Abstract
IgE antibodies are key mediators of antiparasitic immune responses, but their potential for cancer treatment via antibody-dependent cell-mediated cytotoxicity (ADCC) has been little studied. Recently, tumor antigen–specific IgEs were reported to restrict cancer cell growth by engaging high-affinity Fc receptors on monocytes and macrophages; however, the underlying therapeutic mechanisms were undefined and in vivo proof of concept was limited. Here, an immunocompetent rat model was designed to recapitulate the human IgE-Fcε receptor system for cancer studies. We also generated rat IgE and IgG mAbs specific for the folate receptor (FRα), which is expressed widely on human ovarian tumors, along with a syngeneic rat tumor model expressing human FRα. Compared with IgG, anti-FRα IgE reduced lung metastases. This effect was associated with increased intratumoral infiltration by TNFα+ and CD80+ macrophages plus elevated TNFα and the macrophage chemoattractant MCP-1 in lung bronchoalveolar lavage fluid. Increased levels of TNFα and MCP-1 correlated with IgE-mediated tumor cytotoxicity by human monocytes and with longer patient survival in clinical specimens of ovarian cancer. Monocytes responded to IgE but not IgG exposure by upregulating TNFα, which in turn induced MCP-1 production by monocytes and tumor cells to promote a monocyte chemotactic response. Conversely, blocking TNFα receptor signaling abrogated induction of MCP-1, implicating it in the antitumor effects of IgE. Overall, these findings show how antitumor IgE reprograms monocytes and macrophages in the tumor microenvironment, encouraging the clinical use of IgE antibody technology to attack cancer beyond the present exclusive reliance on IgG. Cancer Res; 77(5); 1127–41. ©2017 AACR.
Introduction
Engagement of tumor-specific mAbs via their Fc receptors contributes significantly to the antitumor 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 (8–12).
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. In addition, evidence of negative associations between allergies and reduced risk of gynecologic 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), affected superior tumor cell cytotoxicity and improved survival of tumor-bearing mice compared with IgG1 of equivalent specificity (18–21). Potential roles of monocytes/macrophages were suggested by loss of IgE-conferred survival advantage following monocyte depletion of human peripheral blood mononuclear cells (PBMC) 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). As inflammatory infiltrates of many tumors contain macrophages, repolarizing 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, IL10, and nitric oxide (NO) have been individually reported in these processes (23–25), 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. In addition, 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 anatomic 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; ref. 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 toward tumor cells.
Materials and Methods
Human samples and ethics
Blood and tumor specimens were collected from 6 ovarian carcinoma patients (Supplementary Table S1) and blood was 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.
Cell lines
The CC531tFR cell line, originally derived from a 1,2 dimethyhydrazine–induced colon adenocarcinoma of a WAG-Rij rat (Cell Lines Service; ref. 27), was transfected to express human FRα as described previously and selected on the basis of Geneticin resistance (S. Canevari, M. Colnaghi, Instituto Nazionale Tumori, Milan, Italy; refs. 28, 29). IGROV1 human ovarian carcinoma cells naturally overexpress human FRα (18, 30). A375 (human metastatic melanoma; CRL-11147), SKOV3 (human epithelial ovarian carcinoma, HTB-77), TOV21G (human ovarian clear cell carcinoma, CRL-11730), SKBR3 (human breast carcinoma HTB-30), U937 (human monocytic CRL-1593.2), and THP-1 (human monocytic; TIB 202) cells were from ATCC. FreeStyle 293-F cells (R790-07) were from Invitrogen (Supplementary Materials and Methods). Cell lines from ATCC were authenticated by short tandem repeat profiling. Routine mycoplasma testing was performed by PCR regularly on all cell lines.
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 Laboratories) 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 intravenously with 4 × 106 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/cm2; and % tumor occupancy [total white surface area (mm2)/total lung (black + white) surface area (mm2)].
Assessment of hMOv18 IgE in immunodeficient mouse models.
