Antibodies doi: 10.3390/antib15020034

Authors: Tingting Ji Zhaoyun Zong Ningyuan Gong Minghui Yan Shiyu Chen

Background: Transferrin receptor protein 1 (TfR1) plays a central role in cellular iron uptake and is frequently overexpressed in malignant tumor cells, rendering it an attractive target for tumor-directed therapy and drug delivery. Methods: A fully human single-chain variable fragment (scFv) antibody targeting TfR1, termed T8scFv, was isolated from a human scFv phage display library through three rounds of stringent biopanning and subsequently reformatted into a full-length IgG1 antibody (T8IgG1). Binding kinetics were characterized using Octet biolayer interferometry (BLI), while cellular binding and internalization were assessed by flow cytometry and immunofluorescence microscopy, respectively. T8IgG1 was further conjugated to DT3C, a recombinant truncated diphtheria toxin fusion protein, to evaluate its internalization-dependent cytotoxicity in vitro. Results: T8scFv exhibited nanomolar affinity for TfR1 (KD = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, KD = 18.5 ± 0.1 nM). T8IgG1 specifically recognized TfR1 on the surface of tumor cells and underwent efficient TfR1-mediated internalization. The T8IgG1-DT3C complex significantly reduced cell viability and induced apoptosis in K562 cells in vitro. Conclusions: These findings indicate that T8IgG1 is a moderate-affinity, internalizing anti-TfR1 antibody and highlight its potential as a promising candidate for TfR1-based targeted antitumor drug delivery systems.

Antibodies doi: 10.3390/antib15020033

Authors: Maksymilian Markwitz Natalia Welc Klementyna Kępińska Monika Bowszyc-Dmochowska Marian Dmochowski

Background: Pemphigus diseases are rare autoimmune blistering disorders mediated by pathogenic autoantibodies directed mainly against desmoglein 1 and desmoglein 3. Although most cases are considered idiopathic, external triggers that can disrupt immune tolerance have been described. Vaccination has been discussed as a potential precipitating factor in autoimmune skin diseases. However, the relationship between vaccination and the induction of pemphigus-related autoantibodies has not been comprehensively summarized. Methods: We conducted a narrative review of all available studies published in the last 25 years identified through medical databases, excluding studies on COVID-19 vaccinations. Reports describing either new-onset pemphigus or exacerbation of preexisting pemphigus with a temporal association to vaccination were included. Clinical characteristics, vaccine type, latency period, direct immunofluorescence findings, and ELISA results for desmoglein autoantibodies were analyzed. In addition, we present our own clinical-laboratory experience illustrating this issue. Results: The current evidence consists predominantly of case reports and small case series. Published cases describe pemphigus vulgaris and pemphigus foliaceus occurring after vaccinations against influenza, hepatitis B, tetanus, diphtheria, pertussis, rabies, and other routinely administered immunizations. The latency period most often ranged from several days to a few weeks. Immunopathological findings were consistent with classical pemphigus diseases, including intercellular IgG deposits in the epidermis and circulating autoantibodies against desmoglein 1 and/or desmoglein 3. Our patient was a 78-year-old woman who developed cutaneous form of pemphigus vulgaris, diagnosed with direct immunofluorescence (DIF) and multiplex ELISA, 10 days after diphtheria–tetanus–pertussis vaccination. The patient had a positive family history of autoimmune blistering disease, namely mucous membrane pemphigoid. Conclusions: Based on the currently available evidence, a direct causal relationship between vaccination and pemphigus diseases cannot be established. Nevertheless, accumulated clinical and serological observations suggest that vaccination may act as a triggering factor in genetically or immunologically predisposed individuals, possibly by amplifying pre-existing subclinical autoreactive immune responses. Further population-based and mechanistic studies are required to clarify this association, while the overall benefits of vaccination remain substantial.

Antibodies doi: 10.3390/antib15020032

Authors: Chiara Orlandi Angela Tincani Micaela Fredi Laura Andreoli Francesca Crisafulli Liala Moschetti Cecilia Nalli Maria Grazia Lazzaroni Marco Taglietti Matteo Filippini Sonia Zatti Laura Picciau Franco Franceschini Ilaria Cavazzana

Systemic lupus erythematosus (SLE) is an autoimmune disease that predominantly affects women of childbearing age, and active disease during pregnancy is associated with increased maternal and fetal morbidity. Belimumab is an effective biologic therapy for active SLE; however, its use during pregnancy has long been limited by the scarcity of safety data. Recent evidence and updated international recommendations suggest that belimumab may be considered in selected cases when required to maintain maternal disease control. We report the case of a woman with SLE who experienced three consecutive pregnancies with live births between 2019 and 2024 while receiving belimumab, allowing an intra-individual comparison of different exposure strategies. During the first pregnancy, belimumab was discontinued at conception and was followed by a disease flare in late pregnancy and postpartum. In the second and third pregnancies, belimumab was continued until gestational week 20 following shared decision-making with the patient; nevertheless, disease flares occurred during the third trimester of both pregnancies. All pregnancies resulted in live births at term, with no congenital anomalies, placental insufficiency, or fetal growth restriction. One neonate from the third pregnancy developed early-onset neonatal sepsis and meningitis, which resolved completely after antibiotic treatment. All children are currently growing and developing normally. This case supports a risk-adapted approach to belimumab use during pregnancy. In selected women with SLE at high risk of disease reactivation, continuation of belimumab until mid-gestation may contribute to improved maternal disease control without evident adverse fetal outcomes.

Antibodies doi: 10.3390/antib15020031

Authors: Julia Cieślawska Mariola Pawlaczyk Justyna Gornowicz-Porowska

Human hair performs a number of important physiological and esthetic functions. Hair loss and alopecia are complex disorders which affect people all over the world. Hair loss can be an early manifestation of various autoimmunological disorders. Despite a growing interest of researchers in the role of immune factors—especially autoantibodies—in the etiology of certain types of alopecia, their role in alopecia remains uncertain. Several potential autoantigens of follicular components, mainly derived from keratinocytes and melanocytes of the hair follicles, have been found to play a role in the development of alopecia areata. The list of autoantigens includes trichohyalin, keratin 16, fibroblast growth factor receptor 3, glycoprotein-100, melanoma-associated antigen recognized by T cells 1, dopachrome tautomerase/tyrosinase-related protein 2, tyrosinase, and tyrosine hydroxylase. This narrative review presents different aspects of immunopathogenesis of alopecia, from physiology (hair follicle immune privilege) to pathology (disruption of hair follicle immune privilege) and signaling pathways. Identification of key autoantigens could potentially pave the way for the development of new, effective, and more targeted immunotherapies for alopecia.

Antibodies doi: 10.3390/antib15020030

Authors: Sharon L. Walmsley Leif Erik Lovblom Bryan Boyachuk Curtis Cooper Valérie Martel-Laferrière Mona Loutfy Marie-Louise Vachon Shariq Haider Pamela Aldebes Karen Colwill Anne Claude Gingras Freda Qi Marina B. Klein

Objectives: To determine the incidence and outcomes of SARS-CoV-2 infection and to evaluate seroconversion rates and quantify antibody responses to COVID-19 vaccines in two cohorts of persons living with HIV at a possible higher risk of poor outcomes (HCV coinfection and those over the age of 65 years). Methods: We included participants from two established cohorts of persons living with HIV, those who were older than 65 years of age, and those with hepatitis C (HCV) co-infection. Four hundred and seventy-one participants completed questionnaires on SARS-CoV-2 infection and COVID-19 vaccine doses and submitted peripheral blood specimens for measuring antibody levels to COVID-19 antigens, full-length spike trimer, its receptor binding domain (RBD), and nucleocapsid protein (N) at 6-month intervals up to three visits between February 2021 and December 2024. Logistic and ordinal logistic regression models evaluated predictors of seroconversion and antibody levels. Results: Overall, 51% of participants developed a SARS-CoV-2 infection, but it was mild, with only nine requiring hospital admission and no deaths. Overall, 99% of tested specimens had antibodies above threshold to either spike or RBD proteins. Specimens that did not and those with lower antibody levels had testing earlier in the pandemic, and were from participants with fewer vaccine doses, and did not have natural infection. Age, depression, comorbidity, HCV co-infection, current substance use, CD4 count, or HIV viral load were predictive of antibody level. Those with hybrid immunity had higher antibody responses. Conclusions: In cohorts of persons with HIV-HCV coinfection and those who are ageing, we observed high rates of seroconversion to COVID-19 antigens. Antibody levels were higher among those with more vaccine doses, hybrid immunity, and later in the pandemic waves. Although 51% developed a breakthrough infection, outcomes were mild with no deaths.

Antibodies doi: 10.3390/antib15020029

Authors: Beata Pamuła Martyna Banach Marta Mikońska Karolina Korytkowska Krzysztof Lacek Oliwia Śniadała Małgorzata Marczak Krzysztof Flis Aleksandra Sowińska Damian Kołakowski Jerzy Pieczykolan Beata Zygmunt Maciej Wieczorek Olga Abramczyk

Background/Objectives: Antibodies are a rapidly expanding field in drug discovery, but their monospecificity limits therapeutic applications, particularly in complex inflammatory diseases. Multispecific therapeutics, which combine variable regions targeting two or more antigens, offer potential advantages such as enhanced efficacy, broader target modulation, and reduced side effects. This study aimed to identify and characterize bispecific, VHH-based antibodies simultaneously targeting IL-6 and IL-17A—two key cytokines involved in autoimmune and chronic inflammatory conditions. Methods: A phage display screening was conducted using llama-derived VHH libraries to select binders against human IL-6 and IL-17A. Binding affinities of individual VHHs and assembled bispecific constructs were assessed using Bio-Layer Interferometry (BLI). Functional activity was evaluated using reporter cell lines responsive to IL-6 and IL-17A signaling. Biophysical and quality assessments of selected VHHs and bispecific antibodies were performed using the Uncle screening platform and LabChip capillary electrophoresis. Results: Several high-affinity VHH binders were identified for both IL-6 and IL-17A, and incorporated into bispecific antibody formats. The bispecific candidates exhibited simultaneous inhibition of both cytokine pathways in functional reporter assays. Biophysical characterization confirmed good stability and purity profiles for selected molecules. Conclusions: This study demonstrates the feasibility of generating stable, functional bispecific VHH-based antibodies targeting IL-6 and IL-17A. These constructs show potential as therapeutic agents for treating autoimmune and chronic inflammatory diseases by modulating multiple signaling pathways simultaneously.

Antibodies doi: 10.3390/antib15020028

Authors: Maddalena Calvo Marta Caccamo Dalila Maria Cammarata Laura Trovato

Invasive candidiasis (IC) remains a significant cause of morbidity and mortality among critically ill, hematologic, and neonatal patients worldwide. Rapid and accurate diagnosis is essential to guide timely antifungal therapy and improve outcomes. Among available diagnostic tools, 1,3-β-D-glucan (BDG), a polysaccharide component of the fungal cell wall, has emerged as a key biomarker. BDG assays allow for early detection of probable IC, often preceding positive blood cultures, and offer prognostic information based on serial measurements. Species-specific differences in Candida cell wall composition influence BDG release and diagnostic sensitivity. Candida albicans generally correlates with high BDG levels, whereas Nakaseomyces glabrata, Candida parapsilosis, and Candida auris exhibit variable or lower glucan exposure, limiting assay sensitivity. BDG performance is affected by patient-specific factors, such as prior surgery, transfusions, or coexisting bacterial infections, which may lead to false-positive results. Molecular techniques, including PCR-based assays, provide complementary diagnostic accuracy and species identification, and their combination with BDG testing enhances sensitivity up to 90%. Serial BDG monitoring supports risk stratification and treatment response assessment, with persistent elevations predicting worse outcomes. In neonatal and pediatric populations, optimal cut-off values remain under investigation, highlighting the need for integration with clinical and microbiological data. Overall, BDG represents a valuable adjunct in a multimodal diagnostic workflow, providing both diagnostic and prognostic insights in invasive candidiasis management.

Antibodies doi: 10.3390/antib15020027

Authors: Sanika Naware Saurav Kulkarni Sahil Salvi Dhvani Patel Dhaval K. Shah

Background/Objectives: The neonatal Fc receptor (FcRn) plays a crucial role in extending the systemic half-life of monoclonal antibodies (mAbs), but its influence on ocular distribution remains incompletely understood. This study investigated the impact of FcRn on the ocular disposition of mAbs following systemic administration in rabbits. Methods: New Zealand White rabbits received a single intravenous dose (1 mg/kg) of either wild-type trastuzumab (TS-WT) or its FcRn non-binding variant (IHH). Plasma and ocular tissues (retina, iris–ciliary body, vitreous humor, aqueous humor, cornea, conjunctiva, and tears) were collected at terminal time points up to 336 h for TS-WT and 168 h for IHH. Antibody concentrations were quantified using a validated sandwich ELISA. Pharmacokinetic parameters and antibody biodistribution coefficients (ABC) were calculated to assess the FcRn-mediated effects on ocular distribution. Results: TS-WT demonstrated 2-fold higher systemic exposure compared to IHH. The iris–ciliary body exhibited the highest absolute exposure for both antibodies, with TS-WT showing significantly higher accumulation (ABC0–168h: 14.95% vs. 8.89%). Retinal distribution remained comparable between antibodies (5.96% vs. 5.51%). Both antibodies were detectable in tears, with ABC value of ~4% reported for TS-WT. TS-WT also demonstrated markedly increased distribution in vitreous humor and tear fluid (3.5- and 5.5-fold higher ABC values, respectively) compared to IHH. The cornea (5.76% vs. 5.57%) and conjunctiva (7.71% vs. 7.21%) showed comparable relative distribution between TS-WT and IHH, while aqueous humor showed minimal differences (0.44% vs. 0.52%). Conclusions: This investigation reveals distinct tissue-specific patterns of FcRn-mediated mAb distribution within the eye. FcRn binding significantly enhanced antibody distribution in ocular tissues, such as the iris–ciliary body, and tears, with less pronounced effects on the retina, cornea, conjunctiva and aqueous humor. These findings provide mechanistic insights for optimizing mAb-based therapeutics for ocular disease and understanding the ocular toxicity of mAb-based therapeutics, such as antibody–drug conjugates.

