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The C-C motif chemokine receptor 8 (CCR8) is highly and selectively expressed in regulatory T (Treg) cells and is associated with tumor progression. The massive accumulation of Treg cells into tumors suppresses the effector function of CD8 + cells against tumor cells. Therefore, selective depletion of Treg cells using anti-CCR8 monoclonal antibodies (mAbs) reinvigorates antitumor immune responses and improves responses to cancer immunotherapy. Previously, we developed an anti-mouse CCR8 (mCCR8) mAb, C 8 Mab-2, using the Cell-Based Immunization and Screening method. In this study, the binding epitope of C 8 Mab-2 was investigated using flow cytometry. The mCCR8 extracellular domain-substituted mutant analysis showed that C 8 Mab-2 recognizes the N-terminal region (1-33 amino acids) of mCCR8. Next, 1×alanine (or glycine) scanning and 2×alanine (or glycine) scanning were conducted in the N-terminal region. The results revealed that the 17- DFFTAP -22 sequence is important for the recognition by C 8 Mab-2, and Thr20 is a central amino acid of the epitope. These results revealed the involvement of the N-terminus of mCCR8 in the recognition by C 8 Mab-2.

Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies., (© 2024. The Author(s).)

Precise epitope determination of therapeutic antibodies is of great value as it allows for further comprehension of mechanism of action, therapeutic responsiveness prediction, avoidance of unwanted cross reactivity, and vaccine design. The golden standard for discontinuous epitope determination is the laborious X-ray crystallography method. Here, we present a combinatorial method for rapid mapping of discontinuous epitopes by mammalian antigen display, eliminating the need for protein expression and purification. The method is facilitated by automated workflows and tailored software for antigen analysis and oligonucleotide design. These oligos are used in automated mutagenesis to generate an antigen receptor library displayed on mammalian cells for direct binding analysis by flow cytometry. Through automated analysis of 33930 primers an optimized single condition cloning reaction was defined allowing for mutation of all surface-exposed residues of the receptor binding domain of SARS-CoV-2. All variants were functionally expressed, and two reference binders validated the method. Furthermore, epitopes of three novel therapeutic antibodies were successfully determined followed by evaluation of binding also towards SARS-CoV-2 Omicron BA.2. We find the method to be highly relevant for rapid construction of antigen libraries and determination of antibody epitopes, especially for the development of therapeutic interventions against novel pathogens., (© 2024. The Author(s).)

Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups. This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis. Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD. The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls., Competing Interests: Declaration of competing interest Daniel Nyberg Larsen reports financial support was provided by Innovation Fund Denmark grant number 9065-00215B. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Peptide arrays are a valuable instrument in the characterization of protein-protein interactions (PPIs) and immunogenic regions. New methods were developed to exploit the high-throughput potential of peptide arrays to obtain more in-depth information, replacing traditional resource-intensive experiments. Here, we discuss the recent advances in peptide-array-based technologies and the remaining challenges., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The world has witnessed a revolution in therapeutics with the development of biological medicines such as antibodies and antibody fragments, notably nanobodies. These nanobodies possess unique characteristics including high specificity and modulatory activity, making them promising candidates for therapeutic applications. Identifying their binding mode is essential for their development. Experimental structural techniques are effective to get such information, but they are expensive and time-consuming. Here, we propose a computational approach, aiming to identify the epitope of a nanobody that acts as an agonist and a positive allosteric modulator at the rat metabotropic glutamate receptor 5. We employed multiple structure modeling tools, including various artificial intelligence algorithms for epitope mapping. The computationally identified epitope was experimentally validated, confirming the success of our approach. Additional dynamics studies provided further insights on the modulatory activity of the nanobody. The employed methodologies and approaches initiate a discussion on the efficacy of diverse techniques for epitope mapping and later nanobody engineering.

CD44 is a type I transmembrane glycoprotein and possesses various isoforms which are largely classified into CD44 standard (CD44s) and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 monoclonal antibody, C 44 Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C 44 Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10) and found that C 44 Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C 44 Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C 44 Mab-108 includes Asp280 and Trp281 of CD44v3-10.

B-cell epitope mapping is an approach that can identify and characterise specific antigen binding sites of B-cell receptors and secreted antibodies. The ability to determine the antigenic clusters of amino acids bound by B-cell clones provides unprecedented detail that will aid in developing novel and effective vaccine targets and therapeutic antibodies for various diseases. Here, we discuss conventional approaches and emerging techniques that are used to map B-cell epitopes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Epitope binning, an approach for grouping antibodies based on epitope similarities, is a critical step in antibody drug discovery. However, conventional methods are complex, involving individual antibody production. Here, we established Epitope Binning-seq, an epitope binning platform for simultaneously analyzing multiple antibodies. In this system, epitope similarity between the query antibodies (qAbs) displayed on antigen-expressing cells and a fluorescently labeled reference antibody (rAb) targeting a desired epitope is analyzed by flow cytometry. The qAbs with epitope similar to the rAb can be identified by next-generation sequencing analysis of fluorescence-negative cells. Sensitivity and reliability of this system are confirmed using rAbs, pertuzumab and trastuzumab, which target human epidermal growth factor receptor 2. Epitope Binning-seq enables simultaneous epitope evaluation of 14 qAbs at various abundances in libraries, grouping them into respective epitope bins. This versatile platform is applicable to diverse antibodies and antigens, potentially expediting the identification of clinically useful antibodies., (© 2024. The Author(s).)

