An allergic reaction is rapidly generated when allergens bind and cross-link IgE bound to its receptor Fc«RI on effector cells, resulting in cell degranulation and release of proinflammatory mediators. The extent of effector cell activation is linked to allergen affinity, oligomeric state, valency, and spacing of IgE-binding epitopes on the allergen. Whereas most of these observations come from studies using synthetic allergens, in this study we have used Timothy grass pollen allergen Phl p 7 and birch pollen allergen Bet v 4 to study these effects. Despite the high homology of these polcalcin family allergens, Phl p 7 and Bet v 4 display different binding characteristics toward two human patient-derived polcalcin-specific IgE Abs. We have used native polcalcin dimers and engineered multimeric allergens to test the effects of affinity and oligomeric state on IgE binding and effector cell activation. Our results indicate that polcalcin multimers are required to stimulate high levels of effector cell degranulation when using the humanized RBL-SX38 cell model and that multivalency can overcome the need for high-affinity interactions.

Antibody-Drug conjugates (ADcs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADcs. this approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADc development. Antibody-Drug Conjugates (ADCs) combine the high specificity of antibodies for their target antigens with the cytotoxic effects of payloads to more specifically guide toxic agents towards cancer cells 1. The field of ADCs has been undergoing significant expansion since the first agent gemtuzumab ozogamicin (Mylotarg), targeting CD20-expressing B cell lymphomas, was approved by the Food and Drug Administration (FDA) in 2000 2. However, despite the potential for this therapeutic modality and the launch of many clinical studies, attrition rates are high due to several challenges such as low efficacy and "on-target" toxicity on normal cells. Until now, only eight ADCs along with two immunotoxins have been approved by the regulatory agencies 1. In order to improve