Determination Of Antibodies Research Articles

Allogeneic HLA Humoral Immunogenicity and the Prediction of Donor-Specific HLA Antibody Development.

The development of de novo donor-specific HLA antibodies (dnDSAs) following solid organ transplantation is considered a major risk factor for poor long-term allograft outcomes. The prediction of dnDSA development is a boon to transplant recipients, yet the assessment of allo-HLA immunogenicity remains imprecise. Despite the recent technological advances, a comprehensive evaluation of allo-HLA immunogenicity, which includes both B and T cell allorecognition, is still warranted. Recent studies have proposed using mismatched HLA epitopes (antibody and T cell) as a prognostic biomarker for humoral alloimmunity. However, the identification of immunogenic HLA mismatches has not progressed despite significant improvements in the identification of permissible mismatches. Certainly, the prediction of dnDSA development may benefit permissible HLA mismatched organ transplantations, personalized immunosuppression, and clinical trial design. However, characteristics that go beyond the listing of mismatched HLA antibody epitopes and T cell epitopes, such as the generation of HLA T cell epitope repertoires, recipient's HLA class II phenotype, and immunosuppressive regiments, are required for the precise assessment of allo-HLA immunogenicity.

Mammalian cell display with automated oligo design and library assembly allows for rapid residue level conformational epitope mapping

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.

A Nanoparticle Vaccine Displaying Conserved Epitopes of the Preexisting Neutralizing Antibody Confers Broad Protection against SARS-CoV-2 Variants.

The rapid development of the SARS-CoV-2 vaccine has been used to prevent the spread of coronavirus 2019 (COVID-19). However, the ongoing and future pandemics caused by SARS-CoV-2 variants and mutations underscore the need for effective vaccines that provide broad-spectrum protection. Here, we developed a nanoparticle vaccine with broad protection against divergent SARS-CoV-2 variants. The corresponding conserved epitopes of the preexisting neutralizing (CePn) antibody were presented on a self-assembling Helicobacter pylori ferritin to generate the CePnF nanoparticle. Intranasal immunization of mice with CePnF nanoparticles induced robust humoral, cellular, and mucosal immune responses and a long-lasting immunity. The CePnF-induced antibodies exhibited cross-reactivity and neutralizing activity against different coronaviruses (CoVs). CePnF vaccination significantly inhibited the replication and pathology of SARS-CoV-2 Delta, WIV04, and Omicron strains in hACE2 transgenic mice and, thus, conferred broad protection against these SARS-CoV-2 variants. Our constructed nanovaccine targeting the conserved epitopes of the preexisting neutralizing antibodies can serve as a promising candidate for a universal SARS-CoV-2 vaccine.

Neutralizing antibodies reveal cryptic vulnerabilities and interdomain crosstalk in the porcine deltacoronavirus spike protein

Porcine deltacoronavirus (PDCoV) is an emerging enteric pathogen that has recently been detected in humans. Despite this zoonotic concern, the antigenic structure of PDCoV remains unknown. The virus relies on its spike (S) protein for cell entry, making it a prime target for neutralizing antibodies. Here, we generate and characterize a set of neutralizing antibodies targeting the S protein, shedding light on PDCoV S interdomain crosstalk and its vulnerable sites. Among the four identified antibodies, one targets the S1A domain, causing local and long-range conformational changes, resulting in partial exposure of the S1B domain. The other antibodies bind the S1B domain, disrupting binding to aminopeptidase N (APN), the entry receptor for PDCoV. Notably, the epitopes of these S1B-targeting antibodies are concealed in the prefusion S trimer conformation, highlighting the necessity for conformational changes for effective antibody binding. The binding footprint of one S1B binder entirely overlaps with APN-interacting residues and thus targets a highly conserved epitope. These findings provide structural insights into the humoral immune response against the PDCoV S protein, potentially guiding vaccine and therapeutic development for this zoonotic pathogen.

The Impact of Glycosylation on the Functional Activity of CD133 and the Accuracy of Its Immunodetection.