Covalent labeling in combination with mass spectrometry is a powerful approach used in structural biology to study protein structures, interactions, and dynamics. Recently, the toolbox of covalent labeling techniques has been expanded with fast fluoroalkylation of proteins (FFAP). FFAP is a novel radical labeling method that utilizes fluoroalkyl radicals generated from hypervalent Togni reagents for targeting aromatic residues. This report further demonstrates the benefits of FFAP as a new method for structural characterization of therapeutic antibodies and interaction interfaces of antigen-antibody complexes. The results obtained from human trastuzumab and its complex with human epidermal growth factor receptor 2 (HER2) correlate well with previously published structural data and demonstrate the potential of FFAP in structural biology.

The study of peptide repertoires presented by major histocompatibility complex (MHC) molecules and the identification of potential T-cell epitopes contribute to a multitude of immunopeptidome-based treatment approaches. Epitope mapping is essential for the development of promising epitope-based approaches in vaccination as well as for innovative therapeutics for autoimmune diseases, infectious diseases, and cancer. It also plays a critical role in the immunogenicity assessment of protein therapeutics with regard to safety and efficacy concerns. The main challenge emerges from the highly polymorphic nature of the human leukocyte antigen (HLA) molecules leading to the requirement of a peptide mapping strategy for a single HLA allele. As many autoimmune diseases are linked to at least one specific antigen, we established FASTMAP, an innovative strategy to transiently co-transfect a single HLA allele combined with a disease-specific antigen into a human cell line. This approach allows the specific identification of HLA-bound peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using FASTMAP, we found a comparable spectrum of endogenous peptides presented by the most frequently expressed HLA alleles in the world's population compared to what has been described in literature. To ensure a reliable peptide mapping workflow, we combined the HLA alleles with well-known human model antigens like coagulation factor VIII, acetylcholine receptor subunit alpha, protein structures of the SARS-CoV-2 virus, and myelin basic protein. Using these model antigens, we have been able to identify a broad range of peptides that are in line with already published and in silico predicted T-cell epitopes of the specific HLA/model antigen combination. The transient co-expression of a single affinity-tagged MHC molecule combined with a disease-specific antigen in a human cell line in our FASTMAP pipeline provides the opportunity to identify potential T-cell epitopes/endogenously processed MHC-bound peptides in a very cost-effective, fast, and customizable system with high-throughput potential., Competing Interests: Authors LW, MH, VS, HD, and BV are employees of the company CSL Innovation GmbH. LC is employee of CSL Behring AG as well as PS for CSL Limited. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The work was funded by CSL Innovation GmbH. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Weisbrod, Capriotti, Hofmann, Spieler, Dersch, Voedisch, Schmidt and Knake.)

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

Investigations on binding strength differences of non-covalent protein complex components were performed by mass spectrometry. T4 fibritin foldon (T4Ff) is a well-studied miniprotein, which together with its biotinylated version served as model system to represent a compactly folded protein to which an Intrinsically Disordered Region (IDR) was attached. The apparent enthalpies of the gas phase dissociation reactions of the homo-trimeric foldon F-F-F and of the homo-trimeric triply biotinylated foldon bF-bF-bF have been determined to be rather similar (3.32 kJ/mol and 3.85 kJ/mol) but quite distinct from those of the singly and doubly biotinylated hetero-trimers F-F-bF and F-bF-bF (1.86 kJ/mol and 1.08 kJ/mol). Molecular dynamics simulations suggest that the ground states of the (biotinylated) T4Ff trimers are highly symmetric and well comparable to each other, indicating that the energy levels of all four (biotinylated) T4Ff trimer ground states are nearly indistinguishable. The experimentally determined differences and/or similarities in enthalpies of the complex dissociation reactions are explained by entropic spring effects, which are noticeable in the T4Ff hetero-trimers but not in the T4Ff homo-trimers. A lowering of the transition state energy levels of the T4Ff hetero-trimers seems likely because the biotin moieties, mimicking intrinsically disordered regions (IDRs), induced asymmetries in the transition states of the biotinylated T4Ff hetero-trimers. This transition state energy level lowering effect is absent in the T4Ff homo-trimer, as well as in the triply biotinylated T4Ff homo-trimer. In the latter, the IDR-associated entropic spring effects on complex stability cancel each other out. ITEM-FIVE enabled semi-quantitative determination of energy differences of complex dissociation reactions, whose differences were modulated by IDRs attached to compactly folded proteins.

A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the DVPDY sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen-antibody and other protein-protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated.

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nathalie Norais is a permanent employee of the GSK group of companies. Laura R. Grauslund and Susanne Ständer were employees of the GSK group of companies and PhD students at the University of Copenhagen at the time of this study. The work was funded by the Horizon 2020 EU framework program for research and innovation (VADEMA grant agreement no. 675879) and the European Research Council (ERC) (vAMRes grant agreement no. 787552). Bexsero is a trademark owned by or licensed to the GSK group of companies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We investigate a relatively underexplored component of the gut-immune axis by profiling the antibody response to gut phages using Phage Immunoprecipitation Sequencing (PhIP-Seq). To cover large antigenic spaces, we develop Dolphyn, a method that uses machine learning to select peptides from protein sets and compresses the proteome through epitope-stitching. Dolphyn compresses the size of a peptide library by 78% compared to traditional tiling, increasing the antibody-reactive peptides from 10% to 31%. We find that the immune system develops antibodies to human gut bacteria-infecting viruses, particularly E.coli-infecting Myoviridae. Cost-effective PhIP-Seq libraries designed with Dolphyn enable the assessment of a wider range of proteins in a single experiment, thus facilitating the study of the gut-immune axis., (© 2024. The Author(s).)

