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Passive Immunotherapy is a cornerstone of important therapy regimens in clinical oncology. Notably, all FDA-approved antibodies comprise the IgG subclass, although numerous cancer research articles proposed monoclonal antibodies of the IgM, IgG, IgA and IgE classes directed specifically against tumor-associated-antigens. Especially for IgE, several recent studies could demonstrate high tumoricidic efficacy. This review aims to highlight the latest developments of IgE based immunotherapy of cancer, to discuss possible mechanisms and safety aspects of IgE-mediated tumor cell death with special focus on the attracted immune cells and to give an outlook on how especially comparative oncology could contribute to further development. Due to highly comparable IgE-biology, especially at the interface of allergy and cancer, studies in canine cancer patients could help to translate the recent research findings of allergooncology to clinical veterinary studies with predictive value for the potential of IgE based immunotherapy of cancer for human clinical oncology.
The nascent concept of AllergoOncology (1-2) aims to reveal the inverse associations between atopic and malignant diseases, in particular pancreatic cancer, glioma, and childhood leukemia (3-5) in order to harness allergic mechanisms for therapy of cancer.
Cancer research has aimed for decades to overcome tumor tolerance and instead, engage the immune system in defense of cancer. Strategies that have been pursued cover basically the whole spectrum of the immune repertoire, such as vaccines against tumorigenic viruses (6), vaccinations with tumor cells or tumor-associated antigens (TAA) (7), pulsing of patients antigen presenting cells (8) to passive immunotherapy with monoclonal antibodies (9). More recent experimental approaches purpose to use genetically modified immune cells like natural killer cells to specifically target tumor associated antigens (10) or to engage cytotoxic T-cells for identification and vaccination against TAA-T-cell epitopes (11).
In spite of promising in vitro and in vivo data of several experimental immunotherapeutic trials and numerous immunotherapeutic approaches in the pipeline (http://www.cancer.gov/clinicaltrials), only 2 approaches are at the moment of relevance in public health: prophylactic vaccines against tumorigenic viruses and passive antibody therapy against tumor associated antigens (TAA).
Passive Immunotherapy of Cancer with monoclonal antibodies
Immunotherapy with monoclonal antibodies has found its place in several treatment regimens of malignancies and is at the moment standard of care in e.g. therapy of metastatic breast cancer overexpressing HER-2 (12) or metastatic colon cancer overexpressing EGFR (13). More recent approaches even try to modulate the immune system by attacking immune checkpoint inhibitors such as the anti-CTLA-4 antibody ipilimumab, which displayed encouraging results in clinical studies of advanced metastatic melanoma (14).
The target molecules of the established therapies, however, represent either specific markers of malignantly transformed cells, like CD20, CD33 or CD52 in haematologic malignancies (15) or signal molecules promoting the growth of tumors, like vascular endothelial growth factor (VEGF) (16), as well as growth factor receptors like epidermal growth factor receptor (EGFR) (17) or human epidermal growth factor receptor - 2 (HER-2) (18).
Monoclonal antibodies can thus act in two ways: by interfering via their Fab-regions with binding of growth factors to receptors and thus silencing proliferation signals (19-20) and second by interacting with immune cells via their Fc-regions (21), conferring active tumor cell killing by immune cells via antibody dependent cell mediated cytotoxicity (ADCC) and antibody dependent cell mediated phagocytosis (ADCP).
FcÎ³-receptor mediated tumor cell killing
As all monoclonal antibodies currently applied in clinical oncology comprise the IgG class (22), attracted immune cells are Fc-gamma receptor bearing cells such as, monocytes, macrophages, granulocytes, NK-cells (CD16), dendritic or Langerhans cells (23). These cells lead to ADCC (24) or ADCP (25) of tumor cells, the antigen processing, transport and presentation to the cells.
In humans, three groups of Fc-gamma receptors were identified: CD 64 (FcÎ³RI), CD 32 (FcÎ³RIIa, FcÎ³RIIb, FcÎ³RIIc) and CD 16 (FcÎ³RIIIa, FcÎ³RIV) (24). They can be divided into activating and inhibiting receptors, depending on transmission of their signals via immunoreceptor tyrosine-based activation (ITAM) or inhibitory motifs (ITIM), respectively. In humans only FcÎ³RIIb acts inhibitory, whereas all others are activating receptors (26). In early studies with monoclonal antibodies directed against TAAs, different efficacy of IgG1 or IgG2a could be observed with respect to ADCC (27). This can be explained by the net result of binding capacities to either activating or inhibitory receptors of the two subclasses (28).
