Anti-folate receptor-α IgE but not IgG recruits macrophages to attack tumors via TNF-α/MCP-1 signaling

IgE antibodies are key mediators of anti-parasitic immune responses, but their potential for cancer treatment via antibody-directed 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 monoclonal antibodies 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 intra-tumoral 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 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 anti-tumor 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.


Introduction
Engagement of tumor-specific monoclonal antibodies via their Fc receptors contributes significantly to the anti-tumor effects of the immune system (1).Focusing effector cells such as monocytes/macrophages and natural killer (NK) cells against cancer-associated components may contribute to the functions of therapeutic antibodies such as trastuzumab, cetuximab and the checkpoint inhibitor ipilimumab (2,3).Antibody engineering strategies to optimize antibody-effector cell interactions and to direct these cells against tumors, may therefore improve therapeutic efficacy (4,5).
One strategy to influence these interactions is the exploration of changes to the structure of antibody Fc regions.The IgE immunoglobulin class is characterized by high affinity for cognate interaction with Fcε receptors (100-10,000 times higher than that of IgG for FcγR) on distinct, often tumor-resident, effector cells such as monocytes/macrophages (6,7).Although IgE antibodies play pathogenic roles in allergic inflammation by triggering mast cell degranulation and promoting eosinophil inflammation, they also contribute to the host immune defense against parasitic infections.The potential of IgE to induce inflammatory responses at tumor sites may be harnessed through IgE receptor-expressing effector cells such as monocytes and macrophages in tumors.Strategies to implement this approach include recombinant tumor-associated antigen (TAA)-specific IgEs, and active immunotherapy triggering adaptive IgE responses against cancer (8)(9)(10)(11)(12).
Folate receptor alpha (FRα) is overexpressed by several solid tumors, most significantly by epithelial ovarian carcinomas (13), and is a desirable target for TAA-specific IgE due to overexpression in tumors, and no/low expression and restricted distribution in normal tissues.Additionally, evidence of negative associations between allergies and reduced risk of gynaecological malignancies is reported (14), while little is known about IgE immunity against ovarian carcinoma antigens in patients (15).The chimeric (mouse V/human C) IgE antibody hMOv18 IgE, specific for FRα (16,17), effected superior tumor cell cytotoxicity and improved survival compared with IgG1 of equivalent specificity (18)(19)(20)(21).Potential roles of monocytes/macrophages were suggested by loss of IgE-conferred survival advantage following monocyte depletion of human peripheral blood mononuclear cells (PBMCs) introduced with hMOv18 IgE (20).Monocyte-mediated tumor killing was demonstrated through both known IgE receptors: antibody-dependent cell-mediated cytotoxicity (ADCC) via the high affinity FcεRI, and phagocytosis (ADCP) via the low affinity FcεRII (CD23).Since inflammatory infiltrates of many tumors contain macrophages, re-polarizing these against cancer may constitute an important rationale for developing IgE cancer immunotherapy (22).To-date however, the capacity of IgE to recruit macrophages against cancer in an immunocompetent tumor-bearing setting has not been demonstrated and the mechanisms by which IgE may activate these cells against cancer remain unclear.IgE can rapidly mediate parasite neutralization by FcεR-expressing cells including human macrophages (23,24).Although TNFα, IL-10 and nitric oxide (NO) have been individually reported in these processes (23)(24)(25), the mechanisms engendered through cross-talk between immune cells, IgE antibodies and target cell antigens, including parasite or tumor antigens, have not been elucidated.
Lack of cross-reactivity of human IgE with murine FcεRs and absence of trimeric FcεRI on murine monocytes/macrophages, eosinophils and other subsets have provided challenges for the design of immunologically-relevant models with which to study IgE class antibody functions.Previous immunodeficient mouse models, some reconstituted with human immune cells to provide IgE effector cells, were limited by short lifespans of human effector cells and incomplete representation of human immunity.Additionally, certain human effector cell-secreted cytokines may not interact with the murine immune system.
We investigated whether MOv18 IgE can inhibit tumor progression by recruiting and polarizing macrophages.We constructed a syngeneic rat model of FRα-expressing adenocarcinoma designed to better recapitulate the human IgE-Fcε receptor system and the patient setting.In this model, immune cells are found in their natural anatomical locations, immune cell FcεRI expression and distribution in rats mirrors that of humans, and rat effector cells (e.g.monocytes/macrophages) express trimeric FcεRI (αγ2) (26).We generated anti-FRα IgE and IgG with rat Fc sequences (rMOv18 IgE/IgG2b) to examine alongside antibodies with human Fc (hMOv18 IgE/IgG1).We assessed antibody efficacy and IgE-mediated tissue macrophage migration and activation in vivo, and TNFα and MCP-1 in tumor environments.We evaluated relevance in the human system and dissected the conditions that promote TNFα and MCP-1, by IgE-mediated human monocyte activation.Our findings support the superior therapeutic efficacy of IgE over IgG, and identify a previously-unappreciated tumor antigen-specific IgE-potentiated axis that promotes effector cell polarization and recruitment towards tumor cells.