Natural killer (NK) cells are key players in human innate immunity. Cell engager antibody formats that recruit and activate NK cells more effectively have emerged as a promising immunotherapy approach to target cancer cells through more effective antibody-dependent cell-mediated cytotoxicity (ADCC). Monoclonal antibody drugs with ADCC activity have shown clinical benefit and improved outcomes for patients with certain types of cancer. CD16a, a Fc gamma III receptor, is the major component that is responsible for the ADCC activity of NK cells. Screening AvantGen's yeast displayed human antibody libraries led to the isolation of 2 antibody clones, #1A2 and #2-2A2, that selectively recognize both isoforms (F and V) of CD16a on primary NK cells with high affinity, yet minimally (#1A2) or do not (#2-2A2) cross-react with both allelotypes of CD16b (NA1 and NA2) expressed by neutrophils. Epitope mapping studies revealed that they bind to an epitope dependent on residue Y158 of CD16a, since mutation of Y158 to the corresponding CD16b residue H158 completely abolishes binding to CD16a. When formatted as bispecific antibodies targeting CD16a and a tumor-associated antigen (TAA, e.g. CD19), they exhibit specific binding to NK cells and induce potent NK cell activation upon encountering tumor cells, resulting in effective tumor cell killing. Notably, these bispecific antibody engagers stimulate NK cell cytokine release during co-culture with target cells, resulting in target cell cytotoxicity. These anti-CD16a antibody clones are promising candidates for combination with any TAA of interest, offering the potential for novel NK cell engager-based cancer therapeutics that are minimally affected by the high concentrations of human IgG in the circulation.

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.)

Characterization of peptide antibodies through identification of their target epitopes is of utmost importance, as information about epitopes provide important knowledge, among others, for discovery and development of new therapeutics, vaccines, and diagnostics.This chapter describes a strategy for mapping of continuous peptide antibody epitopes using resin-bound and soluble peptides. The approach combines three different types of peptide sets for full characterization of peptide antibodies; (i) overlapping peptides, used to locate antigenic regions; (ii) truncated peptides, used to identify the minimal peptide length required for antibody binding; and (iii) substituted peptides, used to identify the key residues important for antibody binding and to determine the specific contribution of key residues. For initial screening, resin-bound peptides are used for epitope estimation, while soluble peptides subsequently are used for final epitope characterization and identification of critical hot spot residues. The combination of resin-bound peptides and soluble peptides for epitope mapping provides a time-saving and straightforward approach for characterization of antibodies recognizing continuous epitopes, which applies to peptide antibodies and occasionally antibodies directed to larger proteins as well., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vaccination is an effective means of inducing immune protection to prevent transmissible diseases. During the Covid-19 pandemic, immunizations using traditional and novel vaccine platforms such as the inactivated SARSCo-V-2 vaccine, adenoviral-vectored, and nucleic acid-based mRNA vaccines have been relatively successful in controlling the rates of infection and hospitalizations. Nevertheless, the danger posed by the emergence of SARS-CoV-2 variants would set the stage for the design of next generation vaccines. To overcome the lack of efficacy of current vaccines against emerging SARS-CoV-2 variants, new vaccines must be able to overcome the reduced effectiveness of the current vaccines. Since the current Covid-19 vaccines are dependent on the whole S-protein of Wuhan strain as the antigen, mutations have rendered the current Covid-19 vaccines less effective against variants of concern (VoCs). Instead of using the whole S-protein, peptide-based epitopes could be predicted using immunoinformatic approaches, simulation of the 3D structures, overlapping peptides covering the whole length of the S-protein or peptide arrays based on synthetic peptide combinatorial libraries comprising peptides recognizable by monoclonal antibodies. B-cell epitopes were predicted, and immunogenicity of peptides was validated in mice by immunizing mice with peptides conjugated to keyhole limpet hemocyanin (KLH) mixed with Montanide 51 as an adjuvant. The immunogenicity of epitopes that could elicit peptide specific IgGs was determined by peptide-based ELISA. Neutralizing activities were determined by cPass and pseudovirus-based neutralization assays., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

