mAbs最新文献

{"title":"Structures of clinical antibodies bound to IL-33 uncover two distinct epitopes underlying differential efficacy.","authors":"Jing Chen, Yue Wang, Xinquan Wang","doi":"10.1080/19420862.2026.2639673","DOIUrl":"10.1080/19420862.2026.2639673","url":null,"abstract":"<p><p>Interleukin-33 (IL-33), an alarmin cytokine of the IL-1 family, drives type 2 inflammation through signaling via the ST2 and IL-1RAcP receptors, making it a critical therapeutic target for inflammatory diseases such as asthma and chronic obstructive pulmonary disease. Current therapeutic strategies have primarily focused on antibodies that target IL-33 or ST2 to disrupt their specific interaction. However, the structural mechanisms underlying antibody-mediated neutralization of IL-33 remain poorly understood. Here, we report the structures of three antibodies in clinical trial - etokimab, itepekimab, and tozorakimab - complexed with IL-33, determined by X-ray crystallography and cryo-electron microscopy. Structural analysis reveals two distinct neutralizing epitopes on IL-33, termed Epitope 1 at IL-33/ST2 binding Site 1 and Epitope 2 at IL-33/ST2 binding Site 2. Tozorakimab, which targets Epitope 1, completely blocks ST2 engagement by sterically occluding the ST2 D1-D2 domain-binding interface. In contrast, etokimab and itepekimab, which recognize Epitope 2, interfere with IL-33 recognition of the ST2 D3 domain and thereby only partially inhibit ST2 binding. These structural and biochemical findings provide a molecular explanation for the differential efficacy of the three antibodies in inhibiting IL-33 signaling in cellular assays. Collectively, our results provide valuable insights into the molecular determinants of efficacy for existing IL-33 therapeutics and offer a structural framework for the rational design of next-generation IL-33 targeted inhibitors.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2639673"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12959182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147326608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Beyond sequence similarity: ML-powered identification of pHLA off-targets for TCR-mimic antibodies using high throughput binding kinetics.","authors":"Alexander Sinclair, Stefan Krämer, Christoph Reinhart, Jennifer Stehle, Simon Schuster, Tobias Herz, Hoor Al Hasani, Pranav Hamde, Oliver Selinger, Joerg Birkenfeld","doi":"10.1080/19420862.2025.2601360","DOIUrl":"10.1080/19420862.2025.2601360","url":null,"abstract":"<p><p>T-cell receptor mimic (TCRm) antibodies are an emerging class of tumor-targeting agents used in advanced immunotherapies such as bispecific T-cell engagers and CAR-T cells. Unlike conventional antibodies, TCRms are designed to recognize peptide - human leukocyte antigen (pHLA) complexes that present intracellular tumor-derived peptides on the cell surface. Due to the typically low surface abundance and high sequence similarity of pHLAs, TCRms require high affinity and exceptional specificity to avoid off-target toxicity. Conventional methods for off-target identification such as sequence similarity searches, motif-based screening, and structural modeling focus on the peptide and are limited in detecting cross-reactive peptides with little or no sequence homology to the target. To address this gap, we developed EpiPredict, a TCRm-specific machine learning framework trained on high-throughput kinetic off-target screening data. EpiPredict learns an antibody-specific mapping from peptide sequence to binding strength, enabling prediction of interactions with unmeasured pHLA sequences, including sequence-dissimilar peptides. We applied EpiPredict to two distinct TCRms targeting the cancer-testis antigen MAGE-A4. The model successfully predicted multiple off-targets with minimal sequence similarity to the intended epitope, many of which were experimentally validated via T2 cell binding assays. These findings establish EpiPredict as a valuable tool for lead optimization of TCRms, enabling the identification of antibody-specific off-targets beyond the scope of traditional peptide-centric methods and supporting the preclinical de-risking of TCRm-based therapies.