Heather J. Bax

Targeting folate receptor alpha for cancer treatment

Promising targeted treatments and immunotherapy strategies in oncology and advancements in our understanding of molecular pathways that underpin cancer development have reignited interest in the tumor-associated antigen Folate Receptor alpha (FRα). FRα is a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Its overexpression in tumors such as ovarian, breast and lung cancers, low and restricted distribution in normal tissues, alongside emerging insights into tumor-promoting functions and association of expression with patient prognosis, together render FRα an attractive therapeutic target. In this review, we summarize the role of FRα in cancer development, we consider FRα as a potential diagnostic and prognostic tool, and we discuss different targeted treatment approaches with a specific focus on monoclonal antibodies. Renewed attention to FRα may point to novel individualized treatment approaches to improve the clinical management of patient groups that do not adequately benefit from current conventional therapies.

A tool kit for rapid cloning and expression of recombinant antibodies

Over the last four decades, molecular cloning has evolved tremendously. Efficient products allowing assembly of multiple DNA fragments have become available. However, cost-effective tools for engineering antibodies of different specificities, isotypes and species are still needed for many research and clinical applications in academia. Here, we report a method for one-step assembly of antibody heavy- and light-chain DNAs into a single mammalian expression vector, starting from DNAs encoding the desired variable and constant regions, which allows antibodies of different isotypes and specificity to be rapidly generated. As a proof of principle we have cloned, expressed and characterized functional recombinant tumor-associated antigen-specific chimeric IgE/κ and IgG1/κ, as well as recombinant grass pollen allergen Phl p 7 specific fully human IgE/λ and IgG4/λ antibodies. This method utilizing the antibody expression vectors, available at Addgene, has many applications, including the potential to support simultaneous processing of antibody panels, to facilitate mechanistic studies of antigen-antibody interactions and to conduct early evaluations of antibody functions.

Cytokinergic IgE Action in Mast Cell Activation

Some 10 years ago it emerged that at sufficiently high concentrations certain monoclonal mouse IgEs exert previously unsuspected effects on mast cells. Thus they can both promote survival and induce activation of mast cells without the requirement for antigens. This was a wake up call that appears to have been missed (or dismissed) by the majority of immunologists. The structural attributes responsible for the potency of the so-called “highly cytokinergic” or HC IgEs have not yet been determined, but the events that ensue when such IgEs bind to the high-affinity receptor, FcεRI, on mast cells have been thoroughly studied, and are strikingly similar to those engendered by antigens when they form cross-linked complexes with the receptors. We review the evidence for the cytokinergic activity of IgE, and the structural features and known properties of immunoglobulins, and of IgE in particular, most likely to be implicated in the phenomenon. We suggest that IgEs with cytokinergic activity may be generated by local germinal center reactions in the target organs of allergy. We consider also the important implications that the existence of cytokinergic IgE may have for a fuller understanding of adaptive immunity and of the action of IgE in asthma and other diseases.

Inhibition of allergen-dependent IgE activity by antibodies of the same specificity but different class

IgG4 purified from patients undergoing specific allergen immunotherapy inhibits the activities of the serum IgE in in vitro assays and is thought to reduce the symptoms of the disease. However, it is not known whether this is related to an intrinsic property of this subclass or only the allergen specificity. We tested the hypothesis that allergen specificity is the critical determinant for this activity using a panel of antibodies with identical specificity but different subclasses. The different antibodies were all able to inhibit the activity of IgE to the same extent. We demonstrate that specificity is the dominant factor determining the ability of an antibody to block allergen‐dependent IgE activity.

AllergoOncology – the impact of allergy in oncology: EAACI position paper

Th2 immunity and allergic immune surveillance play critical roles in host responses to pathogens, parasites and allergens. Numerous studies have reported significant links between Th2 responses and cancer, including insights into the functions of IgE antibodies and associated effector cells in both antitumour immune surveillance and therapy. The interdisciplinary field of AllergoOncology was given Task Force status by the European Academy of Allergy and Clinical Immunology in 2014. Affiliated expert groups focus on the interface between allergic responses and cancer, applied to immune surveillance, immunomodulation and the functions of IgE‐mediated immune responses against cancer, to derive novel insights into more effective treatments. Coincident with rapid expansion in clinical application of cancer immunotherapies, here we review the current state‐of‐the‐art and future translational opportunities, as well as challenges in this relatively new field. Recent developments include improved understanding of Th2 antibodies, intratumoral innate allergy effector cells and mediators, IgE‐mediated tumour antigen cross‐presentation by dendritic cells, as well as immunotherapeutic strategies such as vaccines and recombinant antibodies, and finally, the management of allergy in daily clinical oncology. Shedding light on the crosstalk between allergic response and cancer is paving the way for new avenues of treatment.

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

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.