ADCC is one of the most important killing mechanisms harnessed in passive immunotherapy of cancer, underlined by findings that mice deficient for activating receptors FcÎ³RI and FcÎ³RIII were unable to mount protective immune responses against tumor challenge with cells presenting a virus-encoded tumor-specific antigen (29). In contrast, mice deficient for the inhibitory receptor FcÎ³RIIb showed high capacity of ADCC, resulting in tumor growth arrest of subcutaneously grafted BT474 breast cancer cells. Similar effects could be observed in these knock-out mice in a pulmonary metastasis model with B16 melanoma cells, where antibody treatment mediated a 100-fold reduction of pulmonary metastasis load compared to untreated animals (30). In humans binding of IgG1 is affected by a genetic polymorphism of FcÎ³RIIIa on position 158 in the IgG-binding domain (phenylalanine F or valine V, with significantly better binding to FcÎ³RIIIa185V) (31). Accordingly, in a subpopulation analysis of 54 trastuzumab treated breast cancer patients, Musolino et al. could depict that homozygous individuals for FcÎ³RIIIa185V/V showed significantly better objective response rates (ORR) and significantly better progression free survival (PFS) than heterozygous FcÎ³RIIIa185V/F or homozygous for FcÎ³RIIIa185F/F. These findings correlated with significantly higher levels of ADCC in a cytotoxicity assay using peripheral blood mononuclear cells (PBMC) purified from FcÎ³RIIIa185V/V patients. For other polymorphisms of FcÎ³RIIa (histidine H or arginine R on position 131) and FcÎ³RIIb (isoleucine I or threonine T on position 232) no clinical significant difference could be found with only a trend towards better ORR and longer PFS for the FcÎ³RIIa131H/H genotype (32). Similar effects could be demonstrated in 49 follicular non-Hodgkin lymphoma patients treated with the anti-CD20 IgG1 antibody rituximab, where again significantly better ORR were observed in FcÎ³RIIIa158V/V homozygous patients compared to FcÎ³RIIIa158F carriers (33). Also in 69 patients with metastatic irinocetan-refractory colorectal cancer, cetuximab treatment showed significantly better outcome rates in FcÎ³RIIIa158V/V homozygous patients compared to F carriers with respect to PFS; but again this study could not show a significant difference in PFS depending in the FcÎ³RIIa131 genotype (34).
Antibody optimization approaches: trials and pitfalls.
Different approaches to use these observed effects of FcÎ³-receptor polymorphisms therapeutically have been pursued, e.g. modulation of IgG binding to FcÎ³-receptors via site-directed mutagenesis, mediating significantly higher rates of tumor cell lysis via ADCC (35).
Biochemical studies could reveal that variations in posttranslational glycosylation of constant regions in antibodies' heavy chains are also of high relevance for binding to different FcÎ³ receptors (28). So-called "glycoengineering" of monoclonal antibodies like the modification of the N-glycosylation pattern at Asn-297 of the IgG heavy chain into afucosylated oligosaccharides seems to enhance binding to FcÎ³RIIIa resulting in higher ADCC levels of cancer cells (36).
This also indicates that the expression system for such anti-cancer antibodies is of crucial importance. Recently, Platts-Mills et al. observed for cetuximab that alpha-Gal is an immunodominant glyco-epitope derived from SP2/0 cells used as expression system, leading to a risk of anaphylaxis (37).
Anti-cancer IgM, IgA and IgE
Other optimization approaches aim at engaging different classes of immunoglobulins than IgG.
IgM antibodies, physiologically representing the first immune response to foreign antigens, could be one option. Although research in this field is young and not far developed yet, first results are promising. In a model of metastasizing malignant melanoma, a tumor entity with very limited treatment options, Dobroff et al. could demonstrate that monoclonal IgM antibodies reactive to histone 1 can reduce the number of lung nodules in mice (38).