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2601360"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12710896/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145743226","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fine-tuning affinity and spacer design enhances T cell potency in DLL3 and BCMA CAR T cells.","authors":"Nicholas Mazzanti, Ninkka Tamot, Andrea Francese, Jinquan Luo, M Jack Borrok, Julie Rossillo, Joseph Plummer, Gauri Anand Patwardhan, Chi Shing Sum, Michael Ports, Kara L Spiller, Madhusudhanan Sukumar","doi":"10.1080/19420862.2025.2602989","DOIUrl":"10.1080/19420862.2025.2602989","url":null,"abstract":"<p><p>Chimeric antigen receptor (CAR)-modified T cells have garnered substantial attention due to their clinical success, culminating in six Food and Drug Administration-approved therapies for hematological malignancies. Notably, CD19-specific CAR T cell therapies have achieved remarkable clinical efficacy in treating B-cell malignancies, but these profound and durable responses are not observed in CAR T therapies targeting other indications, particularly solid tumors. Key design elements of CAR constructs - namely, antigen binding affinity and spacer length - play critical roles in determining T cell effector function and overall therapeutic effectiveness. Refining CAR designs may enhance T cell functionality, extend clinical application, and potentially apply CAR T cell therapies across a wider array of malignancies. In this study, affinity variant and spacer variant CARs targeting BCMA and DLL3 tumor antigens were evaluated using <i>in vitro</i> measurements of antigen-binding properties and effector function. Each panel of CARs spanned 2-3 logs of antigen binding affinity (BCMA: 181 pM KD to 74 nM KD, DLL3: 417 pM to 407 nM). Additionally, CAR T cells were challenged with tumor spheroids composed of BCMA⁺ H929 and DLL3⁺ SHP77 tumor cells. We show that for both tumor models, higher affinity CARs (KD stronger than approximately 100 nM) paired with an intermediate length spacer (IgG1 Fc, CH2-CH3, 230AA) elicited the strongest levels of tumor killing, CAR⁺ T cell expansion, and proinflammatory cytokine production. These CARs displayed the strongest cellular affinity when measured in a conjugation assay, suggesting a relationship between cellular affinity and T cell functional performance. This study highlights the critical role of CAR design in enhancing T cell functionality, demonstrating that high-affinity CARs combined with intermediate-length spacers yield superior performance in targeting BCMA and DLL3 antigens. This study provides a framework for rational CAR design, informing strategies to broaden the clinical utility of CAR T-cell therapies beyond hematologic cancers.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2602989"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12710886/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145743209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A cell line development vector strategy for improved expression of a trispecific T-cell engager in CHO.","authors":"Rajesh K Mistry, Chendi Nui, Giulia Lambiase, Daniel Ray, Lewis Kearsey, Noah Hitchcock, Luigi Grassi, Ramy Elgendy, James Fleming, Alexandra C Broughton, Peng Zhao, Chi-I Chiang, Pooja Shah, Matthew Cyr, Even Walseng, Yariv Mazor, Diane Hatton, Sarah Dunn","doi":"10.1080/19420862.2026.2632994","DOIUrl":"10.1080/19420862.2026.2632994","url":null,"abstract":"<p><p>Recent advances in trispecific antibody (trisAb) engineering offer great therapeutic potential, but achieving high product yield and quality in cell line development remains a challenge due to complex chain pairing requirements in production cell lines. In this study, three distinct expression vector configurations were evaluated for their ability to support robust, high-level expression of a structurally complex, synapse-gated trisAb T-cell engager (TriMab) in stable Chinese hamster ovary cells. Initial configurations using conventional dual heavy chain (HC) and triple light chain (LC) vectors resulted in poor pool performance characterized by delayed transfection recovery and low titers. By contrast, a redesigned strategy that reversed HC gene order and distributed LCs over separate vectors markedly improved transfection recovery along with product titers and reduced the formation of undesired product variants. Clonal cell lines established with this optimized strategy achieved titers exceeding 2 g/L with correct product quality profiles. Gene copy number and mRNA analyses confirmed that chain order and vector design strongly influenced mRNA levels and thus productivity. These results highlight the critical impact of vector configuration on manufacturability of complex TriMabs, providing a practical framework for the rational design of gene vectors to support next-generation trisAb production.