Patient-derived intraperitoneal human ovarian carcinoma xenografts in female nu/nu mice were described previously (19). Subcutaneous IGROV1 tumors were established in C.B-17 scid/scid (SCID) mice as before (Supplementary Materials and Methods; ref. 18).
Isolation of rat effector cells from peripheral blood and lungs
Rat primary monocytes were prepared from rat peripheral blood leukocytes 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 mAbs (Supplementary Materials and Methods).
Criteria for evaluating immune cell infiltration of tumors
Hematoxylin and eosin–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: percent 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/mm2. 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).
qRT-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-FITC–labeled 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 5-μm pore size (Costar; Supplementary Materials and Methods).
Statistical methods and analyses of publicly available databases
All statistical analyses (Supplementary Materials and Methods) were performed using GraphPad Prism software (version 5.03, GraphPad). P values are represented as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Error bars represent SD and SEM in in vitro figures, and in in vivo figures and histologic analyses, respectively. 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).
Results
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 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 (Fig. 1A and 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− A375 melanoma cells (Fig. 1B).
Rat and human MOv18 antibodies display comparable effector functions. A, Anti-rat FcεRI antibody or rMOv18 IgE binding to primary (CD172+) rat monocytes. B, rMOv18 IgE and IgG2b binding to rat primary monocytes, RBL-2H3 rat mast cells, FRα-expressing rat colon adenocarcinoma CC531tFR cells, and non-FRα–expressing human melanoma A375 cells (gray, nonspecific isotype control or detection antibody control). C, Quantification of ADCC against FRα-expressing tumor (rat CC531tFR and human ovarian carcinoma IGROV1) cells by rat or human MOv18 IgE- or IgG-primed primary monocytes. D, Quantification of ADCC and ADCP against FRα-expressing CC531tFR tumor cells by rat or human MOv18 IgE-primed unstimulated (CD23−) or IL4-stimulated (CD23+) monocytic cells, compared with nonspecific antibody treatments or cells alone (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
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 IL4 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).
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.
Antitumor 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).
Immunofluorescent staining confirmed in situ FRα+ CC531tFR metastases (Fig. 2A). Dose-dependent inhibition of tumors between 5, 10, and 50 mg/kg doses (% tumor occupancy of lungs) was observed with rMOv18 IgE (biweekly; Fig. 2B). Lung metastases and tumor occupancy were significantly lower with rMOv18 IgE compared with rMOv18 IgG2b (P < 0.0001) or PBS (P < 0.0001; Fig. 2C–E) at 10 mg/kg doses. IgE and IgG2b significantly reduced lung metastases at 5 mg/kg compared with PBS (P < 0.0001). Given the longer serum half-life of IgG (14–25 days) compared with IgE (1–2 days; ref. 32), we investigated whether dosing frequency could influence efficacy. rMOv18 IgE dosed weekly induced significantly reduced tumor occupancy at doses as low as 1 mg/kg compared with PBS (P < 0.0001), with a dose-dependent response between 3, 10 (P = 0.0002), and 50 mg/kg (P < 0.001; Fig. 2F). Weekly 3 mg/kg rMOv18 IgE afforded significantly (P = 0.04) superior tumor growth restriction compared with IgG2b (Fig. 2G).