IgA however, either in monomeric (39) or dimeric form (40), can attract a similar panel of effector cells as IgG. NK-cells, granulocytes, monocytes or macrophages express the Fc-alpha receptor CD89 (41) but IgA could lead to diverse effector mechanisms (39, 42-43). IgA can trigger substantial amounts of ADCC via FcÎ±RI, which could be demonstrated elaborately for immature neutrophils, mobilized from the bone marrow upon stimulation with G-CSF (44).
Not only IgA, but especially IgE antibodies could be beneficial in this aspect, as IgE is able to mediate high levels of ADCC. Fu et al. could demonstrate, that IgE antibodies purified from patients suffering of pancreatic cancer act in vitro cytotoxic against pancreatic cancer cell lines (45). Additionally IgE can engage a broad panel of effector cells in tumor defense, with a high cytotoxic and phagocytic potential upon binding to IgE-receptors (46).
FcÎµ-receptor mediated tumor cell killing
In contrast to Fc-gamma receptors, Fc-epsilon receptors comprise only of two classes: FcÎµRI and FcÎµRII (CD23), whereupon FcÎµRI is also termed "high affinity IgE receptor" and CD23 is also known as "low affinity IgE receptor".
Additionally, galectin-3 has IgE-binding properties, but its entire function in context of IgE remains to be determined. So far it is known that it can have proinflammatory functions in a murine asthma model (47), via activating mast cells or basophils by crosslinking receptor-bound IgE (48).
However, both "high affinity" FcÎµRI as well as "low affinity" CD23 show outstanding affinity to the Fc-domains of IgE. For FcÎµRI the affinity is in the range of Ka~1010 M-1. CD23 belongs to the Câ€‘type (calcium dependent) lectin superfamily of receptors and displays three lectin domains each having a Ka~106 - 107M-1 to IgE, thus ranging in the average affinity of FcÎ³-receptors (49). The avidity of the CD23-trimer increases the affinity to a Ka~108-109 M-1 approaching the high-affinity of FcÎµRI (50) and again exceeding the affinity of IgG to its high affinity receptor FcÎ³RI (49).
Using recombinant IgE antibodies specific for folate receptor-Î± on ovarian cancer cells, Karagiannis et al. could demonstrate that monocytic killing of tumor cells via ADCC is FcÎµRI dependent: blocking of IgE binding to FcÎµRI on monocytes with monoclonal antibodies (46) or with a soluble Î±-chain of FcÎµRI (51) resulted in substantially decreased ADCC. Monocytic CD23 however, which is upregulated upon incubation with IL-4 and IL-13, has the function to clear IgE-antigen complexes from the circulation, and it could be demonstrated that this mechanism can lead to IgE-mediated phagocytosis (ADCP) of tumor cells (50). In this ovarian cancer model IgE armed monocytes killed tumor cells via FcÎµRI-mediated cytotoxicity followed by CD23-mediated phagocytosis of the remaining cell fragments (46, 51).
Subsequently, side-by-side comparison studies of ADCC and ADCP of the clinically applied anti-HER-2 antibody trastuzumab (Herceptin®, IgG1) and a trastuzumab-like IgE were performed in a breast cancer model, using HER-2 overexpressing cells as targets and the monocytic cell line U937 as effector cells. In this setting indeed ADCP was the major mechanism of trastuzumab IgG killing, whereas IgE rather triggered monocytes to ADCC of tumor cells (25). The same effect could be observed in a recent study where the clinically used antibody cetuximab (Erbitux®, again IgG1) and cetuximab-like IgE were compared in the same ADCC/ADCP assay using this time EGFR overexpressing A431 cells as targets (Plum et al., manuscript in submission). The classical cetuximab (IgG1) mediated phagocytosis as well as cytotoxicity concentration-dependently. In contrast, cetuximab-like IgE samples caused much less phagocytosis but significantly higher ADCC levels than the IgG, in a concentration dependent manner (52).