The membrane glycoprotein CD133 (prominin-1) is widely regarded as the main molecular marker of cancer stem cells, which are the most malignant cell subpopulation within the tumor, responsible for tumor growth and metastasis. For this reason, CD133 is considered a promising prognostic biomarker and molecular target for antitumor therapy. Under normal conditions, CD133 is present on the cell membrane in glycosylated form. However, in malignancies, altered glycosylation apparently leads to changes in the functional activity of CD133 and the availability of some of its epitopes for antibodies. This review focuses on CD133's glycosylation in human cells and its impact on the function of this glycoprotein. The association of CD133 with proliferation, differentiation, apoptosis, autophagy, epithelial-mesenchymal transition, the organization of plasma membrane protrusions and extracellular trafficking is discussed. In this review, particular attention is paid to the influence of CD133's glycosylation on its immunodetection. A list of commercially available and custom antibodies with their characteristics is provided. The available data indicate that the development of CD133-based biomedical technologies should include an assessment of CD133's glycosylation in each tumor type.

Deep learning of antibody epitopes using positional permutation vectors

BackgroundThe accurate computational prediction of B cell epitopes can vastly reduce the cost and time required for identifying potential epitope candidates for the design of vaccines and immunodiagnostics. However, current computational tools for B cell epitope prediction perform poorly and are not fit-for-purpose, and there remains enormous room for improvement and the need for superior prediction strategies. ResultsHere we propose a novel approach that improves B cell epitope prediction by encoding epitopes as binary positional permutation vectors that represent the position and structural properties of the amino acids within a protein antigen sequence that interact with an antibody. This approach supersedes the traditional method of defining epitopes as scores per amino acid on a protein sequence, where each score reflects each amino acids predicted probability of partaking in a B cell epitope antibody interaction. In addition to defining epitopes as binary positional permutation vectors, the approach also uses the 3D macrostructure features of the unbound protein structures, and in turn uses these features to train another deep learning model on the corresponding antibody-bound protein 3D structures. This enables the algorithm to learn the key structural and physiochemical features of the unbound protein and embedded epitope that initiate the antibody binding process helping to eliminate “induced fit” biases in the training data. We demonstrate that the strategy predicts B cell epitopes with improved accuracy compared to the existing tools. Additionally, we show that this approach reliably identifies the majority of experimentally verified epitopes on the spike protein of SARS-CoV-2 not seen by the model during training and generalizes in a very robust manner on dissimilar data not seen by the model during training. ConclusionsWith the approach described herein, a primary protein sequence and a query positional permutation vector encoding a putative epitope is sufficient to predict B cell epitopes in a reliable manner, potentially advancing the use of computational prediction of B cell epitopes in biomedical research applications.

An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM

In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface. Our results demonstrate that the IAAG grid effectively enriches PA-tagged target proteins and overcomes preferred orientation issues. Furthermore, we demonstrate the utility of our IAAG strategy for on-grid purification of low-abundance target complexes from cell lysates, enabling atomic resolution cryo-EM. This approach greatly streamlines the purification process, reduces the need for large quantities of biological samples, and addresses common challenges encountered in cryo-EM sample preparation. Collectively, our IAAG strategy provides an efficient and robust means for combined sample purification and vitrification, feasible for high-resolution cryo-EM. This approach holds potential for broader applicability in both cryo-EM and cryo-electron tomography (cryo-ET).

Development of an improved blood-stage malaria vaccine targeting the essential RH5-CyRPA-RIPR invasion complex

Reticulocyte-binding protein homologue 5 (RH5), a leading blood-stage Plasmodium falciparum malaria vaccine target, interacts with cysteine-rich protective antigen (CyRPA) and RH5-interacting protein (RIPR) to form an essential heterotrimeric “RCR-complex”. We investigate whether RCR-complex vaccination can improve upon RH5 alone. Using monoclonal antibodies (mAbs) we show that parasite growth-inhibitory epitopes on each antigen are surface-exposed on the RCR-complex and that mAb pairs targeting different antigens can function additively or synergistically. However, immunisation of female rats with the RCR-complex fails to outperform RH5 alone due to immuno-dominance of RIPR coupled with inferior potency of anti-RIPR polyclonal IgG. We identify that all growth-inhibitory antibody epitopes of RIPR cluster within the C-terminal EGF-like domains and that a fusion of these domains to CyRPA, called “R78C”, combined with RH5, improves the level of in vitro parasite growth inhibition compared to RH5 alone. These preclinical data justify the advancement of the RH5.1 + R78C/Matrix-M™ vaccine candidate to Phase 1 clinical trial.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata)

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Human antibody signatures towards the Chlamydia trachomatis major outer membrane protein after natural infection and vaccination