Human Samples and Ethics
Blood and tumor specimens were collected from 6 ovarian carcinoma patients (Supplementary Table S1) and blood drawn from 13 healthy volunteers (Supplementary Table S2), with informed written consent, in accordance with the Helsinki Declaration.Study design was approved by the Guy's Research Ethics Committee, Guy's and St. Thomas' NHS Foundation Trust.

Production of anti-FRα antibodies with human and rat Fc regions
Chimeric mouse/human antibodies MOv18 IgE and IgG1 recognizing human FRα were engineered as before (18).Chimeric mouse/rat MOv18 IgE and IgG2b antibodies were designed with rat constant and mouse variable domains specific for human FRα (Supplementary Materials and Methods).

Tumor cell cytotoxicity and phagocytosis (ADCC/ADCP) assays
Antibody-dependent cell-mediated killing of FRα-expressing tumor cells was quantified by adapting a previously-described flow cytometric method (Supplementary Materials and Methods).

Assessments of antibodies in vivo
Immunocompetent syngeneic WAG rat model of FRα-expressing lung metastases-Female Wistar Albino Glaxo (WAG/RijCrl) rats (Charles River) were maintained and handled in accordance with the Institutional Committees on Animal Welfare of the UK Home Office (The Home Office Animals Scientific Procedures Act, 1986).Rats were injected i.v. with 4x10 6 CC531tFR tumor cells and subsequently treated with MOv18 antibodies (days 1 and 14 or 1, 7, 14 and 21).Lung tumor burden was determined 26 days following CC531tFR inoculation by: mean number of surface-visible metastases/cm 2 ; and % tumor occupancy [total white surface area (mm 2 ) / total lung (black + white) surface area (mm 2 )].

Isolation of rat effector cells from peripheral blood and lungs
Rat primary monocytes were prepared from rat peripheral blood leukocytes (PBL) by flow cytometry cell sorting using a PE-conjugated antibody against CD172 (BD Biosciences) (Supplementary Materials and Methods).

Flow cytometric evaluations of freshly-isolated tumor-infiltrating macrophages
Phenotypic analysis of tumor-infiltrating macrophages from single cell suspensions of tumor-bearing rat lungs was performed with directly-labeled monoclonal antibodies (Supplementary Materials and Methods).

Criteria for evaluating immune cell infiltration of tumors
H&E-stained sections were used to determine the tumor immune cellular infiltrate as a proportion of total tumor areas (Supplementary Materials and Methods).The percentage of tumor occupied by immune cells in each section was derived as follows: % immune cell occupancy = total area occupied by immune cells/total tumor area (Supplementary Fig. S1A).

Evaluations of macrophage infiltration into tumors
Sections double-stained for FRα (AF488, green) and CD68 (AF555, red) were used to determine ratios of within-tumor:peripheral CD68 + cells/mm 2 .CD68 + cells and the area covered by tumor (defined by tissue morphology, density of DAPI staining and FRα staining; Supplementary Fig. S1B) and periphery was calculated.(Supplementary Materials and Methods).