B-cell epitope prediction is key to developing peptide-based vaccines and immunodiagnostics along with antibodies for prophylactic, therapeutic and/or diagnostic use. This entails estimating paratope binding affinity for variable-length peptidic sequences subject to constraints on both paratope accessibility and antigen conformational flexibility, as described herein for the HAPTIC2/HEPTAD User Toolkit (HUT). HUT comprises the Heuristic Affinity Prediction Tool for Immune Complexes 2 (HAPTIC2), the HAPTIC2-like Epitope Prediction Tool for Antigen with Disulfide (HEPTAD) and the HAPTIC2/HEPTAD Input Preprocessor (HIP). HIP enables tagging of residues (e.g., in hydrophobic blobs, ordered regions and glycosylation motifs) for exclusion from downstream analyses by HAPTIC2 and HEPTAD. HAPTIC2 estimates paratope binding affinity for disulfide-free disordered peptidic antigens (by analogy between flexible-ligand docking and protein folding), from terms attributed to compaction (in view of sequence length, charge and temperature-dependent polyproline-II helical propensity), collapse (disfavored by residue bulkiness) and contact (with glycine and proline regarded as polar residues that hydrogen bond with paratopes). HEPTAD analyzes antigen sequences that each contain two cysteine residues for which the impact of disulfide pairing is estimated as a correction to the free-energy penalty of compaction. All of HUT is freely accessible online ( https://freeshell.de/~badong/hut.htm )., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Identifying antigens within a pathogen is a critical task to develop effective vaccines and diagnostic methods, as well as understanding the evolution and adaptation to host immune responses. Historically, antigenicity was studied with experiments that evaluate the immune response against selected fragments of pathogens. Using this approach, the scientific community has gathered abundant information regarding which pathogenic fragments are immunogenic. The systematic collection of this data has enabled unraveling many of the fundamental rules underlying the properties defining epitopes and immunogenicity, and has resulted in the creation of a large panel of immunologically relevant predictive (in silico) tools. The development and application of such tools have proven to accelerate the identification of novel epitopes within biomedical applications reducing experimental costs. This chapter introduces some basic concepts about MHC presentation, T cell and B cell epitopes, the experimental efforts to determine those, and focuses on state-of-the-art methods for epitope prediction, highlighting their strengths and limitations, and catering instructions for their rational use., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The use of precise epitope peptides as antigens is essential for accurate serological diagnosis of viral-infected individuals, but now it remains an unsolvable problem for mapping precise B cell epitopes (BCEs) recognized by human serum. To address this challenge, we propose a novel epitope delimitation (ED) method to uncover BCEs in the delineated human IgG-reactive (HR) antigenic peptides (APs). Specifically, the method based on the rationale of similarities in humoral immune responses between mammalian species consists of a pair of elements: experimentally delineated HR-AP and rabbit-recognized (RR) BCE motif and corresponding pair of sequence alignment analysis. As a result of using the ED approach, after decoding four RR-epitomes of human papillomavirus types 16/18-E6 and E7 proteins utilizing rabbit serum against each recombinant protein and sequence alignment analysis of HR-APs and RR-BCEs, 19 fine BCEs in 17 of 22 known HR-APs were defined based on each corresponding RR-BCE motifs, including the type-specificity of each delimited BCE in homologous proteins. The test with 22 known 16/20mer HR-APs demonstrated that the ED method is effective and efficient, indicating that it can be used as an alternative method to the conventional identification of fine BCEs using overlapping 8mer peptides., (© 2024 Wiley Periodicals LLC.)

Influenza virus infection can cause kidney damage. However, the link between influenza infection and disease is still unclear. The purpose of this study was to analyze the relationship between heterophilic epitopes on H5N1 hemagglutinin (HA) and disease. The monoclonal antibody (mAb) against H5N1 was prepared, mAbs binding to human kidney tissue were screened, and the reactivities of mAbs with five different subtypes of influenza virus were detected. Design and synthesize the peptides according to the common amino acid sequence of these antigens, and analyze the distribution of the epitope on the crystal structure of HA. Immunological methods were used to detect whether the heterophilic epitopes could induce the production of antibodies that cross-react with kidney tissue. The results showed that H5-30 mA b binding to human kidney tissue recognized the heterophilic epitope 191-LVLWGIHHP-199 on the head of HA. The key amino acid were V192, L193, W194 and I196, which were highly conserved in human and avian influenza virus HA. The heterophilic epitope could induce mice to produce different mAbs binding to kidney tissue. Such heterophilic antibodies were also detected in the serum of the patients. It can provide materials for the mechanism of renal diseases caused by influenza virus infection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors declare that they have no conflict of interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In small animal models of severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infection, ferrets ( Mustela putorius furo ) have been used to investigate the pathogenesis. Podoplanin (PDPN) is an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. Monoclonal antibodies (mAbs) against ferret PDPN (ferPDPN) are useful for the pathological analyses of those tissues. We previously established an anti-ferPDPN mAb, PMab-292 using the Cell-Based Immunization and Screening (CBIS) method. In this study, we determined the critical epitope of PMab-292 using flow cytometry. The ferPDPN deletion mutants analysis revealed that the Val34 is located at the N-terminus of the PMab-292 epitope. Furthermore, the PA tag-substituted analysis (PA scanning) showed that Asp39 is located at the C-terminus of PMab-292 epitope. The epitope sequence (VRPEDD) also exists between Val26 and Asp31 of ferPDPN, indicating that PMab-292 recognizes the tandem repeat of the VRPEDD sequence of ferPDPN.

Epitopes are specific regions on an antigen's surface that the immune system recognizes. Epitopes are usually protein regions on foreign immune-stimulating entities such as viruses and bacteria, and in some cases, endogenous proteins may act as antigens. Identifying epitopes is crucial for accelerating the development of vaccines and immunotherapies. However, mapping epitopes in pathogen proteomes is challenging using conventional methods. Screening artificial neoepitope libraries against antibodies can overcome this issue. Here, we applied conventional sequence analysis and methods inspired in natural language processing to reveal specific sequence patterns in the linear epitopes deposited in the Immune Epitope Database (www.iedb.org) that can serve as building blocks for the design of universal epitope libraries. Our results reveal that amino acid frequency in annotated linear epitopes differs from that in the human proteome. Aromatic residues are overrepresented, while the presence of cysteines is practically null in epitopes. Byte pair encoding tokenization shows high frequencies of tryptophan in tokens of 5, 6, and 7 amino acids, corroborating the findings of the conventional sequence analysis. These results can be applied to reduce the diversity of linear epitope libraries by orders of magnitude.

