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Background: Clinical and experimental analyses indicate a pathognomonic role for allergen IgE crosslinking through epitope–paratope interactions as a major initial step in the cascade leading to effector cell activation and clinical manifestations of lgE‐mediated food allergies. We aimed to undertake the initial development and assessment of Ara h 2‐specific IgE epitope‐like peptides that can bind to allergen‐specific IgE paratopes and suppress effector cell activation. Methods: We performed biopanning, screening, IgE binding, selection and mapping of peptides. We generated synthetic peptides for use in all functional experiments. ImmunoCAP inhibition, basophil and mast cell activation tests, with LAD2 cells, a human mast cell line were performed. Twenty‐six children or young adults who had peanut allergy were studied. Results: We identified and selected three linear peptides (DHPRFNRDNDVA, DHPRYGP and DHPRFST), and immunoblot analyses revealed binding to lgE from peanut‐allergic individuals. The peptide sequences were aligned to the disordered region corresponding to the loop between helices 2 and 3 of Ara h 2, and conformational mapping showed that the peptides match the surface of Ara h 2 and h 6 but not other peanut allergens. In ImmunoCAP inhibition experiments, the peptides significantly inhibit the binding of IgE to Ara h 2 (p <.001). In basophil and mast cell activation tests, the peptides significantly suppressed Ara h 2‐induced effector cell activation (p <.05) and increased the half‐maximal Ara h 2 effective concentration (p <.05). Binding of the peptides to specific IgEs did not induce activation of basophils or mast cells. Conclusions: These studies show that the indicated peptides reduce the allergenic activity of Ara h 2 and suppress lgE‐dependent basophil and mast cell activation. These observations may suggest a novel therapeutic strategy for food allergy based on epitope–paratop blocking. [ABSTRACT FROM AUTHOR]

Background: Single domain antibodies (sdAbs) possess unique characteristics that make them highly effective for developing complex therapeutics., Methods: Our process uses a fully synthetic phage display library to generate single domain antibodies that can bind to disease relevant antigen conformations. A human IGHV3 family scaffold makes up the phage display libraries, and these VHO libraries are applied to diverse phage biopannings against target antigens. After NGS processing, unique VHOs undergo automated cloning into expression constructs followed by transfections and purifications. Binding assays were used to determine VHO binding behaviors to the target proteins. Additional VHO interactions are measured against endogenous targets on cells by way of flow cytometry, cell internalization, and activation assays., Results: We show that a fully synthetic phage display library can generate VHOs that bind to disease relevant antigen conformations. The diverse biopanning methods and processing of next-generation sequencing generated many VHO paratopes. These different VHO sequences can be expressed as Fc fusion proteins. Various screening assays resulted in VHOs representing different epitopes or activities. During the hit evaluation, we demonstrate how screening can identify distinct VHO activities that have been used to generate differentiated drug molecules in various bispecific and multispecific antibody formats., Conclusion: We demonstrate how screening can identify distinct VHO activities that have been used to generate differentiated drug molecules in various bispecific and multispecific antibody formats., Competing Interests: The authors whose names are listed are employees of Tavotek Biotherapeutics., (© The Author(s) 2024. Published by Oxford University Press on behalf of Antibody Therapeutics.)

Peptide antibodies have become one of the most important classes of reagents in molecular biology and clinical diagnostics. For this reason, methods for their production and characterization continue to be developed, including basic peptide synthesis protocols, peptide-conjugate production and characterization, conformationally restricted peptides, immunization procedures, etc. Detailed mapping of peptide antibody epitopes has yielded important information on antibody-antigen interaction in general and specifically in relation to antibody cross-reactivity and theories of molecular mimicry. This information is essential for detailed understanding of paratope-epitope dynamics, design of antibodies for research, design of peptide-based vaccines, development of therapeutic peptide antibodies, and de novo design of antibodies with predetermined specificity., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Three soluble single-domain fragments derived from the unique variable region of camelid heavy-chain antibodies (VHHs) against the CMY-2 β-lactamase behaved as inhibitors. The structure of the complex VHH cAb CMY-2 (254)/CMY-2 showed that the epitope is close to the active site and that the CDR3 of the VHH protrudes into the catalytic site. The β-lactamase inhibition pattern followed a mixed profile with a predominant noncompetitive component. The three isolated VHHs recognized overlapping epitopes since they behaved as competitive binders. Our study identified a binding site that can be targeted by a new class of β-lactamase inhibitors designed on the sequence of the paratope. Furthermore, the use of mono- or bivalent VHH and rabbit polyclonal anti-CMY-2 antibodies enables the development of the first generation of enzyme-linked immunosorbent assay (ELISA) for the detection of CMY-2 produced by CMY-2-expressing bacteria, irrespective of resistotype.

[Display omitted] The antigenic epitope regions of pathogens (e.g., viruses) are recognized by antibodies (Abs) and subsequently cleared by the host immune system, thereby protecting us from disease. Some of these epitopes are conserved among different variants or subgroups of pathogens (e.g., Influenza (FLU) viruses, Coronaviruses), hence can be targeted for potential broad-neutralization. Here we report a web-based tool, Epitope Analyzer (EA), that rapidly identifies conformational epitope and paratope residues in an antigen–antibody complex structure. Furthermore, the tool provides the ways and means to analyze broadly neutralizing epitopes by comparing the equivalent epitope residues in similar antigen structures. The similarity in the epitope residues between (multiple) pairs of similar antigen molecules suggest the presence of conserved epitopes that can be targeted by broadly neutralizing antibodies. These details can be used as a guide in developing effective treatments, such as the design of novel vaccines and formulation of cocktail of broadly neutralizing antibodies, against multiple variants or subgroups of viruses. The web application can be freely accessed from the URL, http://viperdb.scripps.edu/ea.php. [ABSTRACT FROM AUTHOR]

Background: B-cell epitope prediction for antipeptide antibody responses enables peptide-based vaccine design and related translational applications. This entails estimating epitopeparatope binding free-energy changes from antigen sequence; but attempts to do so assuming uniform epitope length (e.g., of hexapeptide sequences, each spanning a typical paratope diameter when fully extended) have neglected empirically established variation in epitope length., Objective: This work aimed to develop a sequence-based physicochemical approach to variablelength B-cell epitope prediction for antipeptide paratopes recognizing flexibly disordered targets., Methods: Said approach was developed by analogy between epitope-paratope binding and protein folding modeled as polymer collapse, treating paratope structure implicitly. Epitope-paratope binding was thus conceptually resolved into processes of epitope compaction, collapse and contact, with epitope collapse presenting the main entropic barrier limiting epitope length among nonpolyproline sequences. The resulting algorithm was implemented as a computer program, namely the Heuristic Affinity Prediction Tool for Immune Complexes (HAPTIC), which is freely accessible via an online interface (http://badong.freeshell.org/haptic.htm). This was used in conjunction with published data on representative known peptide immunogens., Results: HAPTIC predicted immunodominant epitope sequences with lengths limited by penalties for both compaction and collapse, consistent with known paratope-bound structures of flexibly disordered epitopes. In most cases, the predicted association constant was greater than its experimentally determined counterpart but below the predicted upper bound for affinity maturation in vivo., Conclusion: HAPTIC provides a physicochemically plausible means for estimating the affinity of antipeptide paratopes for sterically accessible and flexibly disordered peptidic antigen sequences by explicitly considering candidate B-cell epitopes of variable length., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)