IgE effector cells
The IgE-mediated tumoricidic mechanisms of monocytes are also valid for eosinophilic granulocytes, being most classical IgE effector cells. For long they were just known for their role in allergy or defense of helminthic parasitic infections. However, when eosinophils were purified from venous blood and armed with the anti-folate receptor-Î± specific IgE described above, ADCC of ovarian cancer cells could be measured. In contrast to killing by monocytes, no phagocytosis of ovarian cancer cells could be determined (51). This could be due to low constitutive expression of CD23 on the surface of eosinophils (53) or lack of CD23 expression on the surface of the eosinophils used in these assays (51). Upon IgE-activation, eosinophils can release cytotoxic mediators like eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil peroxidase (EPO) and eosinophil-derived neurotoxin (EDN). These proteins are well investigated in their cytotoxic action against bacteria, parasites and viruses but also respiratory epithelium and cancer cells (54). Synthetic eosinophil-derived neurotoxin, slightly modified by adding four extra residues, has even been studied as a therapeutic agent on its own in Kaposi's sarcoma in vitro (55-56). Moreover eosinophilic granulocytes are able to release TNF-Î± (57), and it could be demonstrated in a recent study by Legrand et al. that cytotoxic killing of colon cancer cells by eosinophils can be mediated through TNF-Î± and Granzyme A (58). On the other hand eosinophils could be ambivalent, as they play a role in tissue remodelling in allergic and malignant diseases via mediators such as basic-fibroblast growth factor (b-FGF), IL-6, IL-8, granulocyte macrophage colony stimulating factor (GM-CSF), platelet-derived growth factor (PDGF) or transforming growth factor beta (TGF-¢) (54).
Eosinophilic peroxidase (EPO) is a haloperoxidase enzyme, whose catalyzed metabolites have been shown to promote oxidative stress, and subsequent cell death by apoptosis or necrosis (59). However, even for this eosinophilic enzyme it could be demonstrated at non-cytotoxic levels that it can drive cell cycle progression and proliferation by signalling via the tumor associated receptor tyrosine kinase HER-2 (60).
As eosinophils were described to be found in several cancer entities including malignancies of the head and neck region (61), uterine cervix, esophagus, or the GI-tract (62), the term tumor associated tissue eosinophilia (TATE) was introduced (63). It is not yet clear what TATE means with regard to prognosis; studies in oral squamous cell carcinoma range from higher overall survival (61), across no significant association with respect to tumor differentiation, perineural, vascular, and muscular invasion or locoregional metastasis (64) to unfavorable prognosis for heavy eosinophilic infiltration and expression of HLA-DR antigen (65). What has been accepted so far is that blood eosinophilia (tumor associated blood eosinophilia, TABE (63)) in patients with oral squamous cell carcinoma indicates disseminated carcinoma, resulting in poor outcome (66-67).
Only further studies with recombinant anti-tumor IgG versus IgE antibodies will give a definite picture about the ambivalent role of eosinophils in cancer.
The controversy described above is even bigger for another type of IgE effector cells in and around tumors, mast cells. Mast cells, discovered and named by Paul Ehrlich in the 19th century (68), have been identified as eminent players in allergic and anaphylactic reactions of type I hypersensitivity (69). Upon activation via bi- or multivalent antigen-IgE complex binding leading to crosslinking of FcÎµRI-receptors, mast cells release within minutes preformed histamine, heparin, and other proteoglycans, several proteases, and cytoplasmic-granule-associated cytokines (70), but also a variety of immunomodulatory mediators, such as histamine, serotonine, IL-2, IL-4, IL-21, TNF, G-CSF or prostaglandines (71-72). Clinical symptoms of this mediator release include vasodilatation, increase in vascular permeability, contraction of bronchial smooth muscle, mucus secretion, sneezing, itching or coughing (73). Activation via FcÎµRI-crosslinking also induces the production of cytokines, chemokines, and growth factors, leading to a second wave of allergic symptoms, also-called late-phase reactions that typically develop 2-6 h after allergen encounter and peak after 6-9 h (70, 73). Chronic exposure to allergens results in constitutive activation of mast cells leading to tissue remodelling, e.g. an increase of mucus-producing goblet cells in the airway epithelium, sub-epithelial membrane thickening through increased lung collagen deposition, neoangiogenesis and an increased bronchial smooth muscle mass (70, 74). Similar effects could be demonstrated in a model of human skin, where sonicates of mast cells significantly increased fibroblast proliferation, collagen synthesis and collagen contraction, surrogates for skin remodelling and fibrosis (75). These remodelling effects, especially the induction of angiogenesis and neovascularisation are detrimental in malignant diseases (76).