Chlamydia trachomatis (CT) Major Outer Membrane Protein (MOMP) holds a neutralising epitope in the Variable Domain 4 (VD4), and this region's immune dominance during infection is well known. This study aimed to assess the antibody response induced after infection and compare it for specificity and functionality to the response following vaccination with the vaccine CTH522, which contains VD4's from serovars D, E, F, and G. We assessed the antibody epitopes in MOMP by a high density peptide array. Furthermore, the role of the VD4 epitope in neutralisation was explored by competitive inhibition experiments with a fusion protein holding the neutralising VD4 linear epitope. This was done in two independent groups: 1) MOMP seropositive individuals infected with CT (n=10, from case-control study) and 2) CTH522/CAF®01-vaccinated females (n=14) from the CHLM-01 clinical trial. We identified the major antigenic regions in MOMP as VD4 and the conserved region just before VD3 in individuals infected with CT. The same regions, with the addition of VD1, were identified in vaccine recipients. Overall, the VD4 peptide responses were uniform in vaccinated individuals and led to inhibition of infection invitro in all tested samples, whereas the VD4 responses were more heterogenous in individuals infected with CT, and only 2 out of 10 samples had VD4-mediated neutralising antibody responses. These data provide insights into the role of antibodies against MOMP VD4 induced after infection and vaccination, and show that their functionality differs. The induction of functional VD4-specific antibodies in vaccine recipients mimics previous results from animal models. This work was supported by the European Commission through contract FP7-HEALTH-2011.1.4-4-280873 (ADITEC) and Fonden til Lægevidenskabens Fremme.

Immune Responses to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Vaccines: Updated Insights

Background: Using a multidisciplinary approach, this review explores and investigates the relationship between immune responses, effectiveness, and results of various vaccination strategies among a range of demographics. Moreover, this review aims to aid healthcare professionals in understanding the intricacies of immune responses and their implications for future vaccine development.Methodology: In this systematic review, we employed an electronic online database platform including the National Centre for Biotechnology Information (NCBI), the National Institute for Health and Clinical Excellence (NICE), and the World Health Organization (WHO) for evidence-based material, utilizing the keywords throughout this literature review.Results: This review provides a detailed framework regarding immune responses to the COVID- 19 vaccine, based on demographics, age, sex, types of immunity, and immunization strategies. Such research can provide important academic support for the development of not only COVID- 19 vaccines but also other vaccines.Conclusions: Overall, immune responses tended to dwindle a few months after vaccine administration. This is because B cells block SARS-CoV-2 from infecting cells via antibodies and T cells destroy infected cells. The most efficient tool for providing long-lasting protection against SARS-CoV-2 is vaccination. Humans have developed immunity to SARS-CoV-2, particularly antibody-mediated humoral immunity. The development of vaccines across the whole family of coronaviruses that target antibody epitopes (antigenic determinants) prevents the emergence of mutant strains.

Structural basis for the broad antigenicity of the computationally optimized influenza hemagglutinin X6

Influenza causes significant morbidity and mortality. As an alternative approach to current seasonal vaccines, the computationally optimized broadly reactive antigen (COBRA) platform has been previously applied to hemagglutinin (HA). This approach integrates wild-type HA sequences into a single immunogen to expand the breadth of accessible antibody epitopes. Adding to previous studies of H1, H3, and H5 COBRA HAs, we define the structural features of another H1 subtype COBRA, X6, that incorporates HA sequences from before and after the 2009 H1N1 influenza pandemic. We determined structures of this antigen alone and in complex with COBRA-specific as well as broadly reactive and functional antibodies, analyzing its antigenicity. We found that X6 possesses features representing both historic and recent H1 HA strains, enabling binding to both head- and stem-reactive antibodies. Overall, these data confirm the integrity of broadly reactive antibody epitopes of X6 and contribute to design efforts for a next-generation vaccine.

