 3A). When the cytokine profile of IgE-mediated ADCC was evaluated in vitro,
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Figure 3. Mechanisms of Macrophage-Recruitment and Re-polarization via IgE Antibodies. (A) In vivo and in vitro crosslinking of IgE-bound FcεRI on monocytes and macrophages triggered TNFα upregulation (i). TNFα potentiated increased secretion of the monocyte chemoattractant, MCP-1, by monocytes and tumor cells (ii). MCP-1 then led to further macrophage recruitment to tumors (iii). (B) Cross-linking of IgE-bound FcεRI on M0 and M2 macrophages triggered newly-polarized phenotypes with enhanced secretion of proinflammatory mediators, while M1 macrophage proinflammatory features were retained. (C) FcεRI cross-linking by IgE in vitro altered macrophage signaling. Abbreviations: M0, human quiescent; M1, classically activated; M2, alternatively activated.
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upregulation of the TNFα/MCP-1/IL-10 cytokine signature similar to that found in vivo was seen [13]. In addition, MCP-1 secretion was significantly reduced by TNFα receptor–blocking antibodies, suggesting that MCP-1 production may be dependent on TNFα secretion. Interrogating publicly-available ovarian cancer gene expression datasets, higher expression levels of these mediators is associated with superior 5-year overall patient survival, pointing to a putative clinical relevance of enhancing the TNFα/MCP-1 axis (Figure 4, left) [13,44]. Together, these findings suggested that a TNFα/MCP-1 cascade may play an important role in IgE-mediated functions against tumors (Figure 3A) [13]. Upregulation of TNFα, MCP-1, nitric oxide, and IL-10 have all been detected during parasiticidal activities of macrophages [45] and, similarly, TNFα and MCP-1 appear to be upregulated in tumors following IgE therapy. This suggested that macrophage activity in solid tumors treated with IgE antibodies may mimic physiological antiparasitic activation, rather than an allergic profile (which is normally characterized by upregulation of IL-4 and IL-13).
IgE Stimulation Primes Activatory Cytokine Profile and Gene Expression Signatures by Alternatively Activated Macrophages Studies in rodent models, and in human monocytes suggested that cancer antigen-specific IgE influenced the spatial distribution, maturation, and activation of monocyte lineage cells. It was therefore explored whether IgE engagement may promote differential activation of specific macrophage subsets. Studies of the human macrophage-associated IgE-Fc receptor (FcεR) axis revealed that proportions of human quiescent (M0), alternatively activated (M2), and classically activated (M1) macrophage subsets express both the high-affinity IgE receptor FcεRI, and to a lesser extent the low affinity receptor CD23. When cell surface-bound IgE was cross-
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Figure 4. Immune Activatory Mediators Upregulated with Antitumor IgE May Be Associated with Improved Cancer Patient Survival. Interrogation of a publicly-available database (https://kmplot.com/analysis/index.php?p=service&cancer=ovar) showed that higher TNFα and MCP-1 combined expression (left) or higher Lyn expression (right) in tumors of patients with ovarian cancer were associated with better 5-year overall survival. Therefore, possible clinical outcomes of anticancer IgE therapy may include improved patient survival following upregulation of key mediators such as TNFα, MCP-1, and Lyn.
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linked on M2 macrophages, an increase in the co-stimulatory CD80 M1 macrophage marker was detected, consistent with observations of rat CD80+ TAMs in immunocompetent rats following MOv18 IgE treatment (Figure 2B) [13,25]. This points to a contribution of IgE class antibodies to maturation, polarization, and antigen-presenting cell capacity of the otherwise immunomodulatory M2 subset [16]. Interestingly, cross-linking of cell-bound IgE on M0 and M2 macrophages triggered enhanced secretion of several cytokines such as IL-1b, IL-4, IL-6, IL-10, IL-12, IL-13, IFNγ, TNFα, RANTES, CXCL9, and CXCL11, some of which are normally associated with immunoactivatory functions. Furthermore, while IgE cross-linking on M0 and M2 macrophages triggered enhanced levels of the proinflammatory M1 cytokine TNFα, MCP-1 production was upregulated only by M2 macrophages (Figure 3B) [16]. These suggest that IgE has the capacity to stimulate normally anti-inflammatory M2 macrophages to adopt activatory phenotypes [13,24,46]. Consistent with this and with evidence of IgE-mediated macrophage activation and antitumor functions in rodent models, antitumor IgE triggered cytotoxicity of cancer cells more effectively than the corresponding IgG1 mediated by all (M0, M1, and M2) macrophage subsets. These findings collectively support the notion of IgE-mediated activation of macrophages including those subsets more likely found in the TME. Cross-linking IgE bound on Fc receptors on human M1 macrophages retained both their classically-activated cell phenotype and production of proinflammatory cytokines such as IFNγ and IL-12 [16]. This suggested that IgE engagement and cross-linking may at least preserve M1 macrophages in a manner consistent with known proinflammatory and antigen-presenting functions of this subset. These findings were in concordance with signs of classical immune activation seen with IgE treatment in tumor bearing rats: enhanced TNFα expression by TAMs, elevated TNFα, MCP-1, and IL-10 in the TME and upregulation of in situ proinflammatory immune-associated pathways including IL-12 and NK cell immune activation, in the absence of signs of local or systemic allergic response activation [24].
