Nanoallergens and uses thereof
Inventors
Bilgicer, Zihni Basar • DEAK, Peter Edward • Kiziltepe Bilgicer, Tanyel • STEFANICK, Jared Francis • Ashley, Jonathan Darryl
Assignees
Publication Number
US-11638763-B2
Publication Date
2023-05-02
Expiration Date
2036-09-26
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Abstract
Embodiments of the present disclosure provide a nanoparticle based platform, and nanoallergens for identifying, evaluating and studying allergen mimotopes as multiple copies of a single mimotope or various combinations on the same particle. The nanoparticle is extremely versatile and allows multivalent binding to IgEs specific to a variety of mimotopes, simulating allergen proteins. Nanoparticles can include various molecular ratios of components. For example, the nanoallergens can include about 0.1-40% mimotope-lipid conjugate and about 60-99.9% lipid. The mimotope-lipid conjugate includes a mimotope, a first linker, and lipid molecule. Nanoallergens can be used in in vitro and in vivo applications to identify a specific patient's sensitivity to a set of epitopes and predict a symptomatic clinical response, identify allergen epitopes through blind screening peptide sequences from allergen protein, and in a clinical application similar to a scratch test.
Core Innovation
The invention provides a nanoparticle-based platform, termed nanoallergens, for identifying, evaluating, and studying allergen epitopes as multiple copies of a single epitope or in various combinations on the same particle. Nanoallergens are specifically designed liposomal nanoparticles that display mimotope-lipid conjugates (which can consist of known or suspected allergen epitopes, haptens, or peptide sequences) on their surface in a multivalent fashion. This multivalent display enables the nanoparticles to closely simulate allergen proteins by facilitating multivalent binding to IgEs specific to a variety of mimotopes.
These nanoparticles are composed of about 0.1–40% mimotope-lipid conjugate and about 60–99.9% lipid, with the mimotope-lipid conjugate incorporating a mimotope, specific linkers, and a lipid anchor. The structure enables precise control over particle size, epitope loading, and epitope heterogeneity, allowing for the systematic evaluation of epitope combinations and their capacity to trigger allergic responses. The platform can incorporate different molecular ratios of its components, such as varying mimotope content and epitope series, to study the roles of various allergen epitopes.
The problem addressed by the invention is the inadequacy of existing methods to accurately determine allergen epitope antigenicity in a manner representative of in vivo degranulation, due to limitations such as the inability of current binding assays to measure multivalent interactions and the significant material requirements for such assays. The invention offers a solution by providing a platform that displays multiple epitopes in a multivalent manner, enabling direct assessment of epitope immunogenicity and degranulation potential, both in vitro and potentially in vivo, thereby enhancing the evaluation, ranking, and association of allergen epitopes.
Claims Coverage
The independent claim of this patent covers a novel method for diagnosing peanut allergy using specifically composed nanoallergens, with core inventive features in the nanoparticle composition and its application in a diagnostic assay.
Method of diagnosing a peanut allergy using a nanoallergen-based diagnostic assay
The method comprises: 1. Contacting sera from an allergy sensitive subject with cells in culture. 2. Adding a nanoparticle comprising: - About 0.1 mol % to about 20 mol % of a synmimotope-lipid conjugate; - About 2 mol % to about 10 mol % of a polyethylene glycol-lipid (PEG-lipid) conjugate; - About 80 mol % to about 97 mol % of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 3. The nanoparticle contains a spherical lipid bilayer wherein the PEG-lipid conjugate forms a coating over the exterior surface and one or more synmimotope moieties protrude above this coating. 4. The synmimotope-lipid conjugate is defined by Formula I: - A is a synmimotope moiety of the synmimotope-lipid conjugate (allergen epitope or mimotope comprising one or more of SEQ ID NO: 1-31). - B is a first linker (ethylene glycol6 moiety, covalently attached by amide bonds). - C is a second linker (comprising three lysine amino acids covalently attached by amide bond). - D is a third linker (comprising three ethylene glycol6 moieties attached covalently at distal ends by amide bonds). - E is a tag (tryptophan residue). - F is a palmitoyl moiety covalently attached to the tag by an amide bond. - n is 2. 5. The method evaluates degranulation by measuring secreted beta hexosaminidase or activated granulocytes via assay or FACS, where an increase compared to a pre-nanoparticle level indicates presence of allergy.
The inventive features center on a method of allergy diagnosis using a uniquely structured nanoallergen, defined by specific molecular composition, to reliably detect peanut allergy by assessing cellular degranulation responses.
Stated Advantages
Nanoallergens offer a highly versatile and tunable platform that can display multiple epitopes at precise ratios and particle sizes, allowing for accurate simulation of allergen proteins and their multivalent interactions with IgE.
The platform enables direct assessment of epitope immunogenicity, capturing both high- and low-affinity epitopes that may be overlooked by conventional binding assays such as ELISA.
Nanoallergens can trigger degranulation responses at lower concentrations and with greater sensitivity than existing systems, providing a more physiologically relevant model for allergy research.
The technology allows for the identification of immunodominant IgE binding epitopes in patient serum, facilitating personalized diagnostic and inhibitor design strategies.
It enables accurate, reproducible, and high-throughput screening of allergen epitopes, overcoming limitations of current assays that require large amounts of patient serum and do not measure multivalent binding.
Control over valency and heterogeneity on nanoparticles enables the study of the relative importance of specific epitopes in triggering clinical allergic responses.
Documented Applications
Identifying a specific patient's sensitivity to a set of allergen epitopes using in vitro cell-based assays.
Predicting a symptomatic clinical response to allergens based on detection and quantitation of degranulation responses.
Blind screening of peptide sequences from allergen proteins to identify allergenic epitopes.
Clinical applications analogous to scratch testing for allergies using the nanoparticle platform applied to skin or delivered subcutaneously.
Evaluating degranulation in vitro by measuring beta hexosaminidase release or activated granulocytes in cultured cells exposed to nanoallergens and patient sera.
Testing allergenic epitopes in animal models to evaluate immunological responses to nanoallergens.
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