Quantitative real-time-PCR analysis of TNFα and MCP-1 expression by tumor cells and monocytes
Cells harvested from ADCC and ex vivo stimulation assays were studied for MCP-1 and TNFα relative gene expression.Cells from ADCC assays were CD89-PE-and FRα-FITClabeled and sorted using a FACSAria II Cell Sorter (BD Biosciences).Sorted cells and cells from ex vivo stimulation experiments (Supplementary Materials and Methods) were resuspended in RLT buffer for RNA isolation by RNeasy Kit (Qiagen).

Chemotaxis assay
To analyze the chemotactic properties of MCP-1 on THP-1 cells and human primary monocytes, a chemotaxis assay was performed using Transwell® plates with a polycarbonate membrane insert and 5μm pore size (Costar®) (Supplementary Materials and Methods).

Statistical methods and analyses of publically-available databases
All statistical analyses (Supplementary Materials and Methods) were performed using GraphPad™ Prism software (version 5.03, GraphPad™).P values are represented as Clinical associations of tumor gene expression were assessed using publicly-available data (31), in Kaplan-Meier Plotter (http:/kmplot.com/analysis/)(Supplementary Materials and Methods).

Rat and human IgE and IgG display comparable in vitro properties
We generated surrogate rMOv18 IgE and IgG2b antibodies (equivalent to mouse IgG2a/b and human IgG1, based on their complement-fixing and ADCC functions) recognizing human FRα in order to construct a syngeneic rat model system.Human FRα-expressing WAG rat syngeneic adenocarcinoma CC531tFR cells served as targets.Rat peripheral blood monocytes (CD172+) expressed FcεRI and bound rMOv18 IgE (Figs. 1A-B).rMOv18 IgE bound to rat FcεRI (αβγ2)-expressing rat RBL-2H3 mast cells.rMOv18 IgG2b bound primary rat monocytes (expressing FcγRI), and RBL-2H3 cells (expressing low levels of rFcγRII/FcγRIII).Rat IgE and IgG2b bound to CC531tFR but not to FRα-negative A375 melanoma cells (Fig. 1B).
Rat primary monocytes activated ex vivo with rMOv18 IgE, and rMOv18 IgG2b induced significant CC531tFR cell death compared with isotype control antibodies and equivalent to levels of IGROV1 ovarian tumor cell death triggered by human antibodies (Fig. 1C).Furthermore, monocytic cells stimulated with IL-4 to express the low affinity IgE receptor CD23/FcεRII, and primed with rMOv18 IgE, triggered significant CC531tFR death by ADCP over control antibody, equivalent to levels of ADCP triggered by hMOv18 IgE (Fig. 1D).

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This suggests that rMOv18 antibodies are functionally analogous to their human counterparts, at least with regard to binding FcεR+ and FRα+ cells, and their potentiation of particular effector functions.

Anti-tumor activities of rat MOv18 IgE and IgG2b in vivo
We next examined the in vivo functions of rMOv18 IgE and IgG2b in an immunocompetent rat model of FRα+ CC531tFR lung metastases, designed to better recapitulate the spectrum and functions of human IgE effector cells and the patient setting (Supplementary Fig. S2).
Therefore, rMOv18 IgE and IgG2b were functionally active in vivo.Rat MOv18 IgE at 3mg/kg weekly and 10mg/kg biweekly doses effected superior tumor growth restriction compared with IgG2b.

