262785 Engineering Hetero-Bivalent Peptidic Ligands to Inhibit Mast Cell Degranulation in a Heterotetravalent Allergy Model
An allergic response results from the activation of the adaptive immune system directed against noninfectious environmental substances (allergens). Cross-linking of immunoglobulin E (IgE) bound to its high affinity receptor, FcεR1, on the surface of basophils and mast cells initiates a complex signaling cascade that results in degranulation which is the release of preformed chemical mediators associated with allergic symptoms. The majority of the current therapies used to treat allergies and related adverse immunologic responses, such as autoimmune diseases, involve non-specific suppression of significant parts of the immune system resulting in the increased risk of infection. The development and use of selectively targeted therapeutics would decrease these risks. Our overall goal in this project was to engineer hetero-bivalent peptidic ligands that would competitively and selectively inhibit allergen binding to IgEs on mast cells, thereby inhibiting IgE clustering and preventing an allergic response. This was accomplished via designing hetero-bivalent inhibitors (HBIs) that simultaneously targeted two nearby sites located on the Fv domain of an IgE, the antigen binding site, and the less renowned “unconventional nucleotide binding site”. Simultaneous bivalent binding to both these sites provided the hetero-bivalent inhibitors with enhanced avidity and selectivity for the target IgE, and enabled competitive inhibition of allergen binding to IgE. We previously demonstrated the utility of the HBI design with the most commonly used allergy model – the rat basophilic leukemia cell (RBL)/IgEDNPsystem. In this system, a saturating concentration of a single monoclonal IgE is used to prime the mast cells, then the allergen, typically BSA presenting multiple copies of DNP, is added to the system and the degranulation response is observed. This system has been very useful for studying allergy, but lacks much of the complexity present in the natural system. First, in humans, mast cells are primed with serum derived polyclonal IgE resulting in mast cells presenting IgE specific for a wide range of antigens, of which only 0.1 – 20% is specific for any given allergen. Second, natural allergens typically contain 2 – 9 distinct immunodominant epitopes that possess a range of affinities for the polyclonal IgEs. Third, the method of conjugating multiple copies of a single hapten to scaffolds such as BSA results in poorly defined allergens that contain a wide range in the number of haptens per allergen. A matter that is further complicated as not all the haptens in these synthetic allergens are capable of binding to mast cell bound IgE due to steric constraints.
The limitations in the current model led us to develop a new system to study allergy and determine the effectiveness of our HBIs. The system we developed was based on the design of a series of heterotetravalent allergens (HtTAs) that were used to stimulate the degranulation of mast cells that were primed with 3 monoclonal IgEs each with a different target antigen. The HtTAs contained two sets of two haptens that were specific for a different IgE antibody. The third IgE antibody was used to model the orthogonal IgE (IgE of other specificity) on mast cells. The use of the third IgE allowed us to test the HtTAs with physiological relevant amounts of allergen specific IgE on the mast cell surface. The HtTAs proved to be potent stimulators of degranulation only when the mast cells were primed with both of the IgEs specific to the allergen. This was expected as bivalent allergens are poor stimulators of degranulation.
With our new more advanced allergy model we were able to successfully and specifically inhibit allergen-mediated mast cell degranulation using our HBIs. Specifically, we designed an HtTA possessing two sets of two haptens each specific for a different monoclonal IgE. The first IgE-hapten interaction had a high affinity (Kd = 55 nM) while the second IgE-hapten pair had a weak affinity (Kd = 41 μM). The HtTA stimulated degranulation, following a bell-shaped dose response curve, thus illustrating the importance of weak binding epitopes in allergy. An HBI was designed to inhibit the binding of only 1 of the hapten-IgE pairs by conjugating an antigen binding moiety to a nucleotide binding moiety via a short ethylene glycol linker. The increased avidity and specificity of simultaneous bivalent binding to the antigen binding region and the nucleotide binding site on a single Fv of an IgE, inhibited allergen binding to the IgE. When the HBI was co-incubated with the allergen, degranulation was inhibited in a dose dependent manor. The binding of the HBI to one of the allergen specific IgEs essentially reduced the HtTA to a bivalent allergen which are not effective stimulators of degranulation. This result demonstrated that it is possible to completely inhibit allergen-mediated degranulation through the specific targeting of only a fraction of the allergen specific IgE on the mast cell. The long-term goal of this project is to develop a broadly applicable technology that can selectively inhibit the allergic responses induced by an allergen via competitively inhibiting its binding to the IgE antibodies with engineered hetero-bivalent ligands.
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