Rat MOv18 IgE demonstrates superior tumor growth restriction. A, FRα expression in vivo 26 days post-tumor challenge. Representative immunofluorescent microscopy images from lungs of tumor-bearing and healthy rats (n = 5). B, Percentage (%) tumor occupancy quantified following two biweekly (Q2W) doses of PBS (n = 20), or 1 (n = 3), 3 (n = 2), 5 (n = 11), 10 (n = 11), and 50 (n = 2) mg/kg rMOv18 IgE. C and E, Number of metastases/cm2 and percent tumor occupancy quantified for PBS-treated rats (n = 22) and rats treated with rMOv18 IgE (n = 11) and IgG2b (n = 11) at 10 and 5 mg/kg biweekly. D, Representative images of Indian ink-stained lungs from rMOv18 IgE-, IgG2b- (10 mg/kg), and PBS-treated rats. F, Percentage tumor occupancy quantified following weekly doses of rMOv18 IgE (n = 10 per dose) or PBS (n = 17; mean of 1–3 independent experiments for B, C, E, and F). G, Number of metastases/cm2 and percent tumor occupancy quantified for rats treated weekly with PBS or with rMOv18 IgE or IgG2b at 3 mg/kg. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Therefore, rMOv18 IgE and IgG2b were functionally active in vivo. Rat MOv18 IgE at 3 mg/kg weekly and 10 mg/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. Histologic 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 with 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 IgG2b-treated 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). In addition, 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 with 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; ref. 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 monocyte-enriched cells (Fig. 3F). These findings suggest that the antitumor effects of MOv18 IgE in vivo at least partly reflect immune effector functions of the antibody and enhanced macrophage influx into the tumor mass.
MOv18 IgE treatment induces CD68+ macrophage infiltration into tumors. A and B, Paraffin-embedded lung sections from PBS-, rMOv18 IgG-, and rMOv18 IgE-treated rats stained with hematoxylin and eosin (A) and percentages of immune cell infiltration into tumor foci quantified (B; demarcation strategy, Supplementary Fig. S1; 5 data points/animal, 5 animals/group). Magnification, ×200. White arrow, glandular tumor islet; black arrow, immune and stromal cells. C, Representative cryosections of lung from PBS-, rMOv18 IgG-, and rMOv18 IgE-treated rats costained with anti-human FRα (green) and anti-rat CD68 (red) antibodies. Intratumoral CD68+ cell density/mm2 (right, top) and ratio of tumor:peripheral CD68+ cells (right, bottom) were calculated. IgE (n = 14), IgG (n = 17), PBS (n = 24). D, Correlation of within-tumor CD68+ macrophage density with percent tumor occupancy in rats treated with PBS (n = 9), rMOv18 IgE (n = 13), and rMOv18 IgG2b (n = 9). E, Correlation of within-tumor CD68+ macrophage density with survival of nude mice with intraperitoneal human ovarian carcinoma xenografts, treated with human PBMC and hMOv18 IgE (n = 13). F, Inhibition of subcutaneous IGROV1 tumor growth in SCID mice treated with PBS (n = 4), hMOv18 IgE (n = 4), monocyte-enriched effector cells (n = 5), or monocyte-enriched effector cells plus hMOv18 IgE (n = 5). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
Macrophage polarization and elevated TNFα, MCP-1, and IL10 in lungs of IgE-treated rats
As 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 costimulatory 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 (Fig. 4B and C).
Macrophages infiltrating tumors in IgE-treated rats are differentially polarized. A, Single-cell suspensions, isolated from lungs of rats inoculated with CC531tFR tumor cells 26 days previously, were stained with anti-CD45 and anti-CD68 antibodies. Infiltrating CD68+ macrophages were identified following successive gating on FSChiSSClow-hi and CD45+ populations (Supplementary Fig. S3), and the percentage of CD68+ cells in the CD45+ populations was calculated (right). B and C, Percentages of lung CD68+ macrophages expressing CD80, CD163, TNFα, and IL10 (three independent experiments for A–C). D, Percentages of single- and double-positive (TNFα+/IL10+) cells within the infiltrating CD68+ macrophage populations (n = 3). E, 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 two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant.
A higher proportion of CD68+ macrophages from rMOv18 IgE-treated rats expressed intracellular TNFα, compared with rMOv18 IgG2b- and PBS-treated cohorts (P = 0.04; Fig. 4B and C). In addition, a higher proportion of these cells also expressed intracellular IL10 with rMOv18 IgE treatment, compared with rMOv18 IgG2b- (P = 0.04) and PBS-treated cohorts (P = 0.04). A proportion of macrophages from rMOv18 IgE-treated rats simultaneously expressed TNFα and IL10 (Fig. 4D). In conjunction (Fig. 4E and F), we found significantly elevated TNFα, MCP-1, and IL10 secreted in bronchoalveolar lavage (BAL) fluids of IgE-treated rats compared with IgG (IL10, P = 0.044; TNFα, P = 0.017; MCP-1, P < 0.001). Levels of IFNγ, IL1α, IL1β, IL2, IL4, IL5, IL6, IL8, and IL12 did not differ significantly between treatment groups (Fig. 4E).