Clinician’s Corner Monoclonal antibodies represent one arm of the cancer immunotherapy armamentarium. All currently approved antibodies are of the IgG class. However, Fc-mediated immune effector cell engagement by therapeutic IgGs may be limited by several factors, including the low affinity of IgG for its FcγRs, competition with native IgGs for binding to FcRs, and inhibitory FcRs expressed on cells within the TME. The potential biological advantages of IgE antibodies and the presence of many key FcεR-expressing immune effector cells (including macrophages) in solid tumors, provide a rationale for the development of tumor antigenspecific therapeutic IgE molecules. A MOv18 IgE recognizing the tumorassociated antigen folate receptor alpha (FRα) entered into clinical development in 2015, with the initiation of a first-in-human first-in-class Phase I clinical trial (ClinicalTrials.gov Identifier: NCT02546921). A potential concern of intravenous administration of therapeutic IgE is type I hypersensitivity. Methods to predict and monitor potential hypersensitivity reactions have been developed.
Further investigations revealed that IgE can also influence macrophage signaling. IgE crosslinking on the surface of M2 macrophages enhanced phosphorylation of FcεRI-dependent intracellular pathway molecules Lyn, ERK1/2, Lck, Fyn, and p38 (Figure 3C) [16]. Upregulated Lyn gene expression was detected by M0, M1, and M2 macrophages (Figure 3C). These findings may have clinical significance: elevated Lyn gene expression is associated with improved 5-year overall survival in gastric, lung, and ovarian cancers (Figure 4, right) [16]; transgenic mouse models lacking Lyn have defective macrophage populations and in vivo studies suggest that a loss-offunction of Lyn may predispose to carcinogenesis [47]. Furthermore, the MAPK, PI3K/AKT, Rho GTPase, and other signaling pathways were differentially-activated in M2 versus M1 macrophages with IgE stimulation [16], and IgE cross-linking downregulated expression of the checkpoint molecule T cell, or transmembrane immunoglobulin and mucin domain protein 3 (TIM-3), a tyrosine kinase activated downstream of FcεRI, thought to participate in multiple immune suppressing pathways [48]. Overall, retention and upregulation of Lyn, downregulation of TIM-3, alongside activation of multiple immune mediators and signaling pathways point to significant functions for the FcεR axis in re-educating M2 macrophages towards enhanced immune activating profiles.
Concluding Remarks Findings from disparate rodent models of cancer alongside in vitro and ex vivo functional studies suggest that IgE engagement and Fc receptor cross-linking on TAMs could reactivate these cells against cancer. Antitumor IgE can prime human macrophages of different polarization states to elicit effector function responses and restrict tumor cell growth. Importantly, emerging evidence Trends in Molecular Medicine, Month 2020, Vol. xx, No. xx
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also supports a more prominent role for IgE than for IgG in re-educating M2 macrophages towards activated states, to feature higher expression of M1 macrophage markers such as CD80 and TNFα. These observations suggest that delivery of a tumor-specific IgE may alter the cellular and immunological dynamics in the TME, ultimately leading to tumor regression [13]. In vitro and in vivo functional studies of MOv18 IgE targeting FRα formed the basis for clinical investigation in a first-in-class, first-in-human clinical trial in patients with advanced solid cancers. Further dissection of the molecular interactions between macrophages and IgE in cancer may provide a promising avenue for the development of a new therapeutic strategy for solid tumors (see Outstanding Questions). These observations strengthen the case for the therapeutic potential of antitumor IgE, which seems able not only to mediate killing of cancer cells by ADCC, but also to play a role in re-education of TAMs from an anti-inflammatory profile to an M1-like phenotype. Observations of the TNFα/MCP-1 axis as an IgE-specific cytokine profile in a human context await the clinical development of IgE therapy in patients with cancer. Collectively, despite the frequent tumor-promoting properties of TAMs, new avenues are emerging for their engagement and activation by IgE to induce effective immune surveillance against cancer. Acknowledgments The authors acknowledge support by Cancer Research UK, United Kingdom (C30122/A11527; C30122/A15774); the Medical Research Council, United Kingdom (MR/L023091/1); the Academy of Medical Sciences, United Kingdom; The Inman Charity, United Kingdom; Breast Cancer Now, United Kingdom (147), working in partnership with Walk the Walk; CR UK//NIHR in England/DoH for Scotland, Wales, and Northern Ireland Experimental Cancer Medicine Centre, United Kingdom (C10355/A15587); and the Cancer Research UK King’s Health Partners Centre at King’s College London. J.G. was supported by a grant of the Austrian Science Fund, Austria, CCHD, grant number W1205-B09 (to E.J.J.). The authors also acknowledge support by the European Academy of Allergy and Clinical Immunology (EAACI) (AllergoOncology Task Force). The research was supported by the National Institute for Health Research (NIHR) Biomedical Research Centre (BRC) based at Guy's and St Thomas' NHS Foundation Trust and King's College London, United Kingdom (IS-BRC1215-20006). The authors are solely responsible for study design, data collection, analysis, decision to publish, and preparation of the manuscript. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.
Disclaimer Statement S.N.K. and J.F.S. are founders and shareholders of IGEM Therapeutics Ltd. S.N.K. holds a patent on antitumor IgE antibodies. H.J.B. is funded through a grant by IGEM Therapeutics Ltd. The other authors declare no conflicts of interest.
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