MOv18 IgE treatment is associated with rat macrophage infiltration into tumors
We next investigated spontaneous, endogenous macrophage infiltration into tumors in immunocompetent rats.Histological evaluation of rat lungs showed reduced tumor islet density and increased glandular organization in rMOv18 IgE-compared with rMOv18 IgG2b-treated cohorts (Fig. 3A).Tumor areas occupied by immune and stromal cell infiltration were significantly greater with rMOv18 IgE compared to PBS (P<0.0001) (Fig. 3B).We observed superior rat macrophage infiltration into tumors of rMOv18 IgE-treated rats.CD68+ macrophage clusters (red) surrounded sparse FRα+ (green) tumor islets.Macrophage density within tumor islets was higher in rMOv18 IgE-and rMOv18 IgG2btreated rats compared with PBS (P=0.007) (Fig. 3C).CD68+ cell ratios within tumor islets:tumor periphery were significantly higher with rMOv18 IgE (rMOv18 IgG2b, P=0.03; PBS, P=0.003) (Fig. 3C, Supplementary Fig. S1).Additionally the intensity of macrophage infiltration correlated inversely with tumor occupancy in animals treated with rMOv18 IgE, and rMOv18 IgG2b but not PBS (Fig. 3D).Analyses of patient-derived ovarian carcinoma xenografts, where hMOv18 IgE introduced with human PBMCs prolonged survival of tumor-challenged mice compared to controls, showed that human CD68+ macrophage infiltration into tumors correlated with prolonged survival of hMOv18 IgE-treated mice (r=0.67,P=0.009) (Fig. 3E) (19).Furthermore, in a subcutaneous IGROV1 human ovarian cancer xenograft in immunodeficient mice, significant tumor growth was observed in mice given PBS (P=0.0054),hMOv18 IgE alone (P=0.049) or human monocyte-enriched PBMCs (P=0.0014), but tumor growth was restricted in mice given hMOv18 IgE plus monocyteenriched cells (Fig. 3F).These findings suggest that the anti-tumor effects of MOv18 IgE in vivo, at least partly reflect immune effector functions of the antibody and enhanced macrophage influx into the tumor mass.

Macrophage polarization and elevated TNFα, MCP-1 and IL-10 in lungs of IgE-treated rats
Since antibody treatments were associated with enhanced macrophage infiltration into tumors, we investigated whether IgE was associated with polarization or maturation of tissue-resident macrophages.Consistent with immunohistochemical evaluations (Fig. 3), the mean percentages of freshly-isolated lung CD68+ macrophages within the total CD45+ leukocyte populations (Supplementary Fig. S3) were elevated in rats treated with rMOv18 IgE compared with IgG2b and PBS (Fig. 4A).Macrophages from rMOv18 IgE-treated rats demonstrated enhanced expression of the co-stimulatory molecule CD80, compared with those from rMOv18 IgG2b-or PBS-treated animals (P=0.01).No differences in the alternatively activated (M2) scavenger receptor CD163 (33) expression were found between treatment groups (Figs.4B-C).

Cross-linking of cell surface-bound IgE triggers elevated TNFα by monocytes, and TNFα stimulates MCP-1 by monocytes and tumor cells
We sought to determine whether rMOv18 IgE treatment-associated macrophage activation is relevant in a human setting, and gain insights into how tumor antigen-specific IgE may trigger effector cell activation.Cross-linking of IgE antibodies of different antigen specificities (anti-FRα MOv18, anti-hapten NIP IgE, anti-HER2 against the breast cancer tumor antigen HER2/neu and CSPG4 IgE, recognizing the melanoma tumor associated antigen Chondroitin Sulfate Proteoglycan 4 (34,35)) on monocytes with polyclonal anti-IgE antibody (to mimic engagement by TAA-expressing tumor cells) significantly increased production of mRNA encoding TNFα by human monocytes, compared to IgE alone.Crosslinking of equivalent IgG1s did not trigger TNFα, nor did cross-linking of IgEs on human ovarian IGROV1 tumor cells (Fig. 5A).

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We then investigated whether IgE Fc-mediated TNFα upregulation can promote MCP-1 secretion and therefore, putatively, monocyte mobilization.TNFα stimulation of human monocytic U937 cells and primary human monocytes triggered elevated MCP-1 mRNA expression compared with unstimulated cells (Fig. 5B left; P<0.0001), along with elevated MCP-1 protein secretion compared with unstimulated cells (Fig. 5B right; U937 P=0.002; primary human monocytes P=0.0116).
Therefore, TNFα upregulation, triggered by cross-linking of receptor-bound IgE, but not IgG, on the surface of monocytes, may induce MCP-1 production by human monocytes and a range of tumor cell types, putatively promoting further monocyte chemotaxis and potential accumulation in tumors.These data suggest that IgE-mediated monocyte activation and TNFα upregulation may be relevant in the human setting.