These data demonstrate TNFα-expressing mature macrophage subsets in rMOv18 IgE-treated rat lungs, along with a significantly elevated secreted TNFα/MCP-1/IL10 IgE-associated BAL profile.
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; refs. 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 with IgE alone. Cross-linking of equivalent IgG1s did not trigger TNFα, nor did cross-linking of IgEs on human ovarian IGROV1 tumor cells (Fig. 5A).
IgE cross-linked on monocytes triggers TNFα, which stimulates MCP-1. A, Comparative real-time PCR analysis of TNFα expression by U937 monocytes (left) or FRα+ human ovarian carcinoma IGROV1 tumor cells (right) following cross-linking of human IgEs and IgGs of different antigen specificities by polyclonal antibodies for 1 hour (n = 4). B and C, Effects of TNFα, IL4, and IL10 (20 ng/mL) stimulation on relative mRNA expression and secretion of MCP-1 by U937 monocytes (B; 10-hour stimulation), human primary monocytes (B; 5-hour stimulation) and human ovarian carcinoma IGROV1, SKOV3, and TOV21G, human breast carcinoma SKBR3, and human melanoma A375 tumor cells (C; 10-hour stimulation). D, Quantitation of human primary monocytes (top left) or THP-1 human monocytic cells, (bottom left; representative images, right) migrating through a transwell membrane in response to increasing MCP-1 concentrations (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
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).
Stimulation of ovarian IGROV1, SKOV3 and TOV21G, breast SKBR3, and melanoma A375 cancer cells with TNFα induced significantly elevated MCP-1 mRNA expression and secretion compared with unstimulated cells (Fig. 5C). Furthermore, MCP-1 stimulation could affect human monocyte chemotaxis (human primary monocytes, THP-1 monocytes) in a concentration-dependent manner (Fig. 5D).
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; IL10, P = 0.025), confirming their importance in IgE stimulation (Fig. 6B).
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 incubating with hMOv18 IgE (representative of two independent experiments). D, MCP-1 mRNA expression by IGROV1 cells was elevated following coincubation with U937 monocytes and hMOv18 IgE compared with controls (left), and reduced MCP-1 expression was detected when IGROV1 cells were incubated with blocking antibodies against the human TNFα receptors I and II prior to treatment with hMOv18 IgE (right). E, Kaplan–Meier curves showing higher TNFα/MCP-1, TNFα/MCP-1/IL10, TNFα/MCP-1/IL10+CD68, FcεRIα, or CD23 expression in 1,582 ovarian cancers is significantly associated with improved 5-year overall survival. Ovarian tumor CD68 expression alone does not correlate with patient outcome. F, Model summarizing a proposed mechanism of MOv18 IgE immunotherapy. A TNFα/MCP-1 axis promotes potent recruitment of further macrophages into tumors (1), resulting in enhanced tumor cell–macrophage interactions and subsequent tumor cell death (2). *, P < 0.05; **, P < 0.01.