Abstract 1885: Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment

Abstract ROR1 is a type I transmembrane protein that belongs to the ROR family. It is a receptor for Wnt family signaling molecules Wnt5a, and plays a critical role in various cellular processes, such as cell proliferation, survival, and migration. Being an oncofetal protein, ROR1 exhibits limited expression in most normal tissues. However, it is abnormally expressed in various hematological and solid cancers, contributing to the development and progression of many types of cancer. Due to its overexpression in cancer, ROR1 presents as a highly attractive target for antibody-drug conjugate (ADC) therapy. The current clinical results of ROR1 ADCs have been promising in treating patients with relapsed and/or refractory (R/R) hematologic malignancies. BR111 is anti-ROR1 ADC that consists of a biparatopic antibody, stably conjugated to an antimitotic agent. The biparatopic antibodies demonstrate superior binding affinity to ROR1-expressing cancer cells compared to single epitope antibodies. Notably, our unique and innovative conjugation platform CysX™, not only prevents payload detachment during circulation and reducing the off-target toxicity, but also enables a uniformed drug-to-antibody ratio (DAR) of 4. Both in vitro and In vivo studies have highlighted the remarkable antitumor activity of BR111. Preclinical data have shown great safety profile in cynomolgus monkeys. BR111 demonstrated a wider therapeutic window for cancer treatment, outperforming the lead anti-ROR1 ADC currently in phase II/IIl trial. The promising outcome holds great potential as a therapeutic treatment for hematological and solid cancers, offering a better safety profile. BR111 has great potential in further clinical evaluations. Citation Format: Xiaobei Zhao, Jie Zhu, zhenhua Wu, Yiqun Li, Jing Li, Yaqiong Zhou, Lei Nie, Haibin Wang, Gang Chen. Developing a biparatopic anti-ROR1 antibody drug conjugate BR111 for hematological and solid tumor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1885.

α-catenin middle- and actin-binding domain unfolding mutants differentially impact epithelial strength and sheet migration.

α-catenin (α-cat) displays force-dependent unfolding and binding to actin filaments through direct and indirect means, but features of adherens junction structure and function most vulnerable to loss of these allosteric mechanisms have not been directly compared. By reconstituting an α-cat F-actin-binding domain unfolding mutant known to exhibit enhanced binding to actin (α-cat-H0-FABD+) into α-cat knockout Madin Darby Canine Kidney (MDCK) cells, we show that partial loss of the α-cat catch bond mechanism (via an altered H0 α-helix) leads to stronger epithelial sheet integrity with greater colocalization between the α-cat-H0-FABD+ mutant and actin. α-cat-H0-FABD+ -expressing cells are less efficient at closing scratch-wounds, suggesting reduced capacity for more dynamic cell-cell coordination. Evidence that α-cat-H0-FABD+ is equally accessible to the conformationally sensitive α18 antibody epitope as WT α-cat and shows similar vinculin recruitment suggests this mutant engages lower tension cortical actin networks, as its M-domain is not persistently open. Conversely, α-cat-M-domain salt-bridge mutants with persistent recruitment of vinculin and phosphorylated myosin light chain show only intermediate monolayer adhesive strengths, but display less directionally coordinated and thereby slower migration speeds during wound-repair. These data show α-cat M- and FABD-unfolding mutants differentially impact cell-cell cohesion and migration properties, and suggest signals favoring α-cat-cortical actin interaction without persistent M-domain opening may improve epithelial monolayer strength through enhanced coupling to lower tension actin networks.

Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters.

Characterizing the antibody epitope profiles of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 can aid in elucidating the mechanisms underlying the antibody-mediated immune responses elicited by these vaccines. This study investigated the distinct antibody epitopes toward the SARS-CoV-2 spike (S) protein targeted after a two-dose primary series of mRNA-1273 followed by a booster dose of mRNA-1273 or a variant-updated vaccine among serum samples from clinical trial adult participants. Multiple S-specific epitopes were targeted after primary vaccination; while signal decreased over time, a booster dose after >6 months largely revived waning antibody signals. Epitope identity also changed after booster vaccination in some subjects, with four new S-specific epitopes detected with stronger signals after boosting than with primary vaccination. Notably, the strength of antibody responses after booster vaccination differed by the exact vaccine formulation, with variant-updated mRNA-1273.211 and mRNA-1273.617.2 booster formulations inducing significantly stronger S-specific signals than a mRNA-1273 booster. Overall, these results identify key S-specific epitopes targeted by antibodies induced by mRNA-1273 primary and variant-updated booster vaccination.

Antigen-Heterologous Vaccination Regimen Triggers Alternate Antibody Targeting in SARS-CoV-2-DNA-Vaccinated Mice.