TNFα and MCP-1 are involved in antigen-specific IgE effector functions and may be associated with better patient survival
We investigated whether TNFα/MCP-1 upregulation could be triggered by specific tumor antigen recognition and tumor cell cytotoxicity by IgE in vitro.Human monocytes with hMOv18 IgE induced significantly-elevated IGROV1 cell ADCC compared with nonspecific NIP IgE controls (Fig. 6A).hMOv18 IgE treatment was associated with significantly-elevated expression (relative to control IgE) of the same mediators found in lung extracts of IgE-treated rats in vivo (MCP-1, P=0.002; TNFα, P=0.019; IL-10, P=0.025), confirming their importance in IgE stimulation (Fig. 6B).
In ADCC assays, MCP-1 secretion with hMOv18 IgE was significantly reduced (P=0.004)by TNFα receptor-blocking antibodies (Fig. 6C), and tumor-specific hMOv18 IgE, but not the non-specific anti-NIP IgE, triggered upregulated MCP-1 mRNA expression by IGROV1 tumor cells.This effect was abrogated when TNFα signaling was blocked prior to stimulation with antibody (P=0.032;Fig. 6D).Therefore, MCP-1 production in the context of tumor antigen-specific IgE cytotoxicity is TNFα-dependent.
To gain insights into a potential clinical relevance of the TNFα/MCP-1 axis, we interrogated publicly-available ovarian carcinoma gene expression datasets (Fig. 6E).We found significant associations of improved 5-year overall survival with elevated levels of our herein reported IgE-mediated immune signatures (TNFα/MCP-1, P=0.016; TNFα/MCP-1/IL-10, P=0.022).These signatures were still associated with better overall survival alongside the macrophage marker CD68 (P=0.04) and with the high-and low-affinity IgE Fc receptors (FcεRI, P=0.041; FcεRII/CD23, P=0.035).These findings may suggest that, if enhanced, the TNFα/MCP-1 axis may provide a responsive immune signature with protective potential.
In summary, within tumors, cross-linking of MOv18 IgE on macrophages by FRαexpressing tumor cells induces macrophage TNFα upregulation.TNFα promotes elevated MCP-1 production by both tumor cells and macrophages, putatively acting as a potent chemoattractant, drawing macrophages into tumors.Enhanced tumor cell-macrophage interactions further stimulate production of TNFα and thus MCP-1, forming a selfenhancing circuit of macrophage influx into MOv18 IgE-treated tumors (Fig. 6F).This supports a link between TNFα stimulation and MCP-1 expression in response to effector cell engagement and IgE cytotoxic functions against cancer.

MOv18 IgE triggers anti-tumor ADCC by ovarian cancer patient effector cells
We evaluated the capacity of hMOv18 IgE to trigger anti-tumor ADCC by activating human immune effector cells from patients with ovarian carcinomas.PBMCs from patients with FRα-positive, FRα-negative and FRα-status unknown ovarian carcinomas (by immunohistochemistry, Fig. 7A), together with hMOv18 IgE, induced significantly-elevated ovarian carcinoma IGROV1 ADCC compared with non-specific NIP IgE-treated cells (Fig. 7B), and equivalent to that mediated by healthy volunteer PBMCs (Fig. 7C).Additionally, hMOv18 IgE mediated anti-tumor ADCC against 3 FRα-expressing tumor cell lines (human ovarian carcinoma IGROV1 and SKOV3, and rat colon adenocarcinoma CC531tFR), but minimum ADCC against FRα-dim and FRα-negative cancer cell lines (human ovarian carcinoma TOV21G, and human breast carcinoma SKBR3 respectively, Fig. 7D, Supplementary Fig. S4).Therefore, IgE-engendered anti-tumor functions are target antigenspecific, and have potential application with patient immune effector cells and against different target-expressing tumor cells.