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2632994"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12947573/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147307356","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The making of multispecific immunoglobulins - a clinical perspective.","authors":"Ulrich Brinkmann, Roland E Kontermann","doi":"10.1080/19420862.2026.2613548","DOIUrl":"10.1080/19420862.2026.2613548","url":null,"abstract":"<p><p>Over the past two decades, bi- and multispecific antibodies have emerged as a rapidly advancing class of therapeutic biologics, transforming oncology and immunotherapy. By simultaneously binding two or more distinct antigens or epitopes, these molecules achieve mechanisms of action beyond those of conventional monoclonal antibodies, including immune cell redirection, dual pathway modulation, and enhanced tissue selectivity. Bispecific and multispecific antibodies exhibit considerable structural diversity, encompassing a wide range of molecular architectures covering a steady growing 'zoo' of formats. The therapeutic success and diversity of molecules and formats is reflected in the 2021 revision of the international nonproprietary name system, which introduced the suffix - mig to denote multispecific immunoglobulins. In this review, we provide an overview of multispecific antibodies in clinical development, focusing on format, molecular design, and clinical status. In total, data for 501 multispecific antibodies were compiled and analyzed, identifying 112 different formats. Overall, this analysis highlights the rapid growth, enormous format diversity, and translational potential of multispecific antibodies. It underscores their emerging role as versatile therapeutics not only in oncology, but also in non-cancer indications, reflecting a field that continues to evolve rapidly in response to both scientific innovation and clinical needs.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2613548"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12818813/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145989060","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Using mass spectrometry for robust identity release testing in a global quality control network.","authors":"Sebastien Leibe, Sebastien Violini, Moritz Vollrath, Anja Bathke, Hong Nguyen, Harry Yu, Jack Harris, Christopher Yu, Jan O Stracke, Hilary Pallat, Christian Bell, Elizabeth J Johnson, Cinzia Stella, Patrick Bulau","doi":"10.1080/19420862.2026.2643964","DOIUrl":"10.1080/19420862.2026.2643964","url":null,"abstract":"<p><p>Product identity (ID) testing is a fundamental requirement in pharmaceutical quality control. Roche/Genentech has developed and established a mass spectrometry-based platform ID method for routine release testing of biologics in the global clinical and commercial quality control environment. The established method, using peptide mapping combined with liquid chromatography-mass spectrometry, allows automated data evaluation. Previous experience shows that this approach is highly user-friendly in regulated testing environments, significantly reduces validation work for new products, and, due to its high robustness, results in a reduced error rate during test execution and data evaluation. Due to the specific analysis of product peptides, the method also allows for a sensitive and high-resolution evaluation of further product variants that are attributable to chemical and/or post-translational amino acid modifications. The data already recorded for identity testing purposes can therefore be immediately reevaluated during a quantitative product quality impact assessment following unexpected events in the manufacturing process, thus saving additional analyses during root cause analysis and future failure mitigation. In summary, this technology, established and qualified at all Roche/Genentech quality control sites, enables improved product monitoring beyond the routine release and stability testing methods used.