Efficient identification of epitopes is crucial for drug discovery and design as it enables the selection of optimal epitopes, expansion of lead antibody diversity, and verification of binding interface. Although high-resolution low throughput methods like x-ray crystallography can determine epitopes or protein-protein interactions accurately, they are time-consuming and can only be applied to a limited number of complexes. To overcome these limitations, we have developed a rapid computational method that incorporates N-linked glycans to mask epitopes or protein interaction surfaces, thereby providing a mapping of these regions. Using human coagulation factor IXa (fIXa) as a model system, we computationally screened 158 positions and expressed 98 variants to test experimentally for epitope mapping. We were able to delineate epitopes rapidly and reliably through the insertion of N-linked glycans that efficiently disrupted binding in a site-selective manner. To validate the efficacy of our method, we conducted ELISA experiments and high-throughput yeast surface display assays. Furthermore, x-ray crystallography was employed to verify the results, thereby recapitulating through the method of N-linked glycans a coarse-grained mapping of the epitope., (© 2023 Novo Nordisk A/S and The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Following viral infection, viral antigens bind specifically to receptors on the surface of lymphocytes thereby activating adaptive immunity in the host. An epitope, the smallest structural and functional unit of an antigen, binds specifically to an antibody or antigen receptor, to serve as key sites for the activation of adaptive immunity. The complexity and diverse range of epitopes are essential to study and map for the diagnosis of disease, the design of vaccines and for immunotherapy. Mapping the location of these specific epitopes has become a hot topic in immunology and immune therapy. Recently, epitope mapping techniques have evolved to become multiplexed, with the advent of high-throughput sequencing and techniques such as bacteriophage-display libraries and deep mutational scanning. Here, we briefly introduce the principles, advantages, and disadvantages of the latest epitope mapping techniques with examples for viral antigen discovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Hu and Irving.)

Structural proteomics techniques are useful for the determination of protein interaction interfaces. Each technique provides orthogonal structural information on the structure and the location of protein interaction sites. Here, we have characterized a monoclonal antibody epitope for a protein antigen by a combination of differential photoreactive surface modification (SM), cross-linking (CL), differential hydrogen-deuterium exchange (HDX), and epitope extraction/excision. We found that experimental data from different approaches agree with each other in determining the epitope of the monoclonal antibody on the protein antigens using the HIV-1 p24-mAb E complex as an illustrative example. A combination of these multiple structural proteomics approaches results in a detailed picture of the interaction of the proteins and increases confidence in the determination of the final structure of the protein interaction interface. Data are available via ProteomeXchange with identifier PXD040902.

Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) is widely used for monoclonal antibody (mAb) epitope mapping, which aids in the development of therapeutic mAbs and vaccines, as well as enables the understanding of viral immune evasion. Numerous mAbs are known to recognize N-glycosylated epitopes and to bind in close proximity to an N -glycan site; however, glycosylated protein sites are typically obscured from HDX detection as a result of the inherent heterogeneity of glycans. To overcome this limitation, we covalently immobilized the glycosidase PNGase Dj on a solid resin and incorporated it into an online HDX-MS workflow for post-HDX deglycosylation. The resin-immobilized PNGase Dj exhibited robust tolerance to various buffer conditions and was employed in a column format that can be readily adapted into a typical HDX-MS platform. Using this system, we were able to obtain full sequence coverage of the SARS-CoV-2 receptor-binding domain (RBD) and map the glycosylated epitope of the glycan-binding mAb S309 to the RBD.

The highly pathogenic avian influenza H5 2.3.4.4 and 2.3.2.1c subclades have distinct antigenic properties and are responsible for the majority of human infections. Therefore, it is essential to understand the processes by which antibodies inhibit these subclade viruses to develop effective therapies and vaccines to prevent their escape from neutralizing antibodies. Herein, we report the epitopes of two specific monoclonal antibodies (mAbs) targeting haemagglutinin (HA) of the H5 2.3.4.4b subclade and their neutralizing abilities. The results indicated that the two mAbs provided specific protection against the H5 2.3.4.4b clade viral challenge in MDCK cells and mouse models. Through epitope identification and docking studies, we showed that these novel sites (which are located near the 130-loop (S136, T143) and 190-helix (N199, N205) of HA receptor-binding sites that contribute to the binding affinity of neutralizing mAbs and six residues of the complementarity-determining regions) can be targeted to generate antibodies with enhanced cross-neutralization. This can also help in understanding escape mutations that differ among the H5 2.3.4.4b, h, and 2.3.2.1c subclades. These results provide specific information to facilitate future vaccine design and therapeutics for both subclade viruses, which are dominant and pose a serious threat to humans.

To accurately map the B-cell linear epitopes of lysozyme (LYS) in eggs, five bioinformatics tools were first used to obtain the mimotopes. Afterward, based on the Chinese egg-allergic sera samples screened by the indirect enzyme-linked immunosorbent, the epitopes possessing the capability of binding to IgG/IgE were mapped at both pooled and individual levels by using overlapping peptides covering the complete amino acid sequence of LYS. Six B-cell linear epitopes and two dominant B-cell linear epitopes that could bind to LYS-sIgG were mapped for the first time. Seven IgE-binding epitopes and three dominant IgE-binding epitopes were also obtained. Furthermore, AA31-34 and AA88-91 were the shared dominant epitopes of LYS-sIgG and LYS-sIgE at pooled and individual levels. Overall, the mapped B-cell linear epitopes filled in the gaps in the study of LYS epitopes, and the results may provide theoretical support for the following immunotherapy of egg allergy.

We recently developed a novel anti-human C-C chemokine receptor 9 (hCCR9) monoclonal antibody (mAb), C 9 Mab-11, which is applicable to flow cytometry, western blotting, and enzyme-linked immunosorbent assay (ELISA). This study aims to identify the binding epitope of C 9 Mab-11 by using 1 × and 2 × alanine (or glycine) substituted-hCCR9 peptides (1 × and 2 × Ala-scan) by ELISA. According to the 1 × Ala-scan analysis, the response of C 9 Mab-11 was diminished against M13A of the hCCR9 peptide, but was not eliminated. In the 2 × Ala-scan analysis, the reactions were abolished in the substitution of P11A-N12A, N12A-M13A, and M13A-A14G of hCCR9 N-terminal peptides. The results indicate that the binding epitope of C 9 Mab-11 includes Pro11, Asn12, Met13, and Ala14 of hCCR9, with the region around Met13 being particularly important. The successful identification of the C 9 Mab-11 epitope might be useful for the future pathophysiological analysis of hCCR9.

