Promiscuous antibodies characterised by their physico-chemical properties: From sequence to structure and back

Human B cells produce antibodies, which bind to their cognate antigen based on distinct molecular properties of the antibody CDR loop. We have analysed a set of 10 antibodies showing a clear difference in their binding properties to a panel of antigens, resulting in two subsets of antibodies with a distinct binding phenotype. We call the observed binding multiplicity ‘promiscuous’ and selected physico-chemical CDRH3 characteristics and conformational preferences may characterise these promiscuous antibodies. To classify CDRH3 physico-chemical properties playing a role in their binding properties, we used statistical analyses of the sequences annotated by Kidera factors. To characterise structure-function requirements for antigen binding multiplicity we employed Molecular Modelling and Monte Carlo based coarse-grained simulations. The ability to predict the molecular causes of promiscuous, multi-binding behaviour would greatly improve the efficiency of the therapeutic antibody discovery process

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

IgG(4) 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

RNAi depletion of PRSS21 induces apoptosis in cutaneous squamous cell carcinoma keratinocytes

The mechanisms underlying UVB-induced clearance of psoriasis are incompletely understood. We compared the cellular and molecular effects of a clinically effective wavelength of UVB (311nm) with a clinically ineffective wavelength (290nm) in vivo and in vitro to distinguish bystander effects and utilised a systems biology approach to understand functional significance. 84 adult psoriatic patients were recruited. Biopsies were taken from lesional (plaque) skin before and 4-48h after irradiation with equi-erythemogenic doses of 311 and/or 290nm UVB (0.75-3.0 MED). A significant increase in the numbers of apoptotic epidermal cells (active-caspase-3+ cells) was seen after a single irradiation with 2-3 MEDs 311 nm UVB compared to 2-3 MEDs 290 nm UVB or untreated psoriasis (median 12/1000, 0/1000 and 0/1000 epidermal cells respectively; p<0.001). Routine clinical doses of 311nm also induced apoptosis. Immunochemistry and electron microscopy showed that the vast majority of apoptotic cells were keratinocytes. Live cell imaging of irradiated cultured keratinocytes and mathematical modelling showed that the rate of keratinocyte apoptosis observed in vivo was sufficient to account for clearance of psoriasis. Gene array analysis was used to examine differential gene regulation at these early time-points, prior to clinical response. Apoptosis and control of cell cycle pathways were affected by 311nm but not 290nm UVB. CDKN1A (WAF1/p21) showed the greatest fold change (27 fold up-regulation) following irradiation with 311nm UVB but not 290nm. Together these data provide evidence that epidermal keratinocyte apoptosis is a key mechanism in psoriasis resolution and identifies keratinocyte apoptosis as a potential target for future drug development.1 page(s

Potential Mechanisms for IgG4 Inhibition of Immediate Hypersensitivity Reactions

IgG4 is the least abundant IgG subclass in human serum, representing less than 5 % of all IgG. Increases in IgG4 occur following chronic exposure to antigen and are generally associated with states of immune tolerance. In line with this, IgG4 is regarded as an anti-inflammatory antibody with a limited ability to elicit effective immune responses. Furthermore, IgG4 attenuates allergic responses by inhibiting the activity of IgE. The mechanism by which IgG4 inhibits IgE-mediated hypersensitivity has been investigated using a variety of model systems leading to two proposed mechanisms. First by sequestering antigen, IgG4 can function as a blocking antibody, preventing cross-linking of receptor bound IgE. Second IgG4 has been proposed to co-stimulate the inhibitory IgG receptor FcγRIIb, which can negatively regulate FcεRI signaling and in turn inhibit effector cell activation. Recent advances in our understanding of the structural features of human IgG4 have shed light on the unique functional and immunologic properties of IgG4. The aim of this review is to evaluate our current understanding of IgG4 biology and reassess the mechanisms by which IgG4 functions to inhibit IgE-mediated allergic responses

Harnessing engineered antibodies of the IgE class to combat malignancy:initial assessment of FcɛRI-mediated basophil activation by a tumour-specific IgE antibody to evaluate the risk of type I hypersensitivity

Background IgE antibodies, sequestered into tissues and retained locally by the high-affinity IgE receptor, Fc epsilon RI, on powerful effector cells such as mast cells, macrophages and eosinophils, may offer improvements in the therapy of solid tumours. The chimeric antibody, MOv18 IgE, against the human ovarian carcinoma antigen, folate receptor alpha (FR alpha), is more effective than its IgG1 counterpart in xenograft models of ovarian cancer. Although MOv18 IgE binds to a single epitope on FR alpha and cannot cross-link IgE receptors on basophils, there remains a risk that components in the circulation of ovarian cancer patients might cross-link FR alpha-MOv18-IgE-receptor-Fc epsilon RI complexes on basophils to cause type I hypersensitivity. Objective To assess the propensity for MOv18 used in a therapeutic setting to cause Fc epsilon RI-mediated type I hypersensitivity. Methods As validated readouts of the potential for MOv18 to cause FceRI-mediated type I hypersensitivity we measured release of a granule-stored mediator from a rat basophilic leukaemia cell line RBL SX-38 stably transfected with human tetrameric (alpha beta gamma 2) FceRI, and induction of CD63 on blood basophils from patients with ovarian carcinoma and healthy controls ex vivo. Results Serum FR alpha levels were increased in ovarian cancer patients compared with healthy controls. MOv18 IgE alone, or in the presence of its antigen recombinant human FR alpha, or of healthy volunteer (n = 14) or ovarian carcinoma patient (n = 32) sera, did not induce RBL SX-38 cell degranulation. Exposure to FR alpha-expressing ovarian tumour cells at target-to-effector ratios expected within tumours induced degranulation. MOv18 IgE did not induce expression of CD63 in blood basophils from either healthy volunteers (n = 6), or cancer patients, despite detectable levels of circulating FR alpha (n = 5). Conclusion and Clinical Relevance These encouraging data are compatible with the hypothesis that, when ovarian carcinoma patients are treated with MOv18, Fc epsilon RI-mediated activation of effector cells occurs within the tumour mass but not in the circulation mandating, with due caution, further pre-clinical studies