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2643964"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12977250/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147434056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overcoming claudin family homology: discovery of ARC101, a highly potent CLDN6-specific T-cell engager with a novel CD3 binder for ovarian adenocarcinoma.","authors":"Danlin Yang, Neha Kamran, Gururaj Shivange, Kavita Kumari, Prabhu Srinivas Yavvari, Viduth K Chaugule, Vishal Mahajan, Bridget Larkin, Scott R Brodeur, Joe Erhardt, Sanjaya Singh","doi":"10.1080/19420862.2026.2637299","DOIUrl":"10.1080/19420862.2026.2637299","url":null,"abstract":"<p><p>Claudin-6 (CLDN6) is an oncofetal tight junction protein with minimal to no expression in healthy adult tissues but aberrant upregulation in ovarian malignancies, making it an attractive tumor-selective antigen for T cell-based immunotherapy. The development of CLDN6-targeting antibodies, however, has been challenged by its high homology to CLDN9, which is expressed in normal tissues and differs by only three amino acids within the extracellular domains. Here, we describe the discovery and preclinical development of ARC101, a bispecific CLDN6×CD3 antibody featuring a naturally derived, highly potent CLDN6 binder with no cross-reactivity to CLDN9 or other human membrane proteins. The stringent specificity of ARC101 eliminates off-target binding and distinguishes it from other CLDN6-targeting antibodies in development. The effector arm of ARC101 incorporates a novel conformational CD3 binder, enabling potent T cell-mediated cytotoxicity against CLDN6-expressing tumor cells <i>in vitro</i> and <i>in vivo</i>. ARC101 also demonstrated a favorable pharmacokinetic profile in cynomolgus monkeys, low immunostimulatory responses in <i>ex vivo</i> human donor assays, and robust biophysical properties compatible with standard antibody manufacturing. Collectively, these findings support the clinical advancement of ARC101 as a differentiated, CLDN6-specific bispecific immunotherapy with exceptional tumor selectivity and optimized T cell activity for the treatment of solid tumors.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2637299"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12977278/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147390402","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preclinical characterization of AZD1163, a first-in-class anti-PAD2/4 bispecific antibody for the treatment of rheumatoid arthritis.","authors":"Gary P Sims, Lacie Scaletta, John Andrews, Dorothy A Sims, Martin Strain, Anna Sigurdardottir, Teneema Kuriakose, Elizabeth England, Lichchavi Rajasinghe, Fanyi Jiang, Ximing Xiong, Frances Neal, Lisa Vinall, Philip Newton, Elin Boger, Chong Kim, Helena Dahlbäck, Susanne Prothon, Jacob Leander, Erika Darrah, Mia Collins, Garry Douglas, Nicholas White, Katie Day, Katherine A Vousden, Catherine E Huntington, David Close","doi":"10.1080/19420862.2026.2657629","DOIUrl":"10.1080/19420862.2026.2657629","url":null,"abstract":"<p><p>Anti-citrullinated protein autoantibodies promote inflammation and joint tissue injury and define a poor prognostic group of patients with rheumatoid arthritis (RA). Citrullinated autoantigens that drive this autoimmune response are generated by peptidylarginine deiminase (PAD) enzymes, which are predominately expressed and released by neutrophils and monocytes. Accordingly, blocking the enzymatic activity of PADs to curb the generation of citrullinated autoantigens that drive autoimmunity and tissue injury may provide therapeutic benefit. Herein, we developed a high affinity, bispecific, anti-PAD2/4, effector-null antibody, AZD1163, which potently inhibits recombinant PAD2 and PAD4 activity in both histone H3 and fibrinogen citrullination assays. AZD1163 inhibited all endogenous PAD activity in the serum of patients with RA irrespective of the presence of anti-PAD4 autoantibodies, and neutralized PAD activity in synovial fluid. AZD1163 also bound and internalized PADs expressed on cell membranes into low pH endosomes for degradation, reducing the surface expression and catalytic potential. Binding of AZD1163 to neutrophils and monocytes did not trigger complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, or the production of proinflammatory cytokines, or otherwise impact neutrophil phagocytosis, production of reactive oxygen species, or NETosis. In a non-human primate study of pharmacokinetics (PK) and pharmacodynamics, a single dose of AZD1163 exhibited PK consistent with a half-life extended antibody and a rapid and durable suppression of endogenous PAD activity. AZD1163 has a favorable preclinical safety profile. Collectively, these in vitro and in vivo pharmacology and safety data support the clinical development of AZD1163 as a novel therapeutic strategy for RA by reducing autoantigen load.