Precision medicine requests accurate serological inspections to precisely stratify patients for targeted treatment. Intact transition epitope mapping analysis proved surrogate seroconversion of a model organism's serum when spiked with a monoclonal murine anti-Ovalbumin antibody (mAb) with epitope resolution. Isolation of the IgG fraction from blood serum applied two consecutive protein precipitation steps followed by ultrafiltration and resulted in an ESI-MS analysis-ready IgG preparation. For epitope mapping by epitope extraction, the Ovalbumin antigen was digested with trypsin. After desalting, the peptide mixture was added to the ESI-MS-ready IgG preparation from mAb-spiked serum and the solution was incubated to form an immune complex between the Ovalbumin-derived epitope peptide and the anti-Ovalbumin mAb. Then, the entire mixture of proteins and peptides was directly electrosprayed. Sorting of ions in the mass spectrometer's gas phase, dissociation of the immune complex ions by collision-induced dissociation, and recording of the epitope peptide ion that had been released from the immune complex proved the presence of the anti-Ovalbumin mAb in serum. Mass determination of the complex-released epitope peptide ion with isotope resolution is highly accurate, guaranteeing high specificity of this novel analysis approach, which is termed Intact Transition Epitope Mapping-Serological Inspections by Epitope EXtraction (ITEM-SIX).

Characterization of antibody binding epitopes is an important factor in therapeutic drug discovery, as the binding site determines and drives antibody pharmacology and pharmacokinetics. Here, we present a novel application of carbene chemical footprinting with mass spectrometry for identification of antibody binding epitopes at the single-residue level. Two different photoactivated diazirine reagents provide complementary labeling information allowing structural refinement of the antibody binding interface. We applied this technique to map the epitopes of multiple MICA and CTLA-4 antibodies and validated the findings with X-ray crystallography and yeast surface display epitope mapping. The characterized epitopes were used to understand biolayer interferometry-derived competitive binding results at the structural level. We show that carbene footprinting provides fast and high-resolution epitope information critical in the antibody selection process and enables mechanistic understanding of function to accelerate the drug discovery process.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

With Intact Transition Epitope Mapping-Thermodynamic Weak-force Order (ITEM-TWO) analysis in combination with molecular modeling, the phosphorylation-dependent molecular recognition motif of the anti-HpTGEKP antibody has been investigated with binary and ternary component mixtures consisting of antibody and (phospho-) peptides. Amino acid sequences have been selected to match either the antibody's recognition motif or the cancer-related zinc finger protein mutations and phosphorylations of the respective amino acid residues. Upon electrospraying of all the components of the mixtures, that is, hexapeptides, antibody, and intact immune complexes, the produced ions were subjected to mass spectrometric mass filtering. The antibody ions as well as the immune complex ions traversed into the mass spectrometer's collision chamber, whereas paths of unbound peptide ions were blocked prior to entering the collision cell. After dissociation of the multiply charged immune complexes in the gas phase, the complex-released peptide ions were recorded after having traversed the second mass filter. Complex-released peptides were unambiguously identified by their masses using mass analysis with isotope resolution. From the results of our studies with seven (phospho-) peptides with distinct amino acid sequences, which resembled either the antibody's binding motif or mutations, we conclude the following: (i) A negatively charged phospho group, located near the peptide's N-terminus is mandatory for antibody binding when placed on the peptide surface at a precise distance to the C-terminally located positively charged ε-amino group of a lysinyl residue. (ii) A bulky amino acid residue, such as the tyrosinyl residue at the N-terminal position of the (phospho-) threoninyl residue, abolishes antibody binding. (iii) Two closely spaced phospho groups negatively interfere with the surface polarity pattern and abolish antibody binding as well. (iv) Non-phosphorylated peptides are not binding partners of the anti-HpTGEKP antibody.

An anti-mouse CXC chemokine receptor 6 (mCXCR6) monoclonal antibody (mAb), Cx 6 Mab-1, was developed recently. Cx 6 Mab-1 is applicable for flow cytometry, Western blotting, and enzyme-linked immunosorbent assay. The purpose of this study is to determine the binding epitope of Cx 6 Mab-1 using 2 × alanine mutated mCXCR6. Analysis of flow cytometry revealed that Cx 6 Mab-1 did not recognize S8A-A9G, L10A-Y11A, D12A-G13A, and H14A-Y15A mutants of mCXCR6. The results clearly indicate that the binding epitope of Cx 6 Mab-1 includes Ser8, Ala9, Leu10, Tyr11, Asp12, Gly13, His14, and Tyr15 of mCXCR6. The successful determination of the Cx 6 Mab-1 epitope might contribute to the pathophysiological investigation of mCXCR6.