Discussion
We report the superior anti-tumor efficacy of TAA-specific IgE in an immunocompetent syngeneic rat model of cancer, uniquely suitable for the study of IgE anti-tumor effector functions.We furthermore describe a previously-unappreciated putative contribution of a TNFα/MCP-1 cascade to monocyte and macrophage repolarization and recruitment, delineated in the human IgE and human effector and tumor cell functional context.
The functional significance of this IgE-induced TNFα/MCP-1 axis is supported by: a) enhanced intra-tumor infiltration by macrophages following IgE treatments; b) a TNFαexpressing lung macrophage compartment, and elevated TNFα and MCP-1 concentrations in BAL from IgE-but not IgG-treated rats; c) macrophage recruitment correlating with tumor growth restriction in IgE-treated syngeneic rat and human xenograft mouse models of cancer; d) the ability of human IgE to trigger monocytes to upregulate TNFα in a classspecific manner; e) the induction of MCP-1 production by TNFα in monocytes and tumor cells, in turn shown to promote a monocyte chemotactic response, f) the involvement of TNFα/MCP-1 in antigen-specific human IgE tumor ADCC, abrogated with TNFα receptorspecific blockade on monocyte effector cells and, g) publicly-available ovarian carcinoma gene expression datasets indicating associations between elevated levels of TNFα/MCP-1 with better survival.These findings reveal an immune cascade through which TAA-specific IgE may focus macrophages towards tumor cells.
Rat and human MOv18 IgE showed comparable monocyte-mediated tumor cell killing.In immunocompetent rats, rMOv18 IgE demonstrated potential to compete favorably with IgG in a metastatic setting, and to restrict tumors in highly-vascularized organs like the lung, where IgG (based on its long half-life in the circulation) would have a major advantage.Rat and human MOv18 IgE treatment-associated macrophage infiltration into tumors correlated with tumor growth restriction and better efficacy in the immunocompetent rat and human xenograft mouse models.This supports a function of recruiting macrophages towards tumor cells associated with IgE therapy in vivo.Stromal macrophages can express matrixdegrading, matrix-producing and pro-angiogenic factors (36), allowing regulation of stromal remodeling and neo-vascularization to support tumors.Conversely, macrophage density within tumor islets has been positively associated with patient survival (37,38), and tumor islet-resident macrophages can express IL-1α, IL-1β, IL-6, NOS and TNFα, the latter two thought to be involved in target cell-killing mechanisms of macrophages (39,40).These mechanisms may fail to be effectively deployed against tumors, perhaps partly due to alternatively-polarized humoral immunity in cancers favoring antibody isotypes that fail to activate key effector cells such as macrophages (41)(42)(43)(44)(45).Here we show that tissue-resident macrophage subsets in IgE-treated cohorts are polarized to express the macrophage maturation and co-stimulatory marker CD80, as well as higher levels of pro-inflammatory TNFα.TNFα levels were also enhanced in BAL fluids of IgE-treated but not of IgG-treated rats.TNFα can also be expressed by activated macrophages known to act as effector cells in IgE-mediated parasite control (23)(24)(25).Our findings now directly link tumor-immune cell interactions engendered by IgE with potentiating TNFα production, re-education and recruitment of monocytes and macrophages into tumors.These functions may be further explored in future studies that may include effector cell depletion or enrichment with different polarized phenotypes.Based on expression of TNFα, but not IL-4 in vitro and in vivo in the context of tumor antigen-specific IgE, our findings also suggest that macrophage activatory, but most likely not allergic, mechanisms might be employed by TAA-specific IgE to restrict the growth of tumor metastases.IL-4 has been shown to be a negative factor in IgE/FcεRI cross-presentation by DCs and generation of cytotoxic T lymphocytes (12).It is therefore possible that during Th2-type inflammation, IL-4 may prevent a TNFα/MCP-1 axis-driven macrophage response.
The CC chemokine, M1 macrophage mediator and potent chemoattractant MCP-1 was also elevated in BAL fluid of rMOv18 IgE-treated rats.Cross-linking of macrophage-bound IgE by densely-expressed antigens on the surface of target cells, more likely to occur in tumor lesions, can trigger TNFα, which in turn may stimulate MCP-1.Our findings suggest that cross-linking on the monocyte surface by IgE, but not by IgG, may initiate this TNFα response.Monocytes and cancer cells respond to TNFα signals by producing MCP-1, consistent with increased recruitment of macrophages into tumors following rMOv18 IgE treatment.The high affinity of IgE for FcεRs on tissue-resident monocytes and macrophages may result in sustained antibody retention, thus favoring cross-linking of IgE by multivalent TAAs.This class-specific immune complex formation may potentiate TNFα production in tumor microenvironments and subsequent sustained in situ MCP-1 secretion by different cell subsets, providing an advantage for IgE immunotherapy against cancer.