An in-depth analysis of antibody epitopes following vaccination with different regimens provides important insight for developing future vaccine strategies. B-cell epitopes conserved across virus variants may be ideal targets for vaccine-induced antibodies and therapeutic drugs. However, challenges lie in identifying these key antigenic regions, and directing the immune system to target them. We previously evaluated the immunogenicity of two candidate DNA vaccines encoding the unmodified spike protein of either the SARS-CoV-2 Index strain or the Beta variant of concern (VOC). As a follow-on study, we characterized here the antibody binding profiles of three groups of mice immunized with either the DNA vaccine encoding the SARS-CoV-2 Index strain spike protein only, the Beta VOC spike protein only, or a combination of both as an antigen-heterologous prime-boost regimen. The latter induced an antibody response targeting overlapping regions that were observed for the individual vaccines but with additional high levels of antibody directed against epitopes in the SD2 region and the HR2 region. These heterologous-vaccinated animals displayed improved neutralization breadth. We believe that a broad-focused vaccine regimen increases neutralization breadth, and that the in-depth analysis of B-cell epitope targeting used in this study can be applied in future vaccine research.

HIV-1 Gag, Pol, and Env diversified with limited adaptation since the 1980s.

Global surveillance of HIV-1 sequences is critical for designing relevant prophylactic and therapeutic interventions to infection. We designed an open-source platform, Hervé, for analyzing and visualizing the diversification dynamics of HIV-1 protein sequences. We characterized the evolution of over 12,000 HIV-1 Env, Gag, and Pol protein sequences from 1980-2020 and found that, despite a steady increase in intra-subtype and circulating recombinant form diversity, the most frequent residue at each site, i.e., the consensus, has varied only moderately.

GlcNAc6ST2/CHST4 Is Essential for the Synthesis of R-10G-Reactive Keratan Sulfate/Sulfated N-Acetyllactosamine Oligosaccharides in Mouse Pleural Mesothelium.

We recently showed that 6-sulfo sialyl N-acetyllactosamine (LacNAc) in O-linked glycans recognized by the CL40 antibody is abundant in the pleural mesothelium under physiological conditions and that these glycans undergo complementary synthesis by GlcNAc6ST2 (encoded by Chst4) and GlcNAc6ST3 (encoded by Chst5) in mice. GlcNAc6ST3 is essential for the synthesis of R-10G-positive keratan sulfate (KS) in the brain. The predicted minimum epitope of the R-10G antibody is a dimeric asialo 6-sulfo LacNAc. Whether R-10G-reactive KS/sulfated LacNAc oligosaccharides are also present in the pleural mesothelium was unknown. The question of which GlcNAc6STs are responsible for R-10G-reactive glycans was an additional issue to be clarified. Here, we show that R-10G-reactive glycans are as abundant in the pulmonary pleura as CL40-reactive glycans and that GlcNAc6ST3 is only partially involved in the synthesis of these pleural R-10G glycans, unlike in the adult brain. Unexpectedly, GlcNAc6ST2 is essential for the synthesis of R-10G-positive KS/sulfated LacNAc oligosaccharides in the lung pleura. The type of GlcNAc6ST and the magnitude of its contribution to KS glycan synthesis varied among tissues in vivo. We show that GlcNAc6ST2 is required and sufficient for R-10G-reactive KS synthesis in the lung pleura. Interestingly, R-10G immunoreactivity in KSGal6ST (encoded by Chst1) and C6ST1 (encoded by Chst3) double-deficient mouse lungs was markedly increased. MUC16, a mucin molecule, was shown to be a candidate carrier protein for pleural R-10G-reactive glycans. These results suggest that R-10G-reactive KS/sulfated LacNAc oligosaccharides may play a role in mesothelial cell proliferation and differentiation. Further elucidation of the functions of sulfated glycans synthesized by GlcNAc6ST2 and GlcNAc6ST3, such as R-10G and CL40 glycans, in pathological conditions may lead to a better understanding of the underlying mechanisms of the physiopathology of the lung mesothelium.

P166 Optimizing PD-1 agonist signaling with membrane proximal binding of rosnilimab, a clinical stage PD-1 agonist IgG1 antibody