Antibodies doi: 10.3390/antib15020026

Authors: Jingming Zhang Matthew Larsen Timothy Blanc Babita S. Parekh Ming-Ching Hsieh

Background: Protein A resins are indispensable for monoclonal antibody (mAb) production, yet their condition and performance are traditionally assessed using indirect or qualitative methods. In this study, the multi-attribute method (MAM), previously applied to therapeutic protein characterization, is systematically adapted for the first time as a unified liquid chromatography–mass spectrometry (LC-MS) platform for Protein A resin analysis. Method: Four Cytiva Protein A resins, MabSelect™, MabSelect SuRe™, MabSelect SuRe™ LX, and MabSelect™ PrismA, were evaluated by MAM for resin identity, Protein A ligand integrity, fouling by impurities, and cleaning performance. Results: MAM enables resin-specific peptide fingerprinting and quantitative monitoring of Protein A ligand post-translational modifications (PTMs), including deamidation, isomerization, and fragmentation induced by repeated clean-in-place (CIP) cycles. Comparative analysis of virgin and used resins revealed ligand degradation and fouling despite engineered alkaline stability, with MabSelect™ showing the greatest susceptibility. Importantly, residual monoclonal antibodies (mAbs) and host cell proteins (HCPs) were directly detected and quantified from the resin matrix, providing a molecular-level assessment of resin cleaning effectiveness not achievable with conventional approaches. Conclusions: This work establishes MAM as a novel, sensitive, and comprehensive strategy for Protein A resin lifecycle management, delivering actionable insight for resin selection, cleaning optimization, and downstream process development.

Antibodies doi: 10.3390/antib15020025

Authors: Davide Bagnara Andrea Nicola Mazzarello Monica Colombo Ennio Nano Niccolò Cardente Fabiana Ferrero Nadia Bertola Vanessa Cossu Fabio Ghiotto Adalberto Ibatici Emanuele Angelucci Antonino Neri Massimo Gentile Fortunato Morabito Manlio Ferrarini Giovanna Cutrona Franco Fais

Background/Objectives: The immunoglobulin heavy-chain variable (IGHV) gene repertoire represents a characteristic feature of chronic lymphocytic leukemia (CLL), although its configuration is not well defined at the early disease stages. The IGHV repertoire of a cohort of early CLL patients was analyzed and compared to that of a “real-world” reference cohort. Methods: Patients from the O-CLL1 observational protocol, which enrolled only Binet stage A cases within twelve months from diagnosis, were studied. IGHV/IGHJ rearrangements were sequenced and annotated following ERIC recommendations, and stereotyped subsets were assigned using ARResT/AssignSubsets. The repertoire features were compared with the dataset of a real-world cohort of patients with heterogeneous staging (CTR cohort) and with published early-diagnosis series. Results: IGHV and IGHJ gene distributions and HCDR3-length profiles in O-CLL1 closely mirrored those of CTR, indicating that the BcR IG repertoire at diagnosis is already defined rather than being selected during disease progression. Mutated IGHV (M-CLL) predominated, with a frequency of stereotyped BcR IG comparable to that of other early-diagnosis cohorts. However, within this conserved framework, subset #4 was over-represented among M-CLL from O-CLL without an increased overall IGHV4-34 gene usage, suggestive of a selective expansion rather than a recombinational bias. Subset #4 cases retained canonical HCDR3 motifs and showed time-to-first-treatment like other M-CLL, likely reflecting the younger age structure of O-CLL1. Conclusions: Early-diagnosis CLL displays a biased IGHV repertoire with stereotyped configurations characteristic of CLL, including subsets that are rare in the normal B-cell repertoire. These findings support a central role for antigen-driven selection in shaping CLL evolution.

Antibodies doi: 10.3390/antib15020024

Authors: Maurizio Benucci Elisa Cioffi Francesca Li Gobbi Emanuele Antonio Maria Cassarà Riccardo Terenzi Edda Russo Valentina Grossi Barbara Lari Maria Infantino Mariangela Manfredi

COVID-19 mRNA vaccines activate type I interferon pathways and in genetically or immunologically predisposed individuals may trigger autoimmune responses, including autoantibodies against melanoma differentiation-associated protein 5 (MDA5). Although cases of dermatomyositis (DM), particularly anti-MDA5-positive DM, have been increasingly reported after SARS-CoV-2 vaccination, its clinical spectrum and management remain incompletely defined. We conducted a narrative review of the literature on post-vaccination dermatomyositis, focusing on clinical features, autoantibody profiles, therapeutic approaches, and outcomes. The review was enriched by the inclusion of a new case: a 60-year-old woman who developed anti-MDA5-positive dermatomyositis two weeks after receiving her fourth dose of the BNT162b2 (Pfizer/BioNTech) vaccine. She presented predominantly with cutaneous and articular manifestations in the absence of interstitial lung disease. Treatment with oral prednisone, intravenous alprostadil, and the Janus kinase inhibitor tofacitinib resulted in marked clinical improvement. This case, together with the literature review, illustrates both typical and atypical presentations of vaccine-associated anti-MDA5 DM, highlights diagnostic challenges without lung involvement, and suggests JAK inhibition as a potential therapeutic option, contributing to a more comprehensive understanding of post-vaccination dermatomyositis.

Antibodies doi: 10.3390/antib15020023

Authors: Massimo Papale Carmela Paolillo Loredana Iafelice Tiziana Trivisano Giuseppe Stefano Netti Elena Ranieri Gaetano Corso

Background: Anti-double-stranded DNA (anti-dsDNA) antibodies are a key serological marker for systemic lupus erythematosus (SLE) and are commonly assessed in conjunction with anti-nuclear antibody (ANA) testing by indirect immunofluorescence (IIF) on HEp-2 cells. However, their detection is influenced both by the heterogeneity of the autoimmune response and by the characteristics of the analytical method employed, thereby complicating diagnostic interpretation. Methods: In this retrospective single-center study, 3090 consecutive patients undergoing anti-dsDNA analysis were screened, and 138 positive individuals, with anti-dsDNA levels ≥ 15 IU/mL by fluoroenzyme immunoassay (FEIA), were included in the study. A control group of 29 anti-dsDNA-negative patients was also analyzed. Anti-dsDNA-positive patients were stratified by antibody level (low, mild, high), and the results were correlated with HEp-2 IIF titers and fluorescence patterns. Furthermore, in a subset of 30 positive patients, anti-dsDNA antibodies were evaluated using immunoblotting (IB) and the Crithidia luciliae indirect immunofluorescence test (CLIFT). Statistical analyses assessed associations and concordance among methods. Results: Higher anti-dsDNA levels were generally associated with higher HEp-2 IIF titers. However, a considerable percentage (35%) of patients with positive anti-dsDNA were negative by HEp-2 IIF. Notably, high anti-dsDNA levels were detected in 19% of HEp-2 IIF-negative patients (titer < 1:80), 18% of mildly HEp-2 IIF-positive patients (titer 1:80–1:160), and 25% of HEp-2 IIF-positive patients (titer > 1:320). In the subset of 30 positive patients, FEIA analysis showed high concordance with the immunoblot in both IIF-positive (81%) and -negative (100%) patients, while CLIFT demonstrated lower agreement with both FEIA and IB independently of the IIF. Conclusions: Our findings indicate that anti-dsDNA antibody detection may occur independently of HEp-2 IIF positivity and that FEIA, especially when confirmed by immunoblot, represents a reliable approach for anti-dsDNA assessment. The observed results in this study likely reflect differences in epitope recognition and assay sensitivity among methods, suggesting the use of a multi-step diagnostic strategy in the serological evaluation of SLE.

Antibodies doi: 10.3390/antib15020022

Authors: Philipp D. Kaiser Simon Straß Sandra Maier Evgenia Herbold Bjoern Traenkle Anne Zeck

Background/Objectives: Developability assessment is a critical step in advancing antibody-based molecules toward clinical application. This evaluation typically begins during clinical candidate selection and continues throughout all modifications of the molecule during development. It is guided by the target product profile, which includes the intended administration route and regimen, formulation parameters, and process conditions encountered during manufacturing, storage, and delivery. While developability testing is well established for conventional therapeutic antibodies, strategies for assessing single-domain antibodies (sdAbs) and their conjugates remain underexplored. Here, we present a strategy to test the developability of sdAbs as a case study for two clinical candidates intended as precursors for the production of diagnostic tracers for clinical imaging. Methods: Assays were developed to evaluate chemical and thermodynamic stability, target binding affinity and capacity, and chelation efficiency (“chelatability”). Accelerated stability studies were conducted for both unconjugated sdAbs and their chelator conjugated forms following incubation at two pH conditions, at multiple time points, and after twelve freeze–thaw cycles to simulate process conditions and long-term storage. Analytical assays were applied stepwise in a hierarchical approach to minimize experimental effort and material consumption. Candidates exhibiting critical developability features were selectively addressed by assays with increasing precision. Results: A tailored panel of analytical assays optimized for low molecular weight proteins was established and applied to the two clinical candidates, identifying instability hotspots as well as potential mitigation strategies. Successful engineering of a candidate with an initially critical developability profile was achieved. Conclusions: This study demonstrates the implementation of a structured developability assessment strategy for sdAb conjugates. The approach integrates physicochemical and functional stability evaluations, supporting robust candidate selection, formulation development, and method optimization for this class of molecules.

Antibodies doi: 10.3390/antib15020021

Authors: Marcos García-Ocaña Lorea Legazpi-Olabide Sandra Rodríguez-Rodero Paula Rodríguez-Folgueira Iván Fernández-Vega Marcos Ladreda-Mochales Juan R. de los Toyos Luis J. García-Flórez

Background: Collagen XIα1, encoded by the COL11A1 gene, is a minor fibrillar collagen that is overexpressed in various human cancers, in which its presence correlates with tumor aggressiveness and progression. Methods: In this study, we developed two novel mouse monoclonal antibodies (mAbs)—anti-colXIα1 clone 3 and anti-colXIα1 clone 9—that target the putative C-telopeptide of human collagen XIα1. These antibodies target the RRHTEGMQA sequence, a unique nine-amino-acid stretch within the putative C-telopeptide of human collagen XIα1. Results: Corresponding to nearly identical V(D)J gene segments and complementarity-determining regions (CDRs), the antibodies specifically bound the RRHTEGMQA epitope in ELISAs but did not react with the C-propeptide. This specificity was further confirmed with the purified anti-colXIα1 clone 9 mAb, which demonstrated strong reactivity against recombinant proteins containing the RRHTEGMQA sequence in both ELISAs and Western blot assays. This sequence seems to behave as a linear B-cell neoepitope, in which the RRHT motif is crucial for epitope recognition. Otherwise, no immunodetections were observed, either in cultures and lysates from the COL11A1-highly expressing A204 human cell line or on tissue sections from specimens of human pancreatic ductal adenocarcinoma (PDAC), with strong desmoplastic reactions. Conclusions: Given the lack of precise knowledge of the characteristics of the putative C-telopeptide of human collagen XIα1, the presented antibodies could enhance our understanding of the processing of human procollagen XIα1 and contribute to better characterization of the tumor microenvironment of COL11A1-expressing cancers.

Antibodies doi: 10.3390/antib15020020

Authors: Maja Vilibić Klara Barbić Maja Bogdanić Snježana Židovec-Lepej Ana Matošić Ana Sanković Dalibor Karlović Leona Radmanić Matotek Nataša Kutela Sergej Nadalin Ema Borko Vladimir Savić Ljubo Barbić Marija Santini Hrvojka Janković Vladimir Stevanović Tatjana Vilibić-Čavlek

Background/Objectives: Viral hepatitis A–E represents a significant public health problem. Data on the prevalence of viral hepatitis markers among alcoholics are inconsistent. Methods: The study included 151 patients treated for alcohol abuse in one Croatian center. The control group consisted of 110 individuals from the general population tested for a routine check-up. The prevalence of viral hepatitis markers was assessed using serology and molecular methods. Results: The prevalence rates of hepatitis markers among patients were as follows: anti-HAV, 15.2%; anti-HBs, 11.9%; anti-HBc/anti-HBs, 2.6%; anti-HCV, 4.0%; and anti-HEV, 14.6%. HCV RNA was detected in one patient (0.6%). Compared with the control group, patients showed significantly higher HCV seroprevalence (4.0 vs. 0%), while the prevalence of other hepatitis markers did not differ significantly between the groups. The anti-HAV prevalence was associated with age (from 0% in patients aged <40 years to 42.9% in patients aged 60+ years), employment status (highest among retired individuals at 46.2%), and age of occasional alcohol consumption (highest seroprevalence of 26.3% in those who reported consumption between 22 and 25 years). The association between anti-HEV and educational level was of borderline significance. Logistic regression showed that older and retired patients and those who consumed alcohol occasionally between 22 and 25 years showed higher odds for HAV seropositivity (OR = 11.454–49.400, OR = 6.857, and OR = 4.464, respectively). Patients with university degrees were at lower risk for HEV seroprevalence (OR = 0.083). Conclusions: Alcoholic patients showed a higher HCV seroprevalence than the general population, while the prevalence of other viral hepatitis markers did not differ between the groups. Further studies on a larger cohort of patients are needed to confirm these findings.