Mechanism of the Antigen-Independent Cytokinergic SPE-7 IgE Activation of Human Mast Cells in Vitro

Release of pro-inflammatory mediators by mast cells is a key feature of allergic disease. The ‘dogma’ is that IgE molecules merely sensitise mast cells by binding FcεRI prior to cross-linking by multivalent allergen, receptor aggregation and mast cell activation. However, certain monoclonal IgE antibodies have been shown to elicit mast cell activation in an antigen-independent cytokinergic manner, and DNP-specific murine SPE-7 IgE is the most highly cytokinergic antibody known. We show that both monovalent hapten and recombinant SPE-7 IgE Fab inhibit its cytokinergic activity as measured by mast cell degranulation and TNF-α release. Using SPE-7 IgE, a non-cytokinergic human IgE and a poorly cytokinergic murine IgE, we reveal that interaction of the Fab region of ‘free’ SPE-7 IgE with the Fab of FcεRI-bound SPE-7 IgE is the basis of its cytokinergic activity. We rule out involvement of IgE Fc, Cε1 and Cλ/κ domains, and propose that ‘free’ SPE-7 IgE binds to FcεRI-bound SPE-7 IgE by an Fv-Fv interaction. Initial formation of a tri-molecular complex (one ‘free’ IgE molecule cross-linking two receptor-bound IgE molecules) leads to capture of further ‘free’ and receptor-bound IgEs to form larger clusters that trigger mast cell activation.

Tumour-associated macrophage polarisation and re-education with immunotherapy

Monocytes/macrophages constitute important contributors of cancer-associated inflammation. Through their plasticity and capacity to become polarised by tumours towards less activatory and more immunosuppressive (M2) phenotypes, tumour-associated macrophages (TAM) are thought to support tumour progression. Orchestrated by T helper 2 (Th2)-biased stimuli, macrophage recruitment, activation and polarisation in tumour microenvironments is associated with poorer clinical outcomes. Their key roles in supporting tumour progression and their capacity for plasticity have focused targeted and immunotherapeutic strategies to counteract macrophage pro-tumourigenic activities and to re-ignite their tumour-cytotoxic power. Therapeutic approaches include blockade of macrophage recruitment into tumours, suppression of TAM survival, re-polarisation towards an M1-like phenotype and antibody therapies to enhance TAM anti-tumoural activities. Future immunotherapeutic directions may include monoclonal antibodies with enhanced effector functions. Antibodies of different classes, including those of the IgE class, shown to restrict tumour growth by harnessing monocyte/macrophage cytotoxic properties in pre-clinical cancer models, may synergise or re-educate these potent immune sentinels to destroy rather than support tumours. Opportunities for monitoring monocyte/macrophage polarisation or activatory signatures in patients may inform clinical management.

Mapping of the CD23 Binding Site on Immunoglobulin E (IgE) and Allosteric Control of the IgE-FcϵRI Interaction

Background: Immunoglobulin E (IgE) has two cellular receptors, FcϵRI and CD23, that mediate distinct functional effects. Results: We have identified the CD23 binding site on IgE and show that FcϵRI and CD23 allosterically compete for binding. Conclusion: A mechanism of communication exists within the IgE molecule to prevent simultaneous engagement with the two receptors. Significance: Competition between IgE receptors explains ligand cross-regulation. IgE, the antibody that mediates allergic responses, acts as part of a self-regulating protein network. Its unique effector functions are controlled through interactions of its Fc region with two cellular receptors, FcϵRI on mast cells and basophils and CD23 on B cells. IgE cross-linked by allergen triggers mast cell activation via FcϵRI, whereas IgE-CD23 interactions control IgE expression levels. We have determined the CD23 binding site on IgE, using a combination of NMR chemical shift mapping and site-directed mutagenesis. We show that the CD23 and FcϵRI interaction sites are at opposite ends of the Cϵ3 domain of IgE, but that receptor binding is mutually inhibitory, mediated by an allosteric mechanism. This prevents CD23-mediated cross-linking of IgE bound to FcϵRI on mast cells and resulting antigen-independent anaphylaxis. The mutually inhibitory nature of receptor binding provides a degree of autonomy for the individual activities mediated by IgE-FcϵRI and IgE-CD23 interactions.

IgG subclass switching and clonal expansion in cutaneous melanoma and normal skin.

B cells participate in immune surveillance in human circulation and tissues, including tumors such as melanoma. By contrast, the role of humoral responses in cutaneous immunity is underappreciated. We report circulating skin-homing CD22+CLA+B cells in healthy volunteers and melanoma patients (n = 73) and CD22+ cells in melanoma and normal skin samples (n = 189). Normal and malignant skin featured mature IgG and CD22 mRNA, alongside mRNA for the transiently-expressed enzyme Activation-induced cytidine Deaminase (AID). Gene expression analyses of publically-available data (n = 234 GEO, n = 384 TCGA) confirmed heightened humoral responses (CD20, CD22, AID) in melanoma. Analyses of 51 melanoma-associated and 29 normal skin-derived IgG sequence repertoires revealed lower IgG1/IgGtotal representation compared with antibodies from circulating B cells. Consistent with AID, comparable somatic hypermutation frequencies and class-switching indicated affinity-matured antibodies in normal and malignant skin. A melanoma-associated antibody subset featured shorter complementarity-determining (CDR3) regions relative to those from circulating B cells. Clonal amplification in melanoma-associated antibodies and homology modeling indicated differential potential antigen recognition profiles between normal skin and melanoma sequences, suggesting distinct antibody repertoires. Evidence for IgG-expressing B cells, class switching and antibody maturation in normal and malignant skin and clonally-expanded antibodies in melanoma, support the involvement of mature B cells in cutaneous immunity.