Abstract Background Checkpoint agonism represents a promising class of therapies for the treatment of autoimmune and inflammatory diseases, including ulcerative colitis (UC), where unmet needs persist despite available therapies. Binding to membrane proximal regions of checkpoint receptors, together with Fc interactions with receptors on opposing cells, can contribute to tight immune synapse formation between the immune cell and antigen presenting cell. This has been proposed to improve potency of agonistic signaling by excluding phosphatases such as CD45 from the immune synapse and promoting receptor clustering. Optimization of these characteristics results in improved agonism and depletion, with the potential for restoration of immune balance and broader clinical outcomes. Rosnilimab was engineered to leverage these important characteristics. It is a PD-1 agonist IgG1 antibody designed to optimize inhibitory signaling through the PD-1 receptor and to deplete PD-1high pathogenic T cells. Rosnilimab is in Phase 2 clinical development for UC and rheumatoid arthritis. Methods Mutations to surface exposed regions of the PD-1 extracellular domain were made and surface plasmon resonance was used to infer the epitopes of PD-1 agonist molecules from resulting changes to binding. Membrane proximal and distal binding antibodies were studied in in vitro functional assays to assess T cell proliferation and antibody-dependent cellular toxicity. Results Epitopes of agonistic antibodies were mapped to locations on the PD-1 receptor. The membrane proximal binding epitope of rosnilimab was confirmed and binding epitopes for other reference antibodies (ref) were identified. Rosnilimab and a membrane distally binding antibody (ref 1) were selected for comparison in functional assays. Rosnilimab demonstrated greater reduction of T cell proliferation and depletion of PD-1+ T cells compared to ref 1, consistent with the hypothesis that membrane proximal binding improves agonistic activity and target cell depletion. Conclusion By targeting and leveraging inhibitory immune regulatory mechanisms to modulate the pathogenic T cells driving disease, there is an opportunity to dampen the inflammatory cycle and restore immune balance via agonism. Rosnilimab binds to a membrane proximal region of the PD-1 receptor, resulting in potent reduction of T cell proliferation and depletion of PD-1high T cells. These mechanistic data, translational in vivo and in vitro data, robust Phase 1 healthy volunteer data, and unmet needs in UC provide rationale for an ongoing global Phase 2 study of rosnilimab in UC (NCT06127043).

P193 Fatigued patients with Inflammatory Bowel Disease exhibit distinct systemic antibody epitope repertoires

Abstract Background Patients with inflammatory bowel diseases (IBD) frequently experience fatigue, affecting up to 80% of those with active disease and approximately 50% with quiescent disease. The exact cause of IBD-associated fatigue is often unknown, making clinical management very challenging. In this study we aimed to explore whether patients with quiescent IBD reporting fatigue exhibit specific systemic antibody responses, which could provide insight into immune reactivities underlying fatigue. Methods Systemic antibody epitope repertoires were profiled in 327 patients with IBD (156 Crohn’s disease [CD]; 171 ulcerative colitis [UC]) leveraging phage-display immunoprecipitation sequencing (PhIP-Seq) against 344,000 rationally selected peptide antigens. Fatigue severity was assessed on a 10-point Likert scale, ranging from 1 (no fatigue) to 10 (highest fatigue severity). Quiescent IBD was defined as clinical (Harvey-Bradshaw Index [HBI] <5 or Simple Clinical Colitis Activity Index [SCCAI] <2.5) and biochemical remission (C-reactive protein [CRP] <5 mg/L) at time of sampling. Multivariable logistic regression analyses, allowing adjustment for potential confounding factors e.g. age, sex, and smoking, were performed to identify associations between fatigue and systemic antibody responses. Results A total of 105 different antibody-bound peptides were associated with fatigue (nominal P-value<0.05), albeit none passed adjustment for multiple comparisons. Among these antibodies, 50 (47.6%) were found to be less frequent in highly fatigued patients (fourth quartile, Q4), while 55 (52.4%) were identified as more frequent in highly fatigued patients compared to those with low fatigue scores (first quartile, Q1). Among highly fatigued patients, antibody responses were primarily directed towards viral antigens, notably several antigens from Epstein-Barr virus (EBV), as well as bacterial antigens, including functional proteins from Streptococcus and Staphylococcus species. Fatigued patients with CD exhibited elevated systemic antibody responses against allergens, Staphylococcus, Pseudomonas aeruginosa, and Shigella spp. Fatigued patients with UC showed higher frequencies of antibody responses against herpes simplex virus (HSV), influenza viruses, and few responses against allergens and Streptococcus bacteria. These results remained materially unchanged when repeating analyses in patients with quiescent IBD. Conclusion This study may suggest a potential role of viral antigens, particularly EBV, in the pathophysiology of fatigue in patients with IBD. However, larger confirmatory studies are needed to validate these findings. PhIP-Seq may represent a valuable strategy to approach the investigation of immune responses underlying complex symptoms such as fatigue.