Antibody-based point-of-care diagnostics have become indispensable for modern medicine. In-depth analysis of antibody recognition mechanisms is the key to tailoring the accuracy and precision of test results, which themselves are crucial for targeted and personalized therapy. A rapid and robust method is desired by which binding strengths between antigens and antibodies of concern can be fine-mapped with amino acid residue resolution to examine the assumedly serious effects of single amino acid polymorphisms on insufficiencies of antibody-based detection capabilities of, e.g., life-threatening conditions such as myocardial infarction. The experimental ITEM-FOUR approach makes use of modern mass spectrometry instrumentation to investigate intact immune complexes in the gas phase. ITEM-FOUR together with molecular dynamics simulations, enables the determination of the influences of individually exchanged amino acid residues within a defined epitope on an immune complex's binding strength. Wild-type and mutated epitope peptides were ranked according to their experimentally determined dissociation enthalpies relative to each other, thereby revealing which single amino acid polymorphism caused weakened, impaired, and even abolished antibody binding. Investigating a diagnostically relevant human cardiac Troponin I epitope for which seven nonsynonymous single nucleotide polymorphisms are known to exist in the human population tackles a medically relevant but hitherto unsolved problem of current antibody-based point-of-care diagnostics.

The lack of incorporating epitope information into the selection process makes the conventional antibody screening method less effective in identifying antibodies with desired functions. Here, we developed an epitope-directed antibody selection method by designing a directed library favoring the target epitope and a precise "counter" antigen for clearing irrelevant binders in the library. With this method, we successfully isolated an antibody, pF7_A5, that targets the less conserved region on the FZD2/7 CRD as designed. Guided by the structure of pF7_A5-FZD2 CRD , a further round of evolution was conducted together with the "counter" antigen selection strategy, and ultimately, an FZD2-specific antibody and an FZD7-preferred antibody were obtained. Because of targeting the predefined functional site, all these antibodies exhibited the expected modulatory activity on the Wnt pathway. Together, the method developed here will be useful in antibody drug discovery, and the identified FZD antibodies will have clinical potential in FZD-related cancer therapy., Competing Interests: Declaration of interests The authors have filed a provisional patent application for the antibodies described in this work., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In this paper, we introduce a screening protocol for epitope mapping by hydrogen exchange mass spectrometry (HX-MS) that has higher throughput than a traditional HX-MS epitope mapping. In the screening protocol, three HX labeling times (20, 1000, and 86400 s) are each measured without replicates. The experimental protocol is anchored on a single epitope mapping experiment conducted using the traditional complete protocol (five HX times measured in triplicate) that is used to define HX times and define significance limits. Previously, we reported traditional epitope mapping results on the Borrelia burgdorferi outer surface protein A (OspA) antigen that are in excellent agreement with the X-ray crystallography results. Here, we show that the screening protocol and complete HX-MS identify identical epitopes of OspA but that the screening protocol has a 5-fold higher throughput.

Antibody-mediated neurological diseases constitute an emerging clinical entity that remains to be fully explored. Recent studies identified autoantibodies that directly confer pathogenicity, and it was shown that in these cases immunotherapies can result in profound positive patient responses. These advances highlight the urgent need for improved means to effectively screen patient samples for novel autoantibodies (aAbs) and their subsequent characterization. Here, we discuss challenges and opportunities for peptide microarrays to contribute to the identification, mapping, and characterization of the underlying monospecific disease-defining binding surfaces. We outline control experiments, workflow modifications and bioinformatic filtering methods that enhance the predictive power of array-based studies. Further, we highlight experimental and computer-based display approaches that have the potential to expand the use of synthetic microarrays over the detection of discontinuous epitopes. Knowledge over the autoantibody epitopes in neurological disease will enhance our understanding of the pathological mechanisms and thereby potentially contribute to novel diagnostic approaches or even innovative antigen-specific treatments that avoid the serious adverse effects seen with currently used immunosuppressive therapies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Identifying protein antigenic epitopes that are recognizable by antibodies is a key step in immunologic research. This type of research has broad medical applications, such as new immunodiagnostic reagent discovery, vaccine design, and antibody design. However, due to the countless possibilities of potential epitopes, the experimental search through trial and error would be too costly and time-consuming to be practical. To facilitate this process and improve its efficiency, computational methods were developed to predict both linear epitopes and discontinuous antigenic epitopes. For linear B-cell epitope prediction, many methods were developed, including PREDITOP, PEOPLE, BEPITOPE, BepiPred, COBEpro, ABCpred, AAP, BCPred, BayesB, BEOracle/BROracle, BEST, LBEEP, DRREP, iBCE-EL, SVMTriP, etc. For the more challenging yet important task of discontinuous epitope prediction, methods were also developed, including CEP, DiscoTope, PEPITO, ElliPro, SEPPA, EPITOPIA, PEASE, EpiPred, SEPIa, EPCES, EPSVR, etc. In this chapter, we will discuss computational methods for B-cell epitope predictions of both linear and discontinuous epitopes. SVMTriP and EPCES/EPCSVR, the most successful among the methods for each type of the predictions, will be used as model methods to detail the standard protocols. For linear epitope prediction, SVMTriP was reported to achieve a sensitivity of 80.1% and a precision of 55.2% with a fivefold cross-validation based on a large dataset, yielding an AUC of 0.702. For discontinuous or conformational B-cell epitope prediction, EPCES and EPCSVR were both benchmarked by a curated independent test dataset in which all antigens had no complex structures with the antibody. The identified epitopes by these methods were later independently validated by various biochemical experiments. For these three model methods, webservers and all datasets are publicly available at http://sysbio.unl.edu/SVMTriP , http://sysbio.unl.edu/EPCES/ , and http://sysbio.unl.edu/EPSVR/ ., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