Antibodies doi: 10.3390/antib15020019

Authors: Yunfeng Liu Qiting Huang Dongna Zhang Yingjun Wang Shuaiying Zhao Jianchuan Wen Yingying Kong Jianfeng Xu

Objectives: The epidermal growth factor receptor (EGFR) is a clinically relevant membrane receptor that is frequently overexpressed or dysregulated in multiple types of cancer, making it an important target for antibody-based strategies. Nanobodies, derived from camelid heavy-chain antibodies, possess favorable properties such as small size, high stability, and strong antigen-binding capacity. This study aimed to generate EGFR-specific nanobodies and to systematically characterize their binding properties and initial functional activity. Methodology: Bactrian camels were immunized with a whole-cell antigen prepared from 293F cells transiently transfected to express full-length human EGFR. A high-diversity phage display nanobody library was constructed from peripheral blood lymphocytes. After two rounds of biopanning against EGFR, positive clones were screened and selected. The identified nanobodies were recombinantly expressed in Escherichia coli and purified. Binding specificity, epitope relationships, and kinetic parameters were evaluated using high-performance liquid chromatography (HPLC), bio-layer interferometry (Octet), and flow cytometry. The effect of selected nanobodies on EGF-induced cell proliferation was evaluated using a CCK-8 assay. Results: Two EGFR-specific nanobodies, Nb2H4 and Nb2B6, were successfully isolated. Both nanobodies exhibited specific binding to EGFR and recognized distinct, non-competing epitopes. Kinetic analyses revealed favorable binding affinities, and flow cytometry confirmed their ability to recognize EGFR in its native cellular context. In addition, Nb2H4 significantly suppressed EGF-induced proliferation in an EGFR-overexpression cell model, indicating preliminary functional activity. Conclusions: This study reports on the successful generation and in vitro characterization of EGFR-targeting nanobodies based on the extracellular domain of EGFR. The identified nanobodies provide useful molecular tools for epitope mapping, structural studies, and the further exploration of EGFR-directed antibody engineering strategies.

Antibodies doi: 10.3390/antib15020018

Authors: Isamu Tsuji Kumiko Okada Benjamin Kroppen Tetsufumi Katta Kaori Yamamura Takeshi Nishihama Ayako Miura Hansjörg Götzke Eric Crampon Andrea Bertolotti-Ciarlet

Background/Objectives: The SARS-CoV-2 virus frequently undergoes mutations to evade the human immune system. Vaccines for new strains are developed each season, and an identification test confirming the specific strain is essential for vaccine quality control, as stated by the U.S. Food and Drug Administration. However, a shorter timeline of antibody discovery was required to adjust vaccine development schedules. Therefore, anti-SARS-CoV-2 strain-specific, single-domain antibodies (sdAbs) for SARS-CoV-2 vaccines were discovered using alpaca synthetic libraries without animal immunization. Methods: A synthetic sdAb library was developed based on conserved alpaca sdAb frameworks, with a degree of freedom in the three complementarity-determining regions. Specific and high-affinity sdAb clones were selected from the library by one ribosomal display round, followed by two phage display selections using a biotinylated strain-specific SARS-CoV-2 receptor-binding domain (RBD) of the spike protein as bait and non-biotinylated RBD variants to block. The sdAbs clones were applied to the identification test using Western blotting. The binding epitopes were determined by hydrogen–deuterium exchange mass spectrometry. Results: Five clones of XBB.1.5 and two clones of JN.1-specific sdAbs were discovered. Anti-JN.1 sdAb clone 1B9 detected JN.1 vaccine products but no other previously produced vaccine strains, Wuhan, BA.5 and XBB.1.5, by WB for vaccine identification test. Four binding epitopes for anti-JN.1 sdAb clone 1B9 were identified, including the L455S mutation, a critical amino acid to evade neutralizing antibodies for the JN.1 strain. Conclusions: Anti-XBB.1.5 and JN.1-specific sdAbs were discovered from a synthetic single-domain antibody library within 8–9 weeks, and these sdAbs were applied to vaccine identification testing.

Antibodies doi: 10.3390/antib15010017

Authors: Sébastien Estaran Bernard Hehlen Alain Chavanieu

Background/Objectives: Antibody-dependent cellular cytotoxicity relies on the interaction between the Fc region of immunoglobulin G1 (IgG1) and the CD16a receptor. While removal of core fucosylation on Fc and introduction of the DFTE mutation set (S239D, H268F, S324T, I332E) are known to enhance CD16a binding, the detailed contributions of these engineered sites in solution remain incompletely defined. Methods: Here, we employed 1 µs molecular dynamics simulations to map, at atomic resolution, the interaction networks stabilizing pre-formed Fc-CD16a complexes, including afucosylated Fc-wild-type, DFTE-engineered, Fc-fucosylated, and asymmetrically engineered Fc variants. Results: Our results show that only S239D, present on both Fc chains, and H268F on chain A consistently contribute to stabilizing the CD16a interface, while I332E does not form persistent interactions. Glycan–protein contacts are primarily intrachain, with transient interchain glycan–glycan interactions not contributing significantly to complex stability. Fucosylation on Fc significantly reduces binding stability by disrupting peripheral interactions and critical glycan-mediated contacts. Notably, the asymmetric Fc variant, in which the two heavy chains carry distinct sets of substitutions, retains high-affinity binding despite lacking S239D and carrying core fucose, through a novel hydrophobic cluster and reinforced peripheral electrostatic interactions. Conclusions: Altogether, these findings provide a quantitative framework for how targeted mutations and fucose modifications remodel Fc-CD16a interactions, offering insights for the rational design of next-generation therapeutic antibodies.

Antibodies doi: 10.3390/antib15010016

Authors: Juan Carlos Silva-Espinoza Mauricio I. Rodriguez Rodriguez Claire Eunise Perucho Brian A. Terrazas Carlos Valenzuela Stephany Palos Vargas Andrea Carlin Diana L. Prospero Giulio Francia Manuel Llano

Background/Objectives: Schlafen (SLFN) 8 and SLFN9 are mouse members of the Schlafen protein family, believed to have arisen through a gene duplication event. The physiological roles of these proteins remain poorly defined, in part due to the absence of reliable, commercially available antibodies for their detection. Methods: To develop specific antibodies, we performed an amino acid sequence alignment of these proteins and identified a thirteen amino acids long peptide predicted by AlphaFold modeling and hydropathicity analysis to be surface-exposed in both SLFN proteins. The SLFN8 peptide was conjugated to KLH and used to immunize mice, employing Poly(I:C) as an adjuvant. Results: We verified the anti-SLFN8 antibody specificity in mouse tissues, engineered human cells, and recombinant proteins by different immunodetection techniques, including Western blotting, immunoprecipitation, immunohistochemistry, and ELISA. Furthermore, splenocytes from immunized mice were used to generate hybridomas that secreted IgG antibodies with SLFN8-peptide specificity, as assumed by ELISA. Conclusions: Our results demonstrate that the identified peptide is highly immunogenic and capable of eliciting antibodies that distinguish between these two exceedingly similar proteins in a broad group of immunodetection techniques.

Antibodies doi: 10.3390/antib15010015

Authors: Aya Jalil Nadia Touil Omar Nyabi Elmostafa El Fahime Sara Benlhachemi Jean-Luc Gala Khalid Ennibi Karim Bakkouri Abdelaziz Benjouad Lamiae Belayachi

Background: bacterial pathogens and their toxins present analytical challenges for rapid and specific detection, contributing to over 600 million cases of illness annually and worsening antimicrobial resistance (AMR). Conventional detection methods are useful but limited. Single-domain antibodies (sdAbs) offer alternative recognition elements with unique biochemical and engineering benefits, enabling the development of nanobody-based immunoassays and biosensing platforms that provide fast, highly selective, and reliable detection of bacterial pathogens and toxins in both food and clinical environments. Objectives: this systematic review assesses the analytical and functional performance of nanobody-based immunoassays and sensing formats for detecting bacteria and toxins across food and clinical samples. Methods: following PRISMA guidelines, major scientific databases were used to gather research, resulting in 32 eligible studies published between 2011 and 2025. Results: data collected included assay platforms, target bacteria and toxins, limit of detection, sensitivity, specificity, matrix recovery, and practicality. Risk of bias was evaluated using an adapted QUADAS-2 framework. The review shows that nanobody-based immunoassays have achieved high performance, thermostability, compatibility with genetic engineering, and versatile assay design. When combined with advanced transduction and signal amplification strategies, these systems contribute to the development of highly sensitive and user-friendly bioanalytical platforms for detecting bacteria and toxins. Conclusions: however, most studies relied on spiked samples and lacked large-scale validation, emphasizing the need for standardized benchmarking and real-world testing.

Antibodies doi: 10.3390/antib15010014

Authors: Qiqi Li Zhengyuan Huang Zainab Saeed Orene Greer James A. Harker Nishel M. Shah

The transplacental transfer of maternal immunoglobulin G from the mother to the foetus is central for providing immunity in early life, resulting in full-term newborns having IgG repertoires and levels similar to those of their mothers. The neonatal Fc receptor is recognised as the primary transporter of IgGs across the placental epithelium. Understanding the mechanisms of transplacental antibody transfer and factors that affect them is essential in optimising maternal vaccination strategies, ultimately protecting infants from various environmental pathogens. This review first outlines the biological mechanisms governing transplacental IgG transfer, followed by a discussion of how this process may be disrupted by physiological and pathological conditions during pregnancy, including preterm birth, hypergammaglobulinemia, maternal pathogenic IgG, maternal infections, hyperglycaemia, and exposure to biological therapies. We also summarise currently available models used to study transplacental IgG transfer, highlighting existing knowledge gaps and future directions for research in this field.

Antibodies doi: 10.3390/antib15010013

Authors: Juan González-Fernández Laura Ullate Marta Rodero Alvaro Daschner Carmen Cuéllar

Background/Objectives: Allergic features of anisakiasis, caused by ingestion of third-stage larvae of Anisakis simplex via raw or undercooked fish, manifest clinically as acute gastroallergic anisakiasis (GAA) or chronic urticaria with Anisakis sensitization (CU+). Differentiating these clinical phenotypes remains challenging. This study aimed to evaluate the maturation and avidity of specific antibodies (IgE, IgG4, IgG, and IgA) as biomarkers for discriminating between acute and chronic forms of anisakiasis. Methods: A prospective cohort of 65 patients from Madrid, Spain, was classified into three groups: GAA (n = 22), CU+ (n = 22), and chronic urticaria without sensitization (CU−, n = 21). Serum samples were analyzed for antigen-specific immunoglobulins using ELISA and Western blot. Avidity indices (AIs) were quantified through urea dissociation assays. Statistical comparisons and correlation analyses were performed to associate antibody avidity with clinical phenotype and demographic variables. Results: GAA patients exhibited significantly lower IgE avidity indices compared to CU+ individuals (mean AI: 79.9% vs. 88.5%), indicating a less mature IgE response during acute infection. Conversely, IgG4 and IgG avidity were elevated in GAA relative to CU+, reflecting an active but transient immune response. IgA antibodies were detected in both groups, although avidity differences lacked discriminatory capacity. No sex- or age-related differences in antibody avidity were observed. Longitudinal follow-up of GAA patients demonstrated an increase in IgE avidity over time. Conclusions: Quantitative assessment of antibody avidity, particularly for IgE and IgG4, enhances understanding of A. simplex immunopathogenesis and serves as a valuable biomarker for distinguishing acute from chronic clinical presentations. These findings support the use of avidity indices in the diagnosis, staging, and clinical management of anisakiasis.

Antibodies doi: 10.3390/antib15010012

Authors: Anne-Sophie Kuhlmann Tami Peters Donald K. Hamlin Yawen Li Xinyi Wang Megan Stackhouse Frances M. Cole Jasmin Martinez-Reyes Brenda M. Sandmaier Hans-Peter Kiem D. Scott Wilbur Robert D. Harrington Seth H. Pincus

Background: We are developing cytotoxic immunoconjugates (CICs) to eliminate HIV-infected cells. We investigated the efficacy and kinetics of killing by different forms of CICs targeted by the same monoclonal antibody (mAb), an immunotoxin (IT), antibody-drug conjugate (ADC), and radioimmunoconjugate (RIC). Methods: We compared in vitro effects of CICs made by conjugating anti-gp41 mAb 7B2 to deglycosylated ricin A chain (7B2-dgA), the anthracycline derivative PNU-159682 (7B2-PNU), or the α-emitting isotope actinium-225 (7B2-225Ac). Kinetic analyses of cell growth were performed measuring electrical impedance every 15 min over a 7-day period using cells stably expressing the HIV envelope and Env-negative parent cells. Results: 7B2-dgA and 7B2-225Ac were more potent and acted more rapidly to kill cells than 7B2-PNU. Both the 7B2-PNU and 7B2-225Ac induced bystander-cell killing, whereas the IT did not and consequently allowed the outgrowth of Env-negative cells. Low dose or brief exposure to 7B2-PNU resulted in an increased rate of cell growth. Conclusions: An IT, ADC, and RIC showed substantial differences in the degree of specific toxicity, kinetics, and mechanisms of killing. The results of this side-by-side comparison have implications for the development of CICs to treat HIV, as well as other conditions.

Antibodies doi: 10.3390/antib15010011

Authors: Jilei Wu Chenghua Liu Fenghao Peng Zeyuan Yu Chunxia Qiao Guang Yang Heng Luo Keyi Sun Ziyao Ning Jing Wang Yan Wen Jijun Yu

Background: Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli (EHEC), is a highly potent exotoxin responsible for severe complications such as hemolytic uremic syndrome (HUS). Among its isoforms, Stx2 exhibits stronger cytotoxicity and poses greater clinical risk, yet no effective therapy currently exists. Methods: In this study, two human monoclonal antibodies, YG12-1 and YG12-2, were identified from a phage display library and systematically characterized using an integrated modeling–validation workflow. Results: Structural modeling with ImmuneBuilder and Rosetta revealed that YG12-2 possessed a longer CDRH3 topology, more short-range hydrogen bonds, and stronger electrostatic complementarity, corresponding to lower binding energy and higher apparent affinity in ELISA and SPR. Although YG12-2 had a better affinity, YG12-1 shows better protective activity in a murine model of acute peritoneal infection. This paradox highlights a non-linear relationship between structural affinity and biological efficacy, emphasizing the importance of functional epitope accessibility and pharmacokinetic behavior in determining neutralization outcomes. Conclusions: Overall, these results indicated that targeting Stx2 with YG12-1 and YG12-2 could serve as a promising protective strategy against E. coli O157:H7 infection.