Besides their role in allergy, mast cells are important factors in the defense of parasitic infections, such as nematodes or protozoa (77). Furthermore mast cells contribute to an efficient immune response to bacteria, as it could be demonstrated in an in vivo model of skin infection with Pseudomonas aeruginosa, where mast cell deficient mice showed increased lesions due to impaired neutrophil recruitment and bacterial clearance (78).
With respect to tumors, mast cells were early reported to be found in tumor surrounding tissues of different malignant lesions by Paul Ehrlich himself in the 19th century. He assumed that mast cells directly fulfil nutritional requirements of malignant tissues (68). This is definitely not the case, but the distinct role of mast cells in oncology is still a matter of debate.
One big controversy is whether mast cell derived proteases act tumorpromoting or -inhibiting. On the one hand Aromando et al. could demonstrate in a hamster cheek pouch carcinogenesis model, that tumor growth was stimulated by mast-cell-specific serine protease - 6 (MCP-6, tryptase) through activation of protease activated receptor 2 (PAR-2) on the surface of carcinoma cells (79). This finding is in line with a previous in vitro study, demonstrating that mast cell tryptase stimulates the growth of DLD-1 colon adenocarcinoma cells through PAR-2 and mitogen activated protein kinase (MAPK) -dependent manners (80). Similar tumorpromoting effects could be demonstrated when investigating mast-cell-specific serine protease - 4 (MCP-4, chymase), which can activate progelatinase B, thus acting proangiogenic (81). In contrast, recent findings of a study of chemical cancerogenesis from Siebenhaar et al. could demonstrate that tumor development induced with 7,12-dimethylbenzanthracene and subsequent treatment with the tumor promotor 12-tetradecanoyl-phorbol-13-acetat was significantly increased in mice lacking mast cells. This effect could be outweighed by restitution of mast cells via adoptive transfer. Interestingly, tumor growth in mice lacking MCP-4 did not differ to the situation in wild type animals. Which mast cell released factors exactly protect the animals could not be determined, the authors however could outline that mice lacking mast cells attracted significantly lower numbers of CD8+ T-cells to sites of tumor development. (Siebenhaar F., personal communication). Furthermore it has to be considered that cancerogenesis studies use different chemical compounds for tumor initiation and promotion and different sites, which could also affect the overall susceptibility of the animal to the tumor and efficacy of the immune cells.
Other major players of Th2-driven immune responses as well as possible potent effector cells of IgE based immunotherapies are basophils. Basophils share many features with mast cells, both were initially described by Paul Ehrlich, both express FcÎµRI and both release histamine upon IgE binding. Whereas mast cells are located primarily in the tissue, basophils can be found in circulation, but with less than 1% of leukocytes in healthy human beings, basophils are the least abundant immune cell population (82). Apart from their contribution to allergic and anaphylactic reactions, basophils play a crucial and non-redundant role in defense of endo- and ectoparasites like helminths (83) or ticks (84).
As basophils are one of the major sources of histamine and anaphylactic mediators in the circulation during an anaphylactic shock (85), one of the major concerns of passive immunotherapy of cancer with monoclonal IgE antibodies is that intravenously applied IgE sensitizes FcÎµRI on basophils and could potentially be crosslinked by soluble tumor associated antigens in the circulation, shed by tumors. Therefore it is crucial to target only epitopes, that are not repetitively expressed on the target antigen and that the TAA does not occur complexed in the circulation. Tumor associated antigens that fulfil these requirements are e.g. EGFR and HER-2, for which we could demonstrate that only repetitive antigen display on the surface of cancer cells lead to degranulation of IgE-loaded rat basophilic leukemia cells (RBL-SX38, transfected with human FcÎµRI) whereas the soluble, monomeric protein shows no effect (25, 52). Rudman et al. could demonstrate in a recent study that ovarian cancer patients displayed elevated levels of folate receptor-Î± not only on cancer cells but also in the circulation (up to 35ng/ml). Still, sera of these patients could neither trigger degranulation of RBL-SX38 cells loaded with anti-folate receptor-Î± IgE, nor activate basophils of healthy donors in an ex vivo setting again preloaded with anti-folate receptor-Î± IgE (86-87).