Josephs et al. Page 11 Cancer
Res. Author manuscript; available in PMC 2018 October 05.Europe PMC Funders Author Manuscripts Europe PMC Funders Author ManuscriptsIn human IgE, human monocyte and human tumor cell functional studies, TNFα stimulated tumor cells to upregulate MCP-1, which can trigger monocyte chemotaxis.We confirmed the relevance of both TNFα and MCP-1 in ADCC assays, where cross-linked IgE on the surface of human monocytes upregulated TNFα and MCP-1 production.Loss of MCP-1 production by tumor cells, and significant reduction in IgE-dependent tumor cell cytotoxicity when TNFα signaling was blocked, also suggest that MCP-1 upregulation and IgE anti-tumor functions require TNFα.We and others also demonstrate that tumor cells can be stimulated to produce MCP-1(46)(47)(48)(49).Additionally, potential synergistic effects between MCP-1 and IgE-mediated anti-tumor mechanisms were shown with an anti-MUC1 IgE antibody co-administered with MCP-1, to MUC1-expressing tumor-bearing hFcεRIα transgenic mice(50).Together, our findings thus point to the putative involvement of a TNFα/MCP-1 cascade triggered by IgE-mediated cross-talk between effector cells and tumor cells, and implicated in tumor cell growth restriction in vitro and in vivo.Conversely, we observed elevated levels of IL-10 in rMOv18 IgE-treated rat lungs and in effector cell stimulation assays.In vitro infection of human macrophages with Toxoplasma gondii also upregulated IL-10, inversely correlating with iNOS and NO generation, and IL-10 was found to attenuate IgE-mediated parasite elimination by macrophages(25).IL-10 could therefore provide an in vivo mechanism for restricting IgE-induced effector cell activation, and requires further study.In summary, MOv18 IgE-potentiated tumor-restricting effects are superior to those of IgG in immunocompetent rats.IgE immune complex formation on monocytes and macrophages, initiated through cross-talk with TAA-expressing cancer cells, promotes effector cell polarization, activation and recruitment.This process is driven by TAA IgE-dependent TNFα and MCP-1 upregulation.Our findings draw parallels with physiological roles of IgE in anti-parasitic immune surveillance but not in allergy.Engineering antibodies with Fc regions that confer unique effector cell-polarizing properties against cancer may open a new avenue for addressing tumor resistance to immune clearance.

Figure 6 .
Figure 6.TNFα and MCP-1 are involved in IgE-mediated tumor killing and may be associated with better patient survival.(A) hMOv18 IgE effects significantly-elevated killing of human ovarian carcinoma IGROV1 cells by human U937 monocytes and primary monocytes in vitro, compared with nonspecific anti-NIP IgE controls (n=3).(B) Concentrations of cytokines/chemokines secreted in cultures of human primary monocytes incubated with IGROV1 cells and hMOv18 IgE, control antibody NIP IgE or no antibody.(C) MCP-1 secreted in cultures of U937 monocytes incubated with IGROV1 cells and hMOv18 IgE was reduced when cells were incubated with blocking antibodies against the human TNFα receptors I and II prior to
tumour:peripheral CD68+ cells (right panel, bottom) were calculated.IgE (n=14), IgG (n=17), PBS (n=24).(D) Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Cytokine/chemokine production measured in BAL fluid from tumor-bearing rats given rMOv18 IgE, rMOv18 IgG2b, or PBS (n=3 per group).(F) For selected analytes, fold change values represent mean fluorescence intensity (MFI) for each replicate within the treatment (IgE or IgG) groups, divided by that of the PBS group.Results represent 2 independent experiments.