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 nonspecific 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/IL10, 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 FRα-expressing 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 self-enhancing 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 antitumor ADCC by ovarian cancer patient effector cells
We evaluated the capacity of hMOv18 IgE to trigger antitumor ADCC by activating human immune effector cells from patients with ovarian carcinomas. PBMCs from patients with FRα+, FRα−, and FRα status unknown ovarian carcinomas (by IHC, Fig. 7A), together with hMOv18 IgE, induced significantly elevated ovarian carcinoma IGROV1 ADCC compared with nonspecific NIP IgE-treated cells (Fig. 7B), and equivalent to that mediated by healthy volunteer PBMCs (Fig. 7C). In addition, hMOv18 IgE mediated antitumor ADCC against three FRα-expressing tumor cell lines (human ovarian carcinoma IGROV1 and SKOV3 and rat colon adenocarcinoma CC531tFR), but minimum ADCC against FRα-dim and FRα− cancer cell lines (human ovarian carcinoma TOV21G and human breast carcinoma SKBR3, respectively, Fig. 7D; Supplementary Fig. S4). Therefore, IgE-engendered antitumor functions are target antigen specific and have potential application with patient immune effector cells and against different target-expressing tumor cells.
MOv18 IgE triggers ADCC of different FRα-expressing tumor cell lines by human effector cells from patients with ovarian cancer. A, Paraffin-embedded sections of tumors from patients with ovarian cancer were stained to detect patients with FRα+ (n = 3) and FRα− (n = 1) tumors. B and C, ADCC/ADCP of FRα-expressing human ovarian carcinoma IGROV1 cells by hMOv18 IgE-primed PBMCs from patients with FRα-positive (n = 3), FRα-negative (n = 1), and FRα-status unknown (n = 2) tumors (B) and from healthy volunteers (n = 2; C). D, hMOv18 IgE effects significantly elevated killing of FRα-bright human ovarian carcinoma IGROV1 (n = 3) and SKOV3 (n = 3), and rat colon adenocarcinoma CC531tFR (n = 2) tumor cells by monocytic cells, compared with nonspecific anti-NIP IgE. MFI, mean fluorescence intensity. Minimal killing of FRα-dim human ovarian carcinoma TOV21G (n = 3) and human breast carcinoma SKBR3 (n = 2) tumor cells by hMOv18 IgE was detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001. ns, not significant.
Discussion
We report the superior antitumor efficacy of TAA-specific IgE in an immunocompetent syngeneic rat model of cancer, uniquely suitable for the study of IgE antitumor 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: (i) enhanced intratumor infiltration by macrophages following IgE treatments; (ii) a TNFα-expressing lung macrophage compartment and elevated TNFα and MCP-1 concentrations in BAL from IgE- but not IgG-treated rats; (iii) macrophage recruitment correlating with tumor growth restriction in IgE-treated syngeneic rat and human xenograft mouse models of cancer; (iv) the ability of human IgE to trigger monocytes to upregulate TNFα in a class-specific manner; (v) the induction of MCP-1 production by TNFα in monocytes and tumor cells, in turn shown to promote a monocyte chemotactic response; (vi) the involvement of TNFα/MCP-1 in antigen-specific human IgE tumor ADCC, abrogated with TNFα receptor–specific blockade on monocyte effector cells; and (vii) 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 toward 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 toward tumor cells associated with IgE therapy in vivo. Stromal macrophages can express matrix-degrading, matrix-producing, and proangiogenic factors (36), allowing regulation of stromal remodeling and neovascularization 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 IL1α, IL1β, IL6, 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 (41–45). Here, we show that tissue-resident macrophage subsets in IgE-treated cohorts are polarized to express the macrophage maturation and costimulatory marker CD80, as well as higher levels of proinflammatory 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 (23–25). Our findings now directly link tumor–immune cell interactions engendered by IgE with potentiating TNFα production, reeducation, 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. On the basis of the expression of TNFα, but not IL4 in vitro and in vivo in the context of tumor antigen–specific IgE, our findings also suggest that macrophage-activating, but most likely not allergic, mechanisms might be employed by TAA-specific IgE to restrict the growth of tumor metastases. IL4 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, IL4 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.