This chapter describes an epitope-directed approach to generate antipeptide monoclonal antibodies to multiple nonoverlapping protein sites using a cocktail of fusion peptides as immunogen. It provides a step-by-step protocol on how antigenic peptides on a target protein can be identified by in silico prediction and discusses considerations for final peptide selection. Each antigenic peptide (10-20 amino acids long) is displayed as three-copy inserts on the surface exposed loop of a thioredoxin scaffold protein. The corresponding DNA coding sequence specifying the tripeptide insert flanked by Gly-Ser-Gly-Ser-Gly linkers is cloned in-frame into the Rsr II site of the thioredoxin gene in the pET-32a vector. The presence of a C-terminal polyhistidine tag (His 6 -tag) allows the soluble fusion proteins to be purified by one-step native immobilized metal affinity chromatography (IMAC) to greater than 95% purity. Multiple thioredoxin fusion proteins are mixed in equimolar concentrations and used as an immunogen cocktail for animal immunization. The use of short antigenic peptides of known sequence facilitates direct epitope mapping requiring only small mutagenesis scan peptide libraries in the multipin peptide format., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Monoclonal antibodies have become an important class of therapeutics in the last 30 years. Because the mechanism of action of therapeutic antibodies is intimately linked to their binding epitopes, identification of the epitope of an antibody to the antigen plays a central role during antibody drug development. The gold standard of epitope mapping, X-ray crystallography, requires a high degree of proficiency with no guarantee of success. Here, we evaluated six widely used alternative methods for epitope identification (peptide array, alanine scan, domain exchange, hydrogen-deuterium exchange, chemical cross-linking, and hydroxyl radical footprinting) in five antibody-antigen combinations (pembrolizumab+PD1, nivolumab+PD1, ipilimumab+CTLA4, tremelimumab+CTLA4, and MK-5890+CD27). The advantages and disadvantages of each technique are demonstrated by our data and practical advice on when and how to apply specific epitope mapping techniques during the drug development process is provided. Our results suggest chemical cross-linking most accurately identifies the epitope as defined by crystallography.

The design of optimized protein antigens is a fundamental step in the development of new vaccine candidates and in the detection of therapeutic antibodies. A fundamental prerequisite is the identification of antigenic regions that are most prone to interact with antibodies, namely, B-cell epitopes. Here, we describe an efficient structure-based computational method for epitope prediction, called MLCE. In this approach, all that is required is the 3D structure of the antigen of interest. MLCE can be applied to glycosylated proteins, facilitating the identification of immunoreactive versus immune-shielding carbohydrates., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Understanding antibody specificity and defining response profiles to antigens continue to be essential to both vaccine research and therapeutic antibody development. Peptide scanning assays enable mapping of continuous epitopes in order to delineate antibody-antigen interactions beyond traditional immunoassay formats. We have developed a relatively low-cost method to generate peptide microarray slides for antibody binding studies that allow for interrogation of up to 1536 overlapping peptides derived from the target antigens on a single microslide. Using an IntavisAG MultiPep RS peptide synthesizer and a Digilab MicroGrid II 600 microarray printer robot, each peptide is tagged with a polyethylene glycol aminooxy terminus to improve peptide solubility, orientation, and conjugation efficiency to the slide surface. Interrogation of the surface can then be performed using polyclonal immune sera or monoclonal antibodies, and sensitive detection using an InnoScan 1100 AL scanner with fluorescent-conjugated secondary reagents maximizes conservation of reagents., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Peptide microarrays are a powerful tool to identify linear epitopes of food allergens in a high-throughput manner. The main advantages of the microarray-based immunoassay are as follows: the possibility to assay thousands of targets simultaneously, the requirement of a low volume of serum, the more robust statistical analysis, and the possibility to test simultaneously several immunoglobulin subclasses. Among them, the last one has a special interest in the field of food allergy, because the development of tolerance to food allergens has been associated with a decrease in IgE and an increase in IgG4 levels against linear epitopes. However, the main limitation to the clinical use of microarray is the automated analysis of the data. Recent studies mapping the linear epitopes of food allergens with peptide microarray immunoassays have identified peptide biomarkers that can be used for early diagnosis of food allergies and to predict their severity or the self-development of tolerance. Using this approach, we have worked on epitope mapping of the two most important food allergens in the Spanish population, cow's milk, and chicken eggs. The final aim of these studies is to define subsets of peptides that could be used as biomarkers to improve the diagnosis and prognosis of food allergies. This chapter describes the protocol to produce microarrays using a library of overlapping peptides corresponding to the primary sequences of food allergens and data acquisition and analysis of IgE and IgG4 binding epitopes., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Delineating the precise regions on an antigen that are targeted by antibodies has become a key step for the development of antibody therapeutics. X-ray crystallography and cryogenic electron microscopy are considered the gold standard for providing precise information about these binding sites at atomic resolution. However, they are labor-intensive and a successful outcome is not guaranteed. We used deep mutational scanning (DMS) of the human LAMP-1 antigen displayed on yeast surface and leveraged next-generation sequencing to observe the effect of individual mutants on the binding of two LAMP-1 antibodies and to determine their functional epitopes on LAMP-1. Fine-tuned epitope mapping by DMS approaches is augmented by knowledge of experimental antigen structure. As human LAMP-1 structure has not yet been solved, we used the AlphaFold predicted structure of the full-length protein to combine with DMS data and ultimately finely map antibody epitopes. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one of the two antibodies with a LAMP-1 luminal domain. Finally, we used AlphaFold models of non-human LAMP-1 to understand the lack of mAb cross-reactivity. While both epitopes in the murine form exhibit multiple mutations in comparison to human LAMP-1, only one and two mutations in the Macaca form suffice to hinder the recognition by mAb B and A, respectively. Altogether, this study promotes a new application of AlphaFold to speed up precision mapping of antibody-antigen interactions and consequently accelerate antibody engineering for optimization.