Antibodies doi: 10.3390/antib15010010

Authors: Yukihiro Shoyama

There are a vast number of monoclonal antibodies (MAbs) against biological components; however, the number for natural products is less than 50. MAbs against ginsenosides, i.e., dammarane triterpene glycosides contained in ginseng, were prepared to develop an Eastern blotting method that can estimate the number of bound sugars and pharmacological activity. Meanwhile, as a method for producing ginsenoside Rg3, which is used as an anti-cancer drug, an affinity column for ginsenoside Rb1 was prepared to isolate the raw material ginsenoside Rb1 in a single step, and a method for obtaining ginsenoside Rg3 through fermentation was proposed. A unique MAb capable of detecting all solasodine glycosides contained in Solanum plants was created to prepare an affinity column capable of isolating solasodine glycosides from S. khasianum fruit in a single step. The single-chain variable fragment gene was induced from the MAb against solasodine glycoside and introduced into the hairy root system of S. khasianum, thereby increasing the solasodine glycoside content more than twofold. As a result, we recognized that this method can be used to breed plants with higher concentrations of plant secondary metabolites like solasodine glycosides. The above results collectively demonstrate that solasodine glycoside can be isolated from S. khasianum in high yields and that this compound enables the production of steroids in high yields through a one-step chemical reaction.

Antibodies doi: 10.3390/antib15010009

Authors: Ming-Ching Hsieh Chao Richard Li Margaret A. Velardo Jingming Zhang Babita S. Parekh

Background: This study assesses the robustness of a legacy N-glycan profiling method for the therapeutic antibody MAB1 with different Peptide-N-glycosidase F (PNGase F) enzyme sources, solid phase extraction (SPE) cartridges, and reagent stability, aligning with ICH Q14 lifecycle management principles. Glycosylation profiling is critical for therapeutic antibodies as it influences both function and pharmacokinetics. Method: The legacy N-glycan profiling method, 2-aminobenzoic acid (2-AA) labeling combined with normal-phase HPLC, was re-evaluated to confirm consistent analytical performance in the context of evolving regulatory expectations. The evaluation focused on three key factors: PNGase F enzyme sources, solid-phase extraction (SPE) cartridges, and reagent stability. Results: Commercial PNGase F enzymes showed various performances, with some sources yielding significant differences. Several SPE cartridges were also tested, with certain formats displaying poor recovery and high variability, particularly for sialylated glycans. In addition, reagent stability studies revealed rapid degradation of the labeling reagent within a few days. Conclusions: These results underscore the importance of risk control, continual improvement, and lifecycle management to ensure reliable glycosylation analysis and the sustained robustness of legacy methods.

Antibodies doi: 10.3390/antib15010008

Authors: Stoimen Dimitrov Mihael Tsalta-Mladenov Plamena Kabakchieva Tsvetoslav Georgiev Silva Andonova

Paraneoplastic neurological syndromes (PNSs) are immune-mediated disorders caused by an antitumor response that cross-reacts with the nervous system, leading to severe and often irreversible neurological disability. Once considered exceedingly rare, PNSs are now increasingly recognized owing to the identification of novel neural autoantibodies, wider use of commercial testing, and the emergence of immune checkpoint inhibitor (ICI)-related neurotoxicity that phenotypically overlaps with classic PNS. In this narrative review, we performed a structured search of PubMed/MEDLINE, Scopus, Web of Science, and Google Scholar, without date restrictions, to summarize contemporary advances in the epidemiology, pathogenesis, diagnosis, and management of PNS. Population-based data show rising incidence, largely reflecting improved ascertainment and expanding indications for ICIs. Pathogenetically, we distinguish T-cell-mediated syndromes associated with intracellular antigens from antibody-mediated disorders targeting neuronal surface proteins, integrating emerging concepts of molecular mimicry, tumor genetics, and HLA-linked susceptibility. The 2021 PNS-Care criteria are also reviewed, which replace earlier “classical/non-classical” definitions with risk-stratified phenotypes and antibodies, and demonstrate superior diagnostic performance while underscoring that “probable” and “definite” PNS should be managed with equal urgency. Newly described antibodies and methodological innovations such as PhIP-Seq, neurofilament light chain, and liquid biopsy are highlighted, which refine tumor search strategies and longitudinal monitoring. Management principles emphasize early tumor control, prompt immunotherapy, and a growing repertoire of targeted agents, alongside specific considerations for ICI-associated neurological syndromes. Remaining challenges include diagnostic delays, limited high-level evidence, and the paucity of validated biomarkers of disease activity. Future work should prioritize prospective, biomarker-driven trials and multidisciplinary pathways to shorten time to diagnosis and improve long-term outcomes in patients with PNS.

Antibodies doi: 10.3390/antib15010007

Authors: Muhammad Soyfoo Julie Sarrand

Autoantibodies have long been regarded as passive reflections of immune dysregulation in connective tissue diseases (CTDs). Recent advances in systems immunology and molecular pathology have fundamentally redefined them as active molecular fingerprints that delineate distinct disease endophenotypes with predictive power for clinical trajectories and therapeutic responses. Rather than mere epiphenomena, autoantibodies encode precise information about dominant immune pathways, organ tropism, and pathogenic mechanisms. This review synthesizes emerging evidence that autoantibody repertoires—defined by specificity, structural properties, and functional characteristics—stratify patients beyond traditional clinical taxonomy into discrete pathobiological subsets. Specific signatures such as anti-MDA5 in rapidly progressive interstitial lung disease, anti-RNA polymerase III in scleroderma renal crisis, and anti-Ro52/TRIM21 in systemic overlap syndromes illustrate how serological profiles predict outcomes with remarkable precision. Mechanistically, autoantibody pathogenicity is modulated by immunoglobulin isotype distribution, Fc glycosylation patterns, and tissue-specific receptor expression—variables that determine whether an antibody functions as a biomarker or pathogenic effector. The structural heterogeneity of autoantibodies, shaped by cytokine microenvironments and B-cell subset imprinting, creates a dynamic continuum between pro-inflammatory and regulatory states. The integration of serological, transcriptomic, and imaging data establishes a precision medicine framework: autoantibodies function simultaneously as disease classifiers and therapeutic guides. This endophenotype-driven approach is already influencing trial design and patient stratification in systemic lupus erythematosus, systemic sclerosis, and inflammatory myopathies, and is reshaping both clinical practice and scientific taxonomy in CTDs. Recognizing autoantibodies as endophenotypic determinants aligns disease classification with pathogenic mechanism and supports the transition towards immunologically informed therapeutic strategies.

Antibodies doi: 10.3390/antib15010006

Authors: Giovanni Lasagni Laura Vetrugno Chiara Maria Maggiore Chiara Galassetti Giulia Di Colo Francesco Pavan Andrea Costantino Lorenzo Dagna

Background: Food allergy is a growing public health concern, and oral immunotherapy (OIT) has emerged as a promising approach to induce desensitization and potentially sustained unresponsiveness to allergenic foods. Changes in humoral immunity, particularly in allergen-specific immunoglobulin levels, play a central role in the immunological mechanisms underlying OIT. This review aims to summarize the current evidence on how OIT modulates allergen-specific immunoglobulin E (IgE), G (IgG) and A (IgA) responses in individuals with food allergy. Methods: We conducted a review of original research articles reporting longitudinal data on allergen-specific IgE, IgG, and/or IgA in patients undergoing OIT for common food allergens. Results: OIT was consistently associated with a transient increase in allergen-specific IgE levels during early phases, followed by a gradual decline. In contrast, Allergen-specific IgG4 levels showed a robust and sustained increase, correlating with desensitization and proposed to function as blocking antibodies. Several studies also reported an increase in allergen-specific IgA, particularly secretory IgA at mucosal sites, suggesting a potential role in enhancing mucosal tolerance and immune exclusion of allergens. Conclusions: Humoral immune responses during OIT are characterized by distinct and dynamic changes in immunoglobulin patterns. In particular, the rise in IgG4 and, in some cases, IgA suggests a role in promoting tolerance. Monitoring these biomarkers may offer insights into treatment efficacy and support individualized approaches to OIT.

Antibodies doi: 10.3390/antib15010005

Authors: Hannah Victoria Giles Bhuvan Kishore

Bispecific antibodies (BsAbs) have emerged as an important new class drugs for the treatment of multiple myeloma (MM) over the last few years. Currently, BsAbs are only licensed for use as monotherapy in patients with relapsed/refractory MM who have had at least three prior lines of treatment and are triple class-exposed (patients who have received an anti-CD38 monoclonal antibody, an immunodulatory drug, and a proteasome inhibitor). However, their use in earlier lines, including in the upfront setting, is being explored in multiple ongoing clinical trials with promising early results. The BsAbs have specific toxicities, including a high rate of low-grade cytokine release syndrome and, less commonly, immune effector cell-associated neurotoxicity syndrome. These immune-related toxicities occur almost exclusively during the initiation phase of the BsAbs. This has led to frequent hospitalization of patients for the duration of the initial step-up dosing phase. Strategies that could facilitate outpatient step-up dosing, such as tocilizumab prophylaxis, will become even more critical if BsAbs move into earlier lines of treatment and are used in larger numbers of patients. Optimizing infection prophylaxis is critical for ensuring the safe delivery of BsAbs as infection is the leading cause of non-relapse mortality in patients being treated with BsAbs. Multiple strategies to minimize the infection risk, including antimicrobial prophylaxis, immunoglobulin replacement, vaccination and reduced dosing frequency, have been evaluated. The clinical data on the efficacy of these supportive measures are described in this review article alongside the available strategies for mitigating and managing CRS and ICANS.

Antibodies doi: 10.3390/antib15010004

Authors: Kinsley Wang Kyle Taing Robert Hsu

Background/Objectives: Small-cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid proliferation, early metastasis, and near-universal relapse after initial therapy. While chemo-immunotherapy modestly improves first-line outcomes, survival after progression remains poor and highlights the urgent need for biomarker-directed strategies. Methods: A comprehensive literature search was conducted using major medical databases looking at key relevant studies on SCLC antibody studies. All authors reviewed the literature, assessed study quality, and interpreted the results from each study. Results: Recent advances in antibody–drug conjugates (ADCs) and T-cell engagers (TCEs) have transformed therapeutic development by targeting antigens selectively expressed on SCLC cells, enabling more precise and potentially durable tumor control. DLL3 has emerged as the most clinically relevant target to date, with the bispecific TCE tarlatamab demonstrating meaningful and durable response, manageable cytokine-release toxicity, and ultimately achieving accelerated FDA approval for previously treated extensive-stage SCLC. Concurrently, DLL3-directed ADCs have shown variable efficacy, underscoring the importance of payload selection, linker chemistry, and antigen density. Beyond DLL3, next-generation ADCs targeting TROP2, B7-H3, and SEZ6 have reported encouraging early-phase activity, including response rates exceeding those of existing second-line cytotoxic options, though myelosuppression, interstitial lung disease, and hepatic toxicity remain key considerations. Conclusions: Collectively, these emerging immunotherapies illustrate a shift toward antigen-specific targeting in a disease historically defined by limited therapeutic innovation. Continued optimization of antigen selection, payload and linker engineering, and biomarker-driven trial design will be critical for translating early promise into durable clinical benefit and reshaping the treatment landscape for SCLC.

Antibodies doi: 10.3390/antib15010003

Authors: Anna Balata Katarzyna Pogoda

Breast cancer remains the most common malignancy and one of the leading causes of cancer-related death among women worldwide. Advances in antibody-based therapies have improved outcomes across all biological subtypes: HER2-positive, triple-negative, and luminal breast cancer. Monoclonal antibodies such as trastuzumab and pertuzumab have established HER2-targeted therapy as a standard of care, while immune checkpoint inhibitors have introduced immunotherapy into the treatment of triple-negative breast cancer. The emergence of antibody–drug conjugates (ADCs), including trastuzumab deruxtecan, sacituzumab govitecan, and datopotamab deruxtecan, has further expanded the available therapeutic options. Bispecific antibodies represent a new generation of agents with the potential to overcome resistance and enhance immune activation. Despite impressive progress, important challenges remain, including resistance mechanisms and the management of treatment-related toxicities. This review summarizes the biological rationale, clinical evidence, resistance mechanisms, and safety profiles of therapies based on monoclonal antibodies, bispecific antibodies, and antibody–drug conjugates in breast cancer. The development of these treatment modalities fosters the implementation of personalized, immunologically informed treatment strategies that are redefining precision oncology in breast cancer.

Antibodies doi: 10.3390/antib15010002

Authors: Alexandra Rak Yana Zabrodskaya Pei-Fong Wong Irina Isakova-Sivak

Background/Objectives: Notwithstanding the declaration by the World Health Organization in May 2023 regarding the conclusion of the COVID-19 pandemic, new cases of this potentially lethal infection continue to be documented globally, exerting a sustained influence on the worldwide economy and social structures. Contemporary SARS-CoV-2 variants, while associated with a reduced propensity for severe acute pathology, retain the capacity to induce long-term post-COVID syndrome, including in ambulatory patient populations. This clinical phenomenon may be attributable to potential autoimmune reactions hypothetically triggered by antiviral antibodies, thereby underscoring the need for developing novel, universal vaccines against COVID-19. The nucleocapsid protein (N), being one of its most conserved and highly immunogenic components of SARS-CoV-2, presents a promising target for such investigative efforts. However, the protective role of anti-N antibodies, generated during natural infection or through immunization with N-based vaccines, alongside the potential adverse effects associated with their production, remains to be fully elucidated. In the present study, we aim to identify potential sites of homology in structures or sequences between the SARS-CoV-2 N protein and human antigens detected using hyperimmune sera against N protein obtained from mice, rabbits, and hamsters. Methods: We employed Western blot analysis of lysates from human cell lines (MCF7, HEK293T, THP-1, CaCo2, Hep2, T98G, A549) coupled with mass spectrometric identification to assess the cross-reactivity of polyclonal and monoclonal antibodies generated against recombinant SARS-CoV-2 N protein with human self-antigens. Results: We showed that anti-N antibodies developed in mice and rabbits exhibit pronounced immunoreactivity towards specific components of the human proteome. In contrast, anti-N immunoglobulins from hamsters showed no non-specific cross-reactivity with either hamster or human proteomic extracts because of the lack of autoreactivity or immunogenicity differences. Subsequent mass spectrometric analysis of the immunoreactive bands identified principal autoantigenic targets, which were predominantly heat shock proteins (including HSP90-beta, HSP70, mitochondrial HSP60, and HSPA8), histones (H2B, H3.1–3), and key metabolic enzymes (G6PD, GP3, PKM, members of the 1st family of aldo-keto reductases). Conclusions: The results obtained herein highlight the differences in the development of anti-N humoral responses in humans and in the Syrian hamster model. These data provide a foundational basis for formulating clinical recommendations to predict possible autoimmune consequences in COVID-19 convalescents and are of critical importance for the rational design of future N protein-based, cross-protective vaccine candidates against novel coronavirus infections.