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2657629"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13085943/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147690958","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering of acidic pH-responsive anti-CD3 binding antibodies.","authors":"Grégory La Sala, Katharina B Kroell, Mudita Pincha, Christian Gassner, Lorenzo Deho, Ekkehard Moessner, Xavier Gueripel, Nicole Borin, Moritz Classen, Jörg Benz, Alexander Bujotzek, Christian Klein, Guy Georges, Adrian Hugenmatter, Klaus R Liedl, Anna Vangone","doi":"10.1080/19420862.2026.2658902","DOIUrl":"10.1080/19420862.2026.2658902","url":null,"abstract":"<p><p>The development of anti-CD3 antibody-based T cell engager therapeutics has improved the treatment of various malignancies, yet the challenge of achieving tumor-specific targeting while minimizing on-target off-tumor effects in normal tissues remains a substantial hurdle. One promising strategy to address this issue involves engineering antibodies with conditional pH-dependent binding affinities, capitalizing on the acidic microenvironment characteristics of tumors (pH ~ 6.5-6.8) compared to the neutral pH of healthy tissues (pH ~ 7.4). In this study, we focus on the pH-engineering of antibody binders against the human CD3 antigen, a critical component of T cell activation, to achieve preferential binding at acidic pH. Using molecular dynamics (MD) simulations on the reported CD3ɛ antibody binder 40G5c, we shed light on possible molecular mechanisms of the pH-responsiveness of key mutations and their impact on the overall binder structure at physiological or acidic pH. Our study highlights how MD has emerged as a powerful tool to guide and explain intrinsic pH-dependent molecular mechanisms in antibody engineering. Lastly, we report that our engineered CD3 binders preferentially bind and activate T cells under acidic pH conditions and display favorable affinity and pH-window profiles.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2658902"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13128029/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"RASP: rapid antibody functional screening by pentavalent phage display.","authors":"Manpreet Kaur, Abhishek Dubey, Kartik Chandran","doi":"10.1080/19420862.2026.2627708","DOIUrl":"10.1080/19420862.2026.2627708","url":null,"abstract":"<p><p>The heavy-chain antibody variable domain (VHH) is the smallest antigen-binding domain of such antibodies, which are derived from camelids. In the past three decades, VHHs, which are also called single-domain antibodies, have been extensively used to target pathogens and/or toxins. Conventional screening methods, such as phage display, rely only on antibody-antigen binding as the sole criterion for selection. Despite being robust and high-throughput, such methods often require additional downstream experiments to identify VHH that neutralize their target. Here, we describe an innovative, high-throughput functional screening method, Rapid Antibody functional Screening by Pentavalent phage display (RASP), that incorporates purified antibody-displaying phages for virus neutralization assays, and thus can be used to directly identify neutralizing VHHs. As a proof-of-concept, we first displayed previously identified neutralizing VHHs specific for the spike proteins of SARS-CoV-2 and respiratory syncytial virus on phages and demonstrated a dose-dependent blockade of viral infection. We further improved our method by utilizing the pentavalent display feature of hyperphages. We showed that hyperphage-derived VHH phages were superior to helper phage-derived VHH phages in assaying viral neutralization potential. Thereafter, we applied RASP to identify multiple candidates by screening a semi-synthetic VHH library against recombinant vesicular stomatitis viruses pseudotyped with spike glycoproteins from SARS-CoV-2, Junin virus, and Ebola virus, featuring as case studies in antiviral antibody discovery. Further, we benchmarked RASP against established phage ELISA and next-generation sequencing methods. Overall, we successfully used RASP in the context of the discovery of antiviral VHHs, highlighting its broader applicability as a platform that can be used either in isolation or in conjunction with other functional screening methods to accelerate the discovery of antiviral VHHs.</p>","PeriodicalId":18206,"journal":{"name":"mAbs","volume":"18 1","pages":"2627708"},"PeriodicalIF":7.3,"publicationDate":"2026-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12928633/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146143021","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}