How to approach the translation from bench to bedside:
Clearly, more studies with respect to safety and clinical efficacy are needed to clarify the advantages or complementary effects of IgE based immunotherapy of cancer. Especially side by side comparison studies with "next generation" antibodies like glycoengineered IgG or IgA and IgE antibodies of the same specificity could assess the different potential of antibody classes with particular respect to ADCC and ADCP. There is also great demand for further in vivo studies above preclinical proof-of-concept in mice, given the fact that in vivo efficacy of TAA-specific IgE antibodies has already been demonstrated in severe combined immunodeficiency (SCID) mice with xenografted tumors (46, 88-89). The observed effects do not fully represent the natural picture to be expected in cancer patients with spontaneous tumors. To overcome this experimental limitation is not trivial. In contrast to FcÎ³-receptors, which are similar distributed on human and murine immune cells, the distribution pattern of FcÎµ-receptors differs considerably. Whereas FcÎµRI is expressed on human mast cells, basophils, eosinophils, monocytes, Langerhans cells and Dendritic cells, it could be only found on murine mast cells and basophils (90). Therefore, mouse strains transgenic for human FcÎµRI have been generated by introducing the human Î±-chain of FcÎµRI, which displays the IgE-binding site. Functionality of the receptor could be shown on mast cells (91-92), as well as basophils, eosinophils, monocytes, and epidermal Langerhans cells (90), making these mice strains important models to study the biological function of IgE (93) and models to investigate the effects and potential of passively applied IgE against grafted tumors.
In this context it is important to note that the dog (Canis lupus familiaris) shares a much more similar FcÎµRI expression pattern with humans, with functional FcÎµRI expression not only on mast cells (94), but also on Langerhans cells (95). This results in similar prevalence and pathophysiology of atopic and anaphylactic reactions, underlined by the fact that the historically first described anaphylactic reaction has been described in a dog model by Paul Jules Portier and Charles Robert Richet (68). In recent years the value of the dog as a research model has been rediscovered for food allergy and atopic dermatitis (96-97). As a coincidence, dogs also spontaneously develop tumors, again of striking homology to human disease (98).
Combining both aspects - IgE pathophysiology and cancer - it can thus be anticipated that canine patients would be an ideal model for an AllergoOncology-trial. Dog patients suffering from cancer would offer the same IgE effector cell panel, would therefore have the same risk of side effects, but also potentially the same therapeutic advantage as human oncology patients. Such a study could overcome the limitations of the human-FcÎµRI-mouse.
As dogs live in similar environments like pet owners, dogs nowadays share the same risk factors for cancer like age, ovariectomy, progestagen treatment, obesity in early life and a diet rich of red meat, all associated with higher incidence of mammary carcinoma (99). Furthermore it could be demonstrated, that these malignancies also share biological properties, as canine homologues of the tumor associated antigens EGFR and HER-2 could be detected on canine mammary carcinoma (100-103). This perception that malignancies in companion dogs and humans occur according to very similar biological principles has attracted attention because it offers a chance to speed up drug development for both, humans and animals, for now peaking in the establishment of the comparative oncology trial consortium by the National Cancer Institute (NCI; http://ccr.cancer.gov/resources/cop/COTC.asp). In line with this concept we could show in a recent work that the canine EGFR and HER-2 homologues are susceptible to cetuximab and trastuzumab targeting, leading to growth arrest due to growth signal inhibition (98). Combining both aspects - that dogs resemble similar biologic properties according to the development of malignancies as well as to develop atopic diseases - we suggest the caninization of cetuximab and trastuzumab antibodies to canine IgG and IgE antibodies, respectively, in order to more accurately assess side-by-side the full potential of IgE based immunotherapies against cancer and important therapy-related safety issues.
This work was supported by grant P18238-B19 of the Austrian Science Fund (FWF), by SFB F4606-B19 and JS by the CCHD PhD program, FWF project APW01205FW.