Antibodies doi: 10.3390/antib15010001

Authors: Deepika Godugu Kranthi Gattu Parul Suri Abel B. Daartey Krishna Jadhav Satish Rojekar

Nanobodies (single-domain antibodies, VHHs) have emerged as versatile tools for evaluating and treating Alzheimer’s disease (AD). They offer distinct engineering benefits compared with traditional antibodies and small molecules, including small size, stability, and specificity. In AD, nanobodies have been shown in preclinical models to neutralize toxic amyloid-β oligomers, inhibit tau generation and aggregation, and modulate neuroinflammation, thereby demonstrating significant therapeutic potential. However, all nanobody applications in AD are discussed strictly as preclinical therapeutic potential rather than established clinical therapies, and direct clinical evidence in patients with AD is still lacking. Advanced engineering strategies, including intranasal and intrathecal routes, receptor-mediated transport, plasma protein binding with albumin, and focused ultrasound to facilitate brain penetration. Additionally, to improve nanobody delivery precision, half-life, and efficacy, strategies such as integrating nanobodies with nanoparticles, dendrimers, liposomes, and viral vectors are being employed. In fact, nanobodies are applied beyond monotherapy across multiple technological platforms to optimize brain delivery and target multiple targets. Nanobodies have been used on bispecific and trispecific antibody platforms, as well as in CRISPR/Cas9 editing and AI-driven technologies, to expand their applications. Recently, preclinical evidence has been mounting on the efficacy of nanobodies in clearing Aβ and tau, preserving synapses, and normalizing biomarkers. Comparison with FDA-approved anti-Aβ monoclonal antibodies (aducanumab, lecanemab, and donanemab) highlights opportunities and current translational gaps, including safety testing, half-life extension, and delivery optimization. This review critically delineates the current molecular mechanisms, emerging strategies, and delivery platforms, and emphasizes the potential of nanobodies as promising therapeutic and diagnostic molecules in AD therapeutics.

Antibodies doi: 10.3390/antib14040108

Authors: Jung Gu Lee Inseo Lee Joo-young Kim Suin Kim Woo-jin Jeong Ji-eun Kim

Background: The Fc region of immunoglobulin G (IgG) is a key target in therapeutic and analytical applications, such as antibody purification and site-specific bioconjugation. Although Protein A exhibits strong Fc-binding affinity, its large molecular weight and limited chemical flexibility pose challenges for use in compact or chemically defined systems. To address these limitations, we designed two α-helical peptides, SpA h1 and SpA h2, based on the Fc-binding helices of the Z34C domain from Staphylococcus aureus Protein A. Method: To enhance the structural stability and Fc-binding capability of these peptides, a lactam-based stapling strategy was employed by introducing lysine and glutamic acid residues at positions i and i + 4. Result: The resulting stapled peptides, (s)SpA h1 and (s)SpA h2, exhibited significantly improved α-helical content and IgG-binding performance, as demonstrated by circular dichroism (CD) spectroscopy and fluorescence-based IgG capture assays. Surface plasmon resonance (SPR) analysis confirmed specific, concentration-dependent interactions with the Fc region of human IgG, with (s)SpA h1 consistently showing the binding affinity and stability. Proteolytic resistance assays using α-chymotrypsin revealed that (s)SpA h1 maintained its structural integrity over time, exhibiting markedly enhanced resistance to enzymatic degradation compared to its linear counterpart. Furthermore, (s)SpA h1 exhibited strong Fc selectivity with minimal Fab affinity, confirming its suitability as a compact and Fc-specific binding ligand. Conclusions: These results confirm the successful design and development of structurally reinforced Fc-binding peptides that overcome the inherent limitations of short linear sequences through both high-affinity sequence optimization and lactam-based stapling. Among them, (s)SpA h1 demonstrates the most promising characteristics as a compact yet stable Fc-binding ligand, suitable for applications such as antibody purification and site-specific bioconjugation.

Antibodies doi: 10.3390/antib14040107

Authors: Barbara Colitti Consiglia Longobardi Gabriela Flores-Ramirez Chiara Nogarol Ludovit Skultety Gianmarco Ferrara

Background/Objectives: The detection of anti-Coxiella antibodies using serological methods is essential for identifying exposed ruminants and preventing this important zoonotic disease in livestock. In recent years, numerous attempts have been made to increase diagnostic performance as well as simplify the production of serological assays. Commercially available tests often use whole-cell antigens, which can decrease specificity and require high-level biosafety facilities for manufacturing. The aim of this work was to produce three Coxiella burnetii (C. burnetii) antigens in recombinant form and assess them for the detection of anti-Coxiella antibodies in ruminants. Methods: Three recombinant C. burnetii antigens (Com-1, MceB, AdaA) were selected among immunodominant antigens and produced in a heterologous system (Escherichia coli). Following purification, the proteins were utilized to coat ELISA plates and evaluated for seroreactivity against sera from both negative and positive cattle. Results: Com-1 demonstrated the greatest agreement with the commercial test, albeit moderate. MceB exhibited nonspecific reactivity against a large number of sera, while the AdaA showed reactivity against only a few positive sera. Conclusions: Our findings are consistent with previous research, indicating that utilizing a single antigen to identify exposed animals is unfeasible with current knowledge, most likely due to the complex immunological response following C. burnetii infection in cattle. Consequently, it is critical to continue testing and identifying immunoreactive antigens in order to further investigate them and, potentially, select the most appropriate.

Antibodies doi: 10.3390/antib14040106

Authors: Chia-Hung Chen Yu-Chi Chiu Kai-Yao Huang Hsiao-Hsuan Huang Ta-Wei Kuo Yu-Chi Liu Hui-Ju Kao Chen-Lin Yu Shun-Long Weng Kuang-Wen Liao

Background: Short peptide epitopes are valuable for mechanistic studies, yet their intrinsic low immunogenicity and lack of commercial antibodies hinder rapid antibody generation. Methods: We developed a reproducible, sequence-level workflow combining cross-species/structural triage, independent MHC-I/II prioritization, and conservative heteroclitic-style substitutions to enhance predicted MHC affinity while preserving native epitope features. Using visfatin as a model, two optimized fragments were conjugated to KLH and tested in mice for antibody titers, isotype profiles, and binding kinetics. Results: Mutant peptides improved MHC-binding prediction, elicited stronger antibody titers, and promoted isotype maturation (increased IgG1). Importantly, antibodies maintained measurable binding to wild-type sequences, indicating preserved cross-recognition. Similar effects were reproduced with additional antigens. Conclusions: This proof-of-concept study, based on small exploratory mouse cohorts (n = 3 per group), demonstrates that strategic, minimal sequence edits can significantly enhance peptide immunogenicity while preserving native epitope recognition. This streamlined workflow provides a low-barrier route to generate epitope-directed antibodies when commercial reagents are unavailable.

Antibodies doi: 10.3390/antib14040105

Authors: Yuxin Qian Weiwei Ma Xiao-Ning Xu

Chimeric antigen receptor (CAR)-based immunotherapy has emerged as a transformative strategy in anticancer treatment, driven by advances in CAR construct design, manufacturing platforms, and expansion to diverse immune cell types. The landmark success of CD19-targeted CAR-T cell therapy in B cell malignancies has paved the way for broader clinical applications. As of 2025, the U.S. FDA has approved multiple autologous CAR-T products, underscoring their therapeutic promise. However, challenges persist, including cytokine release syndrome (CRS), neurotoxicity, product inconsistency, and the high cost and complexity of cell manufacturing. Variations in cell source, gene delivery methods, expansion protocols, and CAR design significantly influence the safety, efficacy, and scalability of these therapies. In this review, we comprehensively examine the current advances in manufacturing protocols for CAR-modified T cells, natural killer (NK) cells, and unconventional T cell subsets, including γδ T, invariant natural killer T (iNKT), and mucosal-associated invariant T (MAIT) cells. We also highlight emerging innovations such as in vivo CAR-T generation and off-the-shelf allogeneic approaches. By integrating updated strategies with a critical evaluation of current limitations, this review aims to support the development of standardized, robust, and accessible CAR-based immunotherapies.

Antibodies doi: 10.3390/antib14040104

Authors: Giuseppe Lauletta Cataldo Patruno Claudio Brescia Andrea Cosenza Carolina D’Elia Valentina Ventura Emanuela Martina Maddalena Napolitano

Background: Head and neck dermatitis (HND) represents a challenging phenotype of atopic dermatitis (AD), often showing suboptimal response or paradoxical worsening during biologic therapy. Objective: To review the efficacy and safety of current systemic treatments for HND, with a focus on dupilumab, tralokinumab, lebrikizumab, and janus kinase (JAK) inhibitors. Methods: We conducted a narrative review of randomized controlled trials, post hoc analyses, and real-world studies assessing clinical outcomes in patients with moderate-to-severe AD involving the head and neck. Outcomes included Eczema Area and Severity Index (EASI) H&N subscore, erythema grade, patient-reported measures, and adverse events. Results: Dupilumab shows substantial efficacy for HND in both clinical trials and real-life studies; however, responses are often less pronounced than in other anatomical regions, and facial redness (FR) has emerged as a notable adverse event in up to 9% of patients. Tralokinumab and lebrikizumab demonstrate significant improvements in HND involvement, with low incidence of paradoxical reactions. JAK inhibitors, particularly upadacitinib, provide rapid and marked improvement in refractory cases and in patients developing FR during biologic therapy. Conclusions: Systemic therapy for HND should be individualized, balancing efficacy and tolerability. JAK inhibitors represent a valuable alternative in biologic-refractory phenotypes or in patients experiencing dupilumab-associated FR.

Antibodies doi: 10.3390/antib14040103

Authors: Thais Stelzer Toledo Pauline Martins Cunha Josué da Costa Lima-Junior Monique Paiva De Campos Alinne R. S. Renzetti Fabiano Borges Figueiredo Fernanda Nazaré Morgado Renato Porrozzi Fatima da Conceição-Silva Marta de Almeida Santiago Paula Mello De Luca

Backgroud/Objectives: Canine Visceral Leishmaniasis (CVL), caused by Leishmania infantum, is a significant public health concern due to dogs serving as reservoirs for human infection. An accurate and rapid diagnostic method to distinguish symptomatic and asymptomatic CVL from healthy and vaccinated animals is essential for controlling canine and human disease. Developing innovative antibody detection techniques and exploring new antigens are essential for enhancing CVL testing efficiency. Our study focuses on a multiplex flow cytometry technique to detect Leishmania-specific antibodies in canine serum. This involved conjugating small peptides with carrier proteins or peptide tags, sequences designed to facilitate bead coupling. Methods: A peptide from the L. infantum A2 protein was coupled to beads in three forms: unconjugated, conjugated with BSA, and conjugated with a C-terminal β-alanine–lysine (x4)–cysteine TAG. This TAG was previously designed to enhance peptide solubility, improve binding efficiency, and provide functional groups for covalent attachment to the beads, ensuring stable immobilization in the multiplex assay. Results: Our results suggest that the multiplex approach shows promise as a rapid serological test for CVL, particularly with TAG-conjugated peptides, which optimize bead coupling. However, peptide/BSA conjugation revealed anti-BSA antibodies in samples from healthy and CVL dogs. Conclusions: In conclusion, our findings highlight the potential of multiplex methodologies to enhance CVL diagnostics and caution against using BSA as a bead coupling agent in serological tests for canine samples due to its impact on test specificity and sensitivity.

Antibodies doi: 10.3390/antib14040102

Authors: Eun Seo Choi Sophia Sahota Emily Burnham Yunfeng Ding Eric V. Shusta

Background: Antibodies that cross the blood–brain barrier (BBB) by targeting receptor-mediated transport (RMT) systems can allow efficient drug delivery to the central nervous system (CNS). In order to improve brain uptake of antibodies, their binding properties have been engineered, but it is not always clear what antibody properties dictate BBB transport efficiency. In this study, we therefore developed and employed an in vitro phenotypic screen and a quantitative transcytosis assay in an attempt to identify improved variants of a previously identified BBB transcytosing antibody known as 46.1. Methods: First, a random mutagenic 46.1 antibody phage display library was screened for improved transcytosis through a human induced pluripotent stem cell (iPSC)-derived BBB model. These screens yielded antibody variants that enriched over multiple screening rounds; however, when produced as soluble antibodies, the variants did not display improved in vitro transcytosis over the wild-type (WT) 46.1 antibody. As a second strategy, we performed a targeted histidine point mutation of a solvent-exposed residue in each complementarity-determining region (CDR) and evaluated the in vitro transcytosis capacity of the variants. Results and Conclusions: In this way, we identified a 46.1 variant, R162H, with modestly improved in vitro transcytosis properties. These results show that the iPSC-derived BBB screening insights and evaluation strategies presented here could facilitate the engineering and optimization of lead antibodies for CNS delivery.