In 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 suggests that MCP-1 upregulation and IgE antitumor functions require TNFα. We and others also demonstrate that tumor cells can be stimulated to produce MCP-1 (46–49). In addition, potential synergistic effects between MCP-1 and IgE-mediated antitumor mechanisms were shown with an anti-MUC1 IgE antibody coadministered 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 IL10 in rMOv18 IgE-treated rat lungs and in effector cell stimulation assays. In vitro infection of human macrophages with Toxoplasma gondii also upregulated IL10, inversely correlating with iNOS and NO generation, and IL10 was found to attenuate IgE-mediated parasite elimination by macrophages (25). IL10 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 physiologic roles of IgE in antiparasitic 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.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Disclaimer
The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health.
Authors' Contributions
Conception and design: D.H. Josephs, H.J. Bax, T. Dodev, E. Lever, C.J. Corrigan, F.O. Nestle, H.J. Gould, V. Sanz-Moreno, P.J. Blower, J.F. Spicer, S.N. Karagiannis
Development of methodology: D.H. Josephs, H.J. Bax, T. Dodev, L. Saul, K.M. Ilieva, N. Woodman, M. Bracher, H. Lentfer, E. Lever, H.J. Gould, V. Sanz-Moreno, J.F. Spicer, S.N. Karagiannis
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.H. Josephs, H.J. Bax, T. Dodev, M. Georgouli, M. Nakamura, G. Pellizzari, L. Saul, P. Karagiannis, A. Cheung, C. Herraiz, I. Correa, M. Fittall, P. Gazinska, N. Woodman, S. Mele, G. Chiaruttini, A.E. Gilbert, E. Lever, A. Montes, N. Downes, D. Dombrowicz, A.J. Beavil, S.N. Karagiannis
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.H. Josephs, H.J. Bax, M. Nakamura, A. Cheung, C. Herraiz, M. Fittall, S. Crescioli, G. Chiaruttini, A.E. Gilbert, A. Koers, E. Lever, G. Muirhead, S. Tsoka, N. Downes, C.J. Corrigan, F.O. Nestle, V. Sanz-Moreno, S.N. Karagiannis
Writing, review, and/or revision of the manuscript: D.H. Josephs, H.J. Bax, T. Dodev, P. Karagiannis, A.E. Gilbert, C. Barton, E. Lever, M. Figini, A. Montes, D. Dombrowicz, C.J. Corrigan, P.S. Jones, H.J. Gould, V. Sanz-Moreno, P.J. Blower, J.F. Spicer, S.N. Karagiannis
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.H. Josephs, T. Dodev, S.N. Karagiannis
Study supervision: D.H. Josephs, H. Lentfer, A.J. Beavil, J.F. Spicer, S.N. Karagiannis
Other (supply of antibody): C. Selkirk
Other (development of manufacturing methods for IgE and manufacturing of IgE material for the study): H. Lentfer
Other (generated and provided key reagents and provided expertise on the anti-folate receptor–specific moiety of the final chimeric antibody): S. Canevari, M. Figini
Other (conceived and designed the study, supervised and coordinated the project): S.N. Karagiannis
Grant Support
The authors acknowledge support by Cancer Research UK (C30122/A11527, C30122/A15774, C33043/A12065), The Academy of Medical Sciences, CRUK/EPSRC/MRC/NIHR KCL/UCL Comprehensive Cancer Imaging Centre (C1519/A10331), the Medical Research Council (MR/L023091/1), Breast Cancer Now (147), CRUK/NIHR in England/DoH for Scotland, Wales and Northern Ireland Experimental Cancer Medicine Centre (C10355/A15587), Royal Society (RG110591), and the Federation of European Biochemical Societies. This research was also supported by the National Institute for Health Research (NIHR) BRC based at Guy's and St Thomas' NHS Foundation Trust and King's College London.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Acknowledgments
We thank all volunteers and patients who participated in this study. We acknowledge the Biomedical Research Centre (BRC) Immune Monitoring Core Facility team at Guy's and St Thomas' NHS Foundation Trust for assistance.
Footnotes
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received July 4, 2016.
- Revision received December 15, 2016.
- Accepted December 19, 2016.
- ©2017 American Association for Cancer Research.
References
Volume 77, Issue 5