Antibodies doi: 10.3390/antib14040101

Authors: Garry M. Walsh

Background: Asthma exhibits marked heterogeneity both clinically and at the molecular phenotypic level, requiring specifically targeted treatments to block the key pathways of the disease. Monoclonal-antibody-based biologics targeted at critical inflammatory pathways of T2 inflammation such as IL-5, IL-5R, IL-4, and IL-13 are increasingly regarded as effective treatments for severe refractory eosinophilic asthma. Methods: This review provides an update on the potential of straightforward and reproducible biomarkers to aid in the selection of the biologic-based therapy most likely to be effective in patients with severe or refractory eosinophilic asthma based on English-language original articles in PubMed or MedLine. Results: Monoclonal-antibody-based biologic therapies have revolutionised severe asthma management, enabling reductions in symptoms that include exacerbations, discontinuation of oral corticosteroids, improved lung function, and enhanced quality of life. Significant clinical effects with anti-IL-5 or -IL-4/13 monoclonal antibodies are more likely to be seen when simple predictive biomarkers such as serum periostin, fractional exhaled nitric oxide (FENO), or blood eosinophil counts are used to aid in the identification of those patients with severe refractory eosinophilic asthma who are most likely to benefit from biologic therapies. Conclusions: Biologic-based therapy aimed at T2 inflammation benefits patients with severe eosinophilic asthma, particularly when guided by biomarkers that do not require direct sampling of the airways to target therapy, who are most likely to benefit from these treatments, with good safety profiles for these therapies.

Antibodies doi: 10.3390/antib14040100

Authors: Marta Baselga Javier Sánchez-Prieto Víctor Manuel Medina Pérez Alberto J. Schuhmacher

Background/Objectives: Single-domain antibodies (sdAbs) are derived from camelid heavy-chain antibodies (HCAb). Their small size, high stability, and ease of production, among other properties, makes them highly valuable in biomedical research and therapeutic development. Several sdAb-based molecules are currently progressing through clinical trials, highlighting their translational relevance. As sdAbs originate from HCAb of Camelidae family, they can originate from multiple species including Vicugna pacos, Lama glama, Camelus dromedarius and Camelus bactrianus. Although several reports and databases analyze the structure of sdAbs, comprehensive evaluations on species-dependent structural differences remain scarce. Methods: We assembled MO-IISA, an open-access curated database of sdAbs with known antigen targets by integrating six public resources (iCAN, INDI, SAbDab-nano, sdAb-DB, PLabDab-nano, NbThermo) under harmonized eligibility criteria. Results: The final dataset comprises 2053 sdAbs derived from llamas (Lama glama, n = 1316); alpacas (Vicugna pacos, n = 325), dromedary camels (Camelus dromedarius, n = 377) and Bactrian camels (Camelus bactrianus, n = 35). We quantified region lengths, amino acid frequency, and conservation/entropy across frameworks (FR1–FR4). The average length of all sdAbs was about 124 ± 8 amino acids, with minor interspecies differences. We observed a consistent enrichment of lysines in FR3 (and secondarily FR2) and cysteines primarily in FR1 and FR3, with non-canonical cysteines more frequent in Bactrian and dromedary sdAbs CDRs. CDR2 and, particularly CDR3, contributed most to inter- and intra-species variability, whereas FRs were highly conserved. Conclusions: Species-neutral framework constraints and species-tuned loop adaptations have practical implications for sdAb engineering, species selection, and conjugation strategies. These features are captured in MO-IISA, an open-access database of known-target sdAbs from different species.

Antibodies doi: 10.3390/antib14040099

Authors: Million A. Tegenge

Background: Physiologically based pharmacokinetic (PBPK) modeling is applied to address clinical pharmacology issues including dose selection and exposure assessments for special populations (e.g., pediatrics, and renally or hepatically impaired patients). The objective of this study was to evaluate the predictive performance of a PBPK model for dosing assessment of intravenous immunoglobulin (IVIG) and anti-D immunoglobulin (anti-D Ig) products in pregnant women. Methods: A minimal PBPK (mPBPK) model that incorporates pregnancy-specific physiological parameters and allometric scaling approaches was developed and evaluated for predicting the exposure of IVIG and anti-D Ig in pregnant women. The concentration versus time data were obtained from the published literature. Results: The IVIG (n = 22) and anti-D Ig (n = 29) concentrations were predicted using the mPBPK model with an average fold error of 1.17 and 1.22, respectively. A total of 100% and 95% of IVIG concentrations were predicted within the 0.5–2-fold and 0.5–1.5-fold prediction error ranges, respectively. For anti-D Ig, predictions fell within the 0.5–2-fold and 0.5–1.5-fold ranges for 93% and 76% concentrations, respectively. A mPBPK model-based simulation following administration of 0.5 g/kg IVIG in 100 virtual nonpregnant and pregnant subjects revealed that the maximum plasma concentration (Cmax) was 15% lower and trough concentration (Ctrough) was 8% lower during the third trimester of pregnancy compared to nonpregnant subjects. In contrast, with flat dosing, Cmax and Ctrough were 32% and 26% lower in pregnant subjects, respectively. Overall, the model demonstrated reasonable predictive performance, and bodyweight-based dosing regimen is an acceptable approach that results in minimal change in exposure of IVIG in pregnant women.

Antibodies doi: 10.3390/antib14040098

Authors: Paolo Giannoni Gabriella Pietra Orlando Izzo Giuseppina Fugazza Roberto Benelli Alessandro Poggi Mauro Krampera Chiara Utzeri Monica Marchese Marco Musso Paola Visconti Daniela de Totero

Background/Objectives: Three-dimensional (3D) in vitro cell culture models have recently stimulated great interest since they may have more pre-clinical value than conventional in vitro 2D models. In fact, 3D culture models may mimic the in vivo biophysical 3D structure of tumors and cell-to-cell interaction, thereby representing a more useful approach to testing drug responses. In this study we have developed a 3D culture model of an EBV+/CD30+cell line, D430B, previously characterized as an Anaplastic Large Cell Lymphoma of B phenotype (B-ALCL), to determine the cytotoxic activity of the antibody–drug conjugate Brentuximab Vedotin. Methods: By using of ultra-low attachment plates, we developed D430B spheroids that appeared particularly homogenous in terms of growth and size. Results: Brentuximab Vedotin treatment (1 to 20 μg/mL) turned out to be significantly cytotoxic to these cells, while the addition of the anti-CD20 chimeric antibody Rituximab (10 μg/mL) appeared almost ineffective, even though these cells express CD20. Moreover, when we co-cultured D430B cells with stromal cells (HS5), to re-create a microenvironment representative of neoplastic cell/mesenchymal cell interactions within the lymph node, we observed a significant, although faint, protective effect. Conclusions: This simple and reproducible method of generating D430B-ALCL spheroids to evaluate their response to Brentuximab Vedotin treatment, as here described, may provide a valuable preliminary tool for the future pre-clinical screening of patients’ primary lymphoma cells or the development of novel therapies for this type of pathology and related diseases.

Antibodies doi: 10.3390/antib14040097

Authors: Saravana P. Selvanathan Olivia O. Lansinger David V. Allegakoen Emma J. W. McGuire Ashley R. Gaffey Jeff R. Petro Purushottam B. Tiwari Quinn Tufiño Aykut Üren Jeffrey A. Toretsky

Background: Ewing sarcoma (ES) is a rare tumor that affects children, adolescents, and young adults. ES is associated with high morbidity in all patients and high mortality for those who present with metastatic disease. A chromosomal translocation, either t(11;22)(q24;p12) or t(21;22)(q22;q12) leads to the fusion oncoproteins EWS::FLI1 or EWS::ERG in 95% of ES patients. We recognized a critical need for a stably sourced high-affinity antibody that recognizes EWS::FLI1 with maximal specificity. Understanding EWS::FLI1 protein complexes is a pivotal gap in ES knowledge that necessitates the development of antibodies capable of identifying native proteins in solution. Further, variable epitope sequencing of a monoclonal antibody enables the construction of degraders and nanobody identifiers. Methods: Monoclonal antibodies were produced following informed peptide synthesis, injection, and hybridoma creation. Hybridoma antibodies were validated for specificity and function. Results: Our results indicate that the FLI1 1.2 monoclonal antibody, which recognizes the EWS::FLI1 fusion oncoprotein, can be reliably applied to multiple molecular biology applications like immunoblot, immunoprecipitation, immunofluorescence, and immunohistochemistry. This FLI1 1.2 monoclonal antibody has a high affinity of 0.3 nM KD to EWS::FLI1. In terms of specificity, this antibody is highly specific to EWS::FLI1 and some cross reactivity with ERG. Conclusions: This reagent will provide the research community with valuable tools for further biochemical and genomic interrogation of the oncogenic activity of EWS::FLI1 in ES.

Antibodies doi: 10.3390/antib14040096

Authors: Christina Claus Claudia Ferrara-Koller Johannes Sam Sabine Lang Rosmarie Albrecht Regula B. Buser Esther Bommer Grégory La Sala Valeria G. Nicolini Sara Colombetti Marina Bacac Pablo Umaña Christian Klein

Background/Objectives: T cell bispecific antibodies (TCBs) result in the activation of T cell receptor signaling upon binding to tumor antigens providing signal 1 to T cells. To enhance and sustain their activity, a co-stimulatory signal 2 is required. Here CEACAM5-targeted 4-1BBL antibody fusion proteins for combination with CEA-TCB (cibisatamab, RG7802) are described in an investigation of the relationship between the CEACAM5 epitope and T cell activity. Methods: CEACAM5-targeted bispecific 4-1BBL antibody fusion proteins (CEA-4-1BBLs) were generated based on different CEACAM5 antibodies and characterized in vitro in Jurkat-4-1BB reporter and PBMC cell assays. The impact of shed CEA on in vitro activity and cynomolgus cross-reactivity was studied. In vivo efficacy was assessed in human stem cell humanized NSG mice xenograft models bearing MKN-45 and HPAFII tumors. Results: MFE23-4-1BBL and Sm9b-4-1BBL showed superior functional activity in Jurkat-4-1BB reporter and primary T cell assays when combined with the CD3 antibody V9, whereas T84.66-LCHA-4-1BBL and A5B7-4-1BBL performed better when combined with CEA-TCB. In humanized NSG mice MKN-45 and HPAFII xenograft models, T84.66-LCHA-4-1BBL mediated the best anti-tumor efficacy. Conclusions: For the assessment of the combination of CEA-TCB with CEA-4-1BBL, co-stimulatory antibody fusion protein in vitro assays are not sufficient to fully capture the complex relationships affecting efficacy. Thus, screening with different cell assays and in vivo efficacy studies in combination with CEA-TCB are essential to select the best candidate. Based on the totality of data on the T84.66-LCHA-4-1BBL antibody fusion protein comprising the CEACAM5 antibody, T84.66-LCHA was selected as the optimal combination partner for CEA-TCB.

Antibodies doi: 10.3390/antib14040095

Authors: Marina Fernández-González Santiago Llorente José Antonio Galián Carmen Botella Rosana González-López María José Alegría Alicia Hita María Rosa Moya-Quiles Helios Martinez-Banaclocha Manuel Muro-Pérez Javier Muro Alfredo Minguela Isabel Legaz Manuel Muro

B cells have attracted increasing interest in the field of organ transplantation due to their newly discovered immunoregulatory properties in alloimmune responses. Traditionally, B cells have been primarily associated with adaptive immunity to foreign substances and alloreactive immune response to allografts, differentiating into antibody-producing plasma cells or memory cells upon antigen recognition and T cell collaboration. However, the existence of B cells with regulatory functions (Bregs) in humans has been widely confirmed, highlighting the presence of this subset, which has immunosuppressive properties and which might contribute to allograft tolerance, within the B cell compartment in humans and mice. In this mini review, we summarize all the information available in the published reports about the role of regulatory B cells in solid organ transplantation.

Antibodies doi: 10.3390/antib14040094

Authors: Alexandra Anatolieva Atanasova Ginka Ilieva Cholakova Alexandra Panagiotis Kapogianni Vancho Donev Delina Ivanova Anna Dimitrova Yordanova Vanya Petkova Bogoeva Ivanka Georgieva Tsacheva

Background and Aims: C1q is an autoantigen in different autoimmune disorders, Systemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN) among them. The two functional domains of C1q, the collagen-like region (CLR) and the globular head region (gC1q), are frequently recognized by autoantibodies in SLE and LN when C1q is immobilized. We studied whether autoantibodies to C1q in SLE and LN patients recognized C1q as a soluble autoantigen and whether the act of immobilization was a prerequisite for the recognition of C1q autoepitopes localized on gC1q domains. Methods: The interaction of soluble C1q and its globular fragments ghA, ghB, and ghC with immobilized IgG autoantibodies (and vice versa) from sera of 48 patients with SLE and LN was studied with ELISA. Data were compared using Spearman correlation coefficient. Fluorescence spectroscopy was used to study the interaction between C1q and LN IgG autoantibodies both presented in solution. Results: We found that anti-C1q autoantibodies from SLE and LN patients specifically bound C1q and gC1q fragments, ghA, ghB, and ghC, both as immobilized and soluble antigens. Correlation analysis indicated a negative correlation between the levels of autoantibodies against immobilized and soluble C1q and immobilized and soluble gC1q fragments which indicates different epitopes when these proteins were recognized as autoantigens in soluble and immobilized conformations. Conclusions: Serum C1q in patients with SLE is a target molecule for binding from anti-C1q autoantibodies. The gC1q region undergoes a conformational change in an immobilized and a soluble form, thus exposing different epitope-binding sites.

Antibodies doi: 10.3390/antib14040093

Authors: Jessica Castañeda-Casimiro Luis Vallejo-Castillo Eliud S. Peregrino Alejandro Hernández-Solis Luis Vázquez-Flores Rommel Chacón-Salinas Isabel Wong-Baeza Jeanet Serafín-López

Antibodies are produced by cells of the adaptive immune response and recognize epitopes of microbial structures with high affinity and specificity. Antibodies are recognized by Fc fragment receptors (FcRs) found on the surface of phagocytic cells (neutrophils, monocytes, macrophages) and NK cells, among others. Hence, antibodies link the adaptive immune response with the innate immune response. The functions of antibodies are related to the N-glycosylation profile of these proteins. In this review, we describe how N-glycosylation of the Fc fragment of the different antibody classes is carried out, and which oligosaccharides are most commonly found in these antibodies. Subsequently, we summarize the biological effects of N-glycosylation of antibodies: on the binding of antibodies to FcRs (which affects various functions, such as antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and the production of pro- or anti-inflammatory chemokines and cytokines), on the ability of antibodies to activate complement and on the ability of some antibodies to directly neutralize the adhesion of bacteria and viruses to host cells (independently of Fab recognition). We describe how the N-glycosylation profile of antibodies is modified during certain infections (such as tuberculosis, COVID-19, influenza and dengue) and in response to vaccination, and the potential use of this profile to identify the stage and severity of an infection. Finally, we review the importance of N-glycosylation for the pharmacokinetic, pharmacodynamic and safety profiles of therapeutic monoclonal antibodies.

Antibodies doi: 10.3390/antib14040092

Authors: Imran Gani Usman Baig Ahmad Mirza Shais Jallu Abrar Ahad Chawdhary

Rabbit antithymocyte globulin is one of the most commonly used agents for induction immunosuppression in renal transplantation. It has contributed significantly to improved allograft survival and has a favorable safety profile. Despite its advantages, rabbit antithymocyte globulin carries a rare but potentially life-threatening risk of anaphylaxis, which can lead to severe morbidity and mortality. Anaphylaxis is an acute and dramatic complication that requires prompt recognition and immediate management. In this review, we discuss the pathophysiology, clinical features, and management of rabbit antithymocyte globulin-associated anaphylaxis. We have also included practical insights from our clinical experience to guide early recognition and management, aiming to help clinicians safely manage this critical adverse event.

Antibodies doi: 10.3390/antib14040091

Authors: Avery Koi Trine Engebretsen Alfred S. Lea Daniel Arango Heather L. Stevenson Michael L. Kueht

Introduction: Simultaneous liver–kidney (SLK) transplant recipients are considered at lower immunologic risk than kidney-alone recipients, so steroid-only induction is often used. However, some centers continue to include basiliximab induction in their protocols. This study compared graft and infectious outcomes in SLK recipients receiving basiliximab (Bas) induction versus those without basiliximab (No Bas). Methods: Using TriNetX, we conducted a retrospective, propensity-score-matched study of SLK recipients comparing 3-, 6-, and 12-month graft and infectious outcomes. Patients receiving alemtuzumab or anti-thymocyte globulin were excluded; steroid induction was permitted but not required in either cohort. Maintenance immunosuppression included tacrolimus, mycophenolate, and prednisone. Cohorts were matched on 71 variables, including demographics, disease etiology, severity markers, and cPRA. Results: After matching, 292 patients were included per cohort (mean age 56.9 ± 10.1 years; 61% male). Kidney and liver rejection rates were similar. The No Bas cohort had more liver biopsies (25.5% vs. 18.2% at 1 year, p = 0.04). Kidney biopsy, graft failure, re-transplantation, delayed graft function, and mortality were comparable. Liver primary non-function was more frequent in Bas (2.8% vs. 0.4%, p = 0.04). The No Bas cohort had higher CMV at 3 months (13.4% vs. 6.7%, p = 0.008) and higher EBV at all time points (4.0% vs. 0.4% at 1 year, p = 0.004). Conclusions: SLK recipients without basiliximab induction had comparable rejection outcomes but more viral infections, potentially from greater steroid exposure, and more liver biopsies, which may reflect higher clinical suspicion for rejection or incomplete capture of rejection events in EMR data.

Antibodies doi: 10.3390/antib14040090

Authors: Adi Aharon Rachel Birnboim-Perach Omer Grotto Adi Amir Daniel Diadko Nitzan Beltran Limor Nahary Itai Benhar

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint inflammation that leads to tissue damage and disability. RA affects approximately 0.5–1% of the global population and is driven by a complex interplay of genetic susceptibility, environmental factors, and immune dysregulation. While biologic and targeted synthetic DMARDs improved RA treatment, they have limitations in efficacy, safety, and accessibility. B-cell-targeting therapies, such as anti-CD20, have shown effectiveness, but only with broad immunosuppression, which can increase infection risk and compromise humoral immunity. Therefore, there is an unmet need for more selective therapeutic strategies that modulate pathogenic immune pathways while preserving protective immune functions. It has been suggested that targeting the BAFF pathway may offer a more favorable therapeutic approach compared to targeting CD20. Objectives: In this study, we evaluated the therapeutic potential of V3-46s mIgG2a, an anti-BAFF-R (BR3) antibody in a mouse RA model, hypothesizing that it would offer a more selective and effective strategy. Methods: We expressed and purified four antibody variants and assessed their binding and neutralizing activity in vitro. V3-46s mIgG2a was selected for in vivo evaluation in a collagen-induced arthritis (CIA) model. Results: Treatment with this antibody delayed disease onset and reduced arthritis severity, spleen index, and B-cell populations. Conclusions: These findings highlight the potential of BAFF-R-targeting antibodies as a therapeutic approach for RA treatment. This preclinical work lays the groundwork for future development of BAFF-R blockade as a complementary or alternative strategy to current biologic treatments.

Antibodies doi: 10.3390/antib14040089

Authors: Ming-Ching Hsieh Kristiina Dorofejeva Jingming Zhang Diane L. Vy Jun Qian Alice M. Matathia Timothy Blanc Chao Richard Li Babita S. Parekh

Background: Antibody-dependent cellular cytotoxicity (ADCC) is an immune response where antibodies bind to target cells and activate effector cells through Fcγ receptors, ultimately leading to the destruction of the target cells. Methods: This study examined the ADCC activities of charge variants of a therapeutic IgG1, MAB1, using an internally developed reporter gene assay. In this assay, the proprietary target was expressed on DiFi cells, while FcγRIIIa was expressed on Jurkat effector cells. Results: The results revealed that different charge variants had varying levels of ADCC activity, with variants containing C-terminal lysine residues showing enhanced activity. The charge variants arose from modifications such as the presence of sialic acid at the glycan moiety, deamidation, and C-terminal lysine truncation, including K2 (two C-terminal lysine residues), K1 (one C-terminal lysine residue), and K0 (no C-terminal lysine residues) variants. Notably, the K1 and K2 variants demonstrated higher ADCC activity compared to the K0 and acidic variants. However, the observed increase was attributed not to the lysine residue itself, but rather to the MAN5 glycan associated with the lysine-containing variants. Conclusion: These findings challenge previous assumptions about the role of C-terminal lysine in ADCC, suggesting a shift in understanding the functional significance of charge variants and emphasizing the critical influence of glycan composition in therapeutic antibody efficacy.

Antibodies doi: 10.3390/antib14040088

Authors: Wenli Sun Keke Huang Yaping Cheng Ailing Huang Yu Kong Jun Lu Tianlei Ying Yanling Wu

Background: Antibodies have revolutionized therapeutics and diagnostics, but their applications are largely restricted to extracellular targets due to challenges in intracellular delivery and stability. Nanobodies, with their small size and lack of disulfide bonds, hold great promise for intracellular use but face challenges such as aggregation and rapid degradation in the cytosol. Methods: To overcome this, we engineered nanobodies by fusing them with subcellular localization motifs to redirect their localization within cells, including the mitochondrial surface, endoplasmic reticulum surface, endomembrane system, and cytoskeleton. Results: Our results demonstrate that nanobodies located in the cytoskeleton or endomembrane exhibit significantly reduced degradation rates and enhanced stability, while maintaining their target-binding capacity. Mechanistically, these modifications lowered ubiquitination levels and prolonged functional activity. Conclusions: This work provides a novel strategy to enhance the intracellular stability and efficacy of nanobodies, expanding their potential applications in functional proteomics, disease research, and therapeutic development.

Antibodies doi: 10.3390/antib14040087

Authors: Lauren Gebhardt Molica Abel Jing Zhou Audrey M. Vogt Bo Hee Shin Sarah L. Herrick Wagman Ana Santos Jerome Puginier Florian M. Wurm Maria J. Wurm Guoying Grace Yan Adedolapo Adeniyi Sean K. H. Lim Will Somers Laura Lin Aaron M. D’Antona Xiaotian Zhong

Background: Recent advances in antibody discovery technologies, especially progress in de novo synthesis through machine learning, have imposed a significant production challenge for the generation of a large diversity of antibodies against nearly any target of interest. There is a demand for the rapid production of dozens of purified antibodies in 10-milligram quantities sufficient for functional screening and molecular assessment studies. Objectives: To meet this requirement, a semi-automated production methodology and workflow was developed to bridge the miniaturized high-throughput screenings (HTSs) and the conventional custom-scale workflow by taking advantage of four new technology applications. Methods: First, it exploited a novel, simple, high-titer transient expression system, “CHO4Tx®”, which could achieve high yields in the range of 200 mg/L and above, across a variety of antibody constructs, including challenging targets. The consistently high yields from this transient CHO platform enabled the delivery of ~20 mg of crude material per 100 mL scale flask production with a throughput capacity of nineteen constructs in a single run. Secondly, we established a magnetic ProA bead in-culture antibody-capturing process, which significantly shortened the production timeline by eliminating the steps of cell centrifugation, filtration, and medium column loading. Third, we utilized the GenScript AmMag™ SA Plus semi-automation, which could handle magnetic ProA bead elution for 12 constructs within less than 1 h. Lastly, we transformed the AKTA PureTM system into an automated buffer exchange purification system with a capacity of processing 19 samples in a single run. Results and Conclusions: This new production platform was proven to be robust and could be applied for the routine production of antibodies of sufficient quality and quantity in support of cell-based assays and biophysical characterization.

Antibodies doi: 10.3390/antib14040086

Authors: Federico Francisco Marsili Fernanda Bittencourt de Aquino Hiam Rodrigo da Silva Arruda Mayra Amorim Marques Katia Maria dos Santos Cabral Marcius da Silva Almeida Guilherme Augusto Piedade de Oliveira Andrea Queiroz Maranhão Renato Sampaio Carvalho Leda dos Reis Castilho

Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light and heavy chain coding genes, employing a wild-type Encephalomyocarditis virus (EMCV) IRES functional element to drive expression of the second gene. Methods: Using two neutralizing anti-SARS-CoV-2 IgG1 antibodies as model molecules, we conducted transient transfections in the commercially available ExpiCHOTM platform. Following protein A affinity purification and quantification, vectors positioning the light chain as the first cistron consistently yielded higher expression levels than those with the heavy chain upstream. To confirm the quality attributes of the mAbs, we applied a comprehensive analytical workflow, including SDS-PAGE and Western blot for molecular mass and purity, circular dichroism for secondary structure, intrinsic tryptophan fluorescence for tertiary structure, and SEC-HPLC for quaternary structure and aggregate detection. Additionally, we assessed binding affinity to the target using spot blot and surface plasmon resonance, analyzed N-glycosylation profiles by HILIC-HPLC and mass spectrometry, and examined molecular structure by transmission electron microscopy. Results and Conclusions: Together, these results provide insight into the impact of gene positioning within bicistronic vectors on mAb expression efficiency and quality, supporting optimization strategies for scalable recombinant antibody production.

Antibodies doi: 10.3390/antib14040085

Authors: Christopher R. Thornton Genna E. Davies

Background: The frequency of necrotising cutaneous and soft tissue infections caused by the Mucorales fungi Apophysomyces and Sakasenaea is increasing. The absence of sophisticated diagnostic technologies in low- and middle-income countries (LMICs) means that detection of cutaneous mucormycosis continues to rely on culture of the infecting pathogens from biopsy and their differentiation based on morphological characteristics. However, Apophysomyces and Sakasenaea are notorious for their failure to sporulate on standard mycological media used for the identification of human pathogenic fungi. Differentiation of these pathogens and their discrimination from Aspergillus fumigatus, the most common mould pathogen of humans, is essential due to their differing sensitivities to the antifungal drugs used to treat mucormycosis. Methods: A murine IgG1 monoclonal antibody, JD4, has been developed that is specific to Apophysomyces species. In Western blotting and enzyme-linked immunosorbent assay (ELISA), mAb JD4 is shown to bind to an extracellular 15 kDa protein, readily detectable in crude antigen extracts from non-sporulating cultures of Apophysomyces. Results: When combined with a Mucorales-specific lateral-flow immunoassay (LFIA), mAb JD4 allows the differentiation of Apophysomyces from Saksenaea species and discrimination from Aspergillus fumigatus. Monoclonal antibody JD4 enables the detection and differentiation of Apophysomyces species from other fungal pathogens that cause rapidly progressive cutaneous and soft tissue mycoses in humans. When this is combined with a rapid LFIA, improvements are offered in the sensitivity and specificity of Mucorales detection based on mycological culture, which remains a gold-standard procedure for mucormycosis detection in LMICs lacking access to more sophisticated diagnostic procedures.

Antibodies doi: 10.3390/antib14040084

Authors: Alessia Muzi Roberto Arriga Giovanni Bulfaro Francesca Fata Antonella Costanzo Valerio Chiarini Manuela Cappelletti Fabiana Fosca Ferrara Federica Bucci Linda Celeste Montemiglio Carmelinda Savino Emanuele Marra Gennaro Ciliberto Luigi Aurisicchio Beatrice Vallone Giuseppe Roscilli

Background/Objectives: The ErbB protein family plays a critical role in the progression of various solid tumors, and HER3 has been implicated in resistance mechanisms to multiple cancer therapies due to its ability to form heterodimers with other ErbB receptors, thereby activating pathways that promote tumor growth and survival. This study aimed to generate and characterize humanized monoclonal antibodies against HER3 to inhibit its function and evaluate their potential as therapeutic agents. Methods: Murine monoclonal antibodies TK-A3 and TK-A4 were humanized and tested for binding to ErbB3 and competition with neuregulin-1β (NRG). Specificity was assessed by ELISA, and epitope identified by X-ray crystallography. Downstream signaling was analyzed by western blot for phosphorylated ErbB3, Akt, and MAPK. Antitumor activity was evaluated in vitro and in a pancreatic cancer xenograft model. A toxicology study was also conducted. Results: TK-hu A3 and TK-hu A4 bound specifically to ErbB3 without cross-reactivity to other ErbB receptors. The ErbB3-TK-hu A3 Fab structure revealed the binding epitope. Both antibodies competed with NRG, inhibiting ErbB3, Akt, and MAPK phosphorylation in a dose-dependent manner. They suppressed cancer cell survival in vitro, and TK-hu A3 significantly delayed tumor growth in vivo. The toxicology study indicated good tolerability. Conclusions: TK-hu A3 emerged as the lead candidate, showing specific HER3 targeting, strong pathway inhibition, and antitumor efficacy in vivo. Beyond standalone use, it could support novel strategies such as T-cell engagers, ADCs, CAR-T, and bispecific antibodies. These findings highlight TK-hu A3 as a promising therapy for HER3-positive, treatment-resistant cancers, meriting further development.

Antibodies doi: 10.3390/antib14040083

Authors: Tristan Mangeat Célestine Mairaville Myriam Chentouf Madeline Neiveyans Martine Pugnière Giang Ngo Vincent Denis Corentin Catherine Alexandre Pichard Emmanuel Deshayes Margaux Maurel Matthieu Gracia Anne Bigot Vincent Mouly Sébastien Estaran Alain Chavanieu Pierre Martineau Bruno Robert

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As tumors generate an acidic microenvironment, we investigated whether we could generate pH-dependent antibodies to increase their tumor specificity. For this proof-of-concept study, we selected the tyrosine kinase receptor AXL because we already developed several antibodies against this target. Methods: To generate a pH-dependent anti-AXL antibody, we performed classical panning of a single-chain variable fragment (scFv) library using phage display at an acidic pH throughout the process. Results: After the third round of panning, 9 scFvs, among the 96 picked clones, bound to AXL at acidic pH and showed very low binding at a neutral pH. After reformatting them into IgG, two clones were selected for further study due to their strong pH-sensitive binding. Using molecular docking and alanine scanning, we found that their binding strongly depended on two histidine residues present on AXL at positions 61 and 116. Conclusions: To conclude, we set-up an easy process to generate pH-dependent antibodies that may increase their tumor-binding specificity and potentially decrease toxicity towards healthy tissues.

Antibodies doi: 10.3390/antib14040082

Authors: Edward John Filippone John L. Farber

Primary podocytopathies, including minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), are caused by a circulating factor or factors injurious to the podocyte. An immunologic origin seems likely based on responsiveness to corticosteroids or other immunosuppressive agents, including calcineurin inhibitors targeting T-cells and rituximab targeting B-cells. Potential non-antibody-mediated circulating factors have been identified, including cardiotrophin-like cytokine 1, soluble urokinase plasminogen activator receptor, and angiopoietin-like 4, among others. More recent research supports a primary antibody pathogenesis, with anti-nephrin antibodies found in a significant percentage of cases. Such antibodies also predict recurrence after transplantation. Other potential antigenic targets besides nephrin include annexin, the proteosome, podocin, and CD40. Additionally, high-resolution confocal microscopy has identified punctate immunoglobulin deposits along the slit diaphragm and podocyte cell body that may or may not colocalize with abnormal punctate nephrin staining and may correlate with detectable circulating antibodies. The success of rituximab in observational studies in both native kidneys and transplants supports a primary role for autoantibodies. We discuss in detail the data supporting putative non-antibody circulating factors, as well as the recent data supporting antibody pathogenesis, which may provide some clues on treating the individual patient.

Antibodies doi: 10.3390/antib14040081

Authors: Enrique Adrián Herrera-Aguirre Diana León-Núñez Jaime Marcial-Quino Saúl Gómez-Manzo César Augusto Reyes-López Yolanda Medina-Flores Olga Mata-Ruíz Lizbeth Xicotencatl-García Hector Luna-Pastén Luz Belinda Ortiz-Alegría Nury Pérez-Hernández Magdalena Escorcia Dolores Correa Fernando Gómez-Chávez

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite responsible for toxoplasmosis, a disease with significant health implications for humans and animals. The surface antigen 1 (SAG1) of T. gondii is a major immunodominant protein that facilitates host cell invasion, making it an ideal target for diagnostic and therapeutic interventions. Immunoglobulin Y (IgY), the primary antibody in avian species, offers unique advantages over mammalian IgG, including easier animal care, lower costs, high-yield production, and potential passive immunization. Objectives: This study aimed to induce, purify, and characterize IgY antibodies targeting T. gondii SAG1 from hen egg yolks. Methods: The coding region of the mature portion of T. gondii SAG1 was amplified by PCR, cloned into the pET32a(+) vector for heterologous expression in E. coli. The recombinant SAG1 (rSAG1) was purified by affinity chromatography and used to immunize hens. IgY was extracted from egg yolks using PEG. SDS-PAGE and spectrophotometry were used to evaluate purity and concentration. By ELISA, Western blot, and flow cytometry, the specificity of IgY was assessed against recombinant and endogenous, native, and denatured SAG1. Results: Purified IgY demonstrated strong recognition of both recombinant and native SAG1 in ELISA and Western blot, and against T. gondii tachyzoites by flow cytometry. Conclusions: SAG1-specific IgY was produced in a pure form; it could be helpful in research, diagnosis, and treatment at low costs on a larger production scale, with minimal animal harm.

Antibodies doi: 10.3390/antib14030080

Authors: Sun Lee Seoryeong Park Hyunji Yang Geummi Cho Seung Youn Lee Donggeun Lee Nara Tae Dae Hee Kim Junho Chung

Background/Objectives: Calsequestrin (CSQ) is a calcium-binding protein that is highly soluble and can serve as a solubility-enhancing fusion tag in recombinant protein expression. Its unique property of calcium-induced precipitation followed by EDTA-mediated resolubilization enables efficient purification. However, the broader application of CSQ-tagged proteins in research have been hampered by the lack of reliable anti-CSQ detection reagents. This study aimed to develop single-domain antibodies (sdAbs) against CSQ for use in diverse immunoassays and cell-based analyses. Methods: Single-domain antibodies were selected from phage-displayed chicken VH libraries generated from CSQ-immunized chickens. After biopanning, CSQ-specific VH sdAb clones were isolated and expressed as VH–human kappa light chain constant region (VH-Cκ) fusion proteins in E. coli. The PE06 clone was chosen for further characterization and conjugated to horseradish peroxidase (HRP) and Alexa Fluor 647 for assay applications. Results: PE06 VH-Cκ fusion protein demonstrated specific binding to CSQ-tagged proteins and enabled reliable detection in enzyme-linked immunosorbent assay (ELISA), immunoblotting, and flow cytometry. These results validated its utility as a chemically defined detection reagent for CSQ fusion proteins expressed in E. coli. Conclusions: This study establishes a CSQ-specific chicken VH sdAb as a versatile detection tool for CSQ-tagged proteins. The approach expands the utility of CSQ as a protein fusion tag and enables the development of recombinant antibodies fused with CSQ, such as scFv-CSQ constructs, for broad application in research and assay systems.

Antibodies doi: 10.3390/antib14030079

Authors: Francois Rosset Nadia Sciamarrelli Luca Mastorino Valentina Pala Sara Boskovic Eleonora Bongiovanni Orsola Crespi Yingying Liao Simone Ribero Pietro Quaglino

Background/Objectives: Lichen planus (LP) is a chronic inflammatory disease of autoimmune origin, affecting the skin and mucous membranes. While corticosteroids and immunosuppressants are traditionally used, many cases remain refractory or intolerant to standard therapies. Recent advances in immunopathogenesis have led to the exploration of targeted therapies, including biologic agents and small-molecule inhibitors. Methods: This review synthesizes current evidence from case reports, case series, and observational studies on the use of monoclonal antibodies (anti-TNF-α, anti-IL-17, anti-IL-23, anti-IL-6) and JAK inhibitors in LP. A structured literature search was conducted across PubMed, Scopus, and Web of Science, focusing on studies published between 2010 and 2025. Data on mechanisms, clinical efficacy, safety, and research limitations were extracted and summarized. Results: Promising therapeutic responses were reported for IL-17 inhibitors (secukinumab, ixekizumab) and JAK inhibitors (tofacitinib, baricitinib) in mucosal and recalcitrant LP. Anti-TNF agents showed variable efficacy, while emerging targets such as BTK and IFN-γ are under investigation. Adverse events were generally mild to moderate, but long-term safety data are lacking. The absence of randomized controlled trials and standardized outcome measures limits generalizability. Conclusions: Biologic and small-molecule therapies represent a potential paradigm shift in the treatment of LP, offering targeted immunomodulation with promising efficacy in refractory cases. Further collaborative research, including randomized studies and biomarker-driven approaches, is urgently needed to validate these treatments and establish personalized care strategies.

Antibodies doi: 10.3390/antib14030078

Authors: Masayuki Tasaki Kazuhide Saito Kota Takahashi

Background: The accurate evaluation of anti-ABO antibodies is essential for risk stratification in ABO-incompatible (ABOi) transplantation. Historically, hemagglutination-based titration has been the cornerstone of such an assessment; however, different tools are being evaluated in this context. In recent years, several novel methods have been reported. Methods: A narrative review was conducted using PubMed, Scopus, and Google Scholar, focusing on recent studies evaluating anti-ABO antibody measurement techniques in the context of ABOi organ transplantation. Results: In addition to the conventional tube method, techniques such as column agglutination technology, flow cytometry, and enzyme-linked immunosorbent assay are utilized for anti-ABO antibody assessment. However, any particular technique, significant interinstitutional and interoperator variabilities have been reported due to differences in the detailed protocols and the inherently subjective nature of some techniques. Moreover, these assays are based on the antibody binding to ABO antigens expressed on red blood cells, which might not accurately reflect the clinical context of organ transplantation. In recent years, technological advances have enabled the development of novel assays evaluating antibody responses specifically against the ABO antigens expressed on vascular endothelial cells. These include glycan microarrays, which differentiate responses by ABO antigen subtypes, and CD31-based microarrays, wherein recombinant CD31 proteins expressing ABO antigens are immobilized. These approaches are applied to assess clinically relevant anti-ABO antibodies in the context of ABOi organ transplantation. Conclusions: The objective evaluation of antibody titers against ABO antigens on vascular endothelial cells might not only enable a more accurate risk assessment but also facilitate meaningful comparisons between institutions.

Antibodies doi: 10.3390/antib14030077

Authors: Hiroshi Yoshida Ayumi Sugitani Mayumi Kobayashi-Kato Masaya Uno Mitsuya Ishikawa

Background: p16 immunohistochemistry (IHC) serves as a surrogate marker for high-risk human papillomavirus (hrHPV) and is widely used in gynecologic pathology. However, few studies have directly compared the staining performance and reproducibility of different p16 antibody clones in this context. Methods: We retrospectively evaluated 176 gynecologic tumor specimens including 42 whole slide sections and 134 tissue microarray cores from the cervix, endometrium, vulva, and ovary using three fully automated p16 IHC assays: E6H4 (Ventana/Roche), JC8 (Agilent/Dako), and 6H12 (Leica). Two pathologists independently reviewed each case, and concordance and interobserver agreement were analyzed. Sensitivity, specificity, and Cohen’s κ statistics were calculated, with E6H4 serving as the reference. Results: All three antibody clones demonstrated excellent staining performance with preserved tissue morphology and minimal background artifacts. Concordance for p16 positivity/negativity was 100% across all clone pairings (95% CI: 97.9–100%). Interobserver reproducibility was also perfect, with a κ coefficient of 1.00 (95% CI: 0.94–1.00). Minor non-block staining patterns did not impair interpretability. Conclusions: Our findings indicate that E6H4, JC8, and 6H12 clones yield comparable staining results when used in conjunction with standardized automated protocols. These results support the practical interchangeability of these clones in clinical and research settings, particularly when cost, availability, or risk management require substitution. Laboratories should continue to perform internal validation and utilize external quality assurance programs when implementing p16 IHC.

Antibodies doi: 10.3390/antib14030076

Authors: Brigita Šemeklienė Brigita Gradauskienė

Infertility is a multifactorial condition with a wide range of potential causes, including anatomical, hormonal, genetic, and lifestyle-related factors. Among these, immunological mechanisms have increasingly been recognized as important contributors. The immune system plays a critical role in maintaining reproductive health, and its dysregulation can impair fertility in both men and women. Recent scientific studies suggest that altered immune responses, particularly those involving autoimmune reactions, may negatively affect fertility by disrupting the complex immunological balance required for successful conception and pregnancy maintenance. This review focuses on the most common autoantibodies, such as antinuclear, antisperm, antiendometrial, antiovarian, antiphospholipid, and antithyroid antibodies. Treatment options, including immunomodulatory therapy, hormone replacement therapy, and lifestyle interventions, are also reviewed.

Antibodies doi: 10.3390/antib14030075

Authors: Luca Bettolini Stefano Bighetti Silvia Mariel Ferrucci Angelo Valerio Marzano Francesca Barei Alessandra Narcisi Matteo Bianco Andrea Carugno Nicola Zerbinati Simone Ribero Michela Ortoncelli Elena Pezzolo Maddalena Napolitano Martina Maurelli Giampiero Girolomoni Zeno Fratton Enzo Errichetti Caterina Foti Giacomo Dal Bello Ilaria Trave Anna Balato Dario Didona Niccolò Gori Federica Veronese Giovanni Paolino Franco Rongioletti Mario Bruno Guanti Laura Calabrese Riccardo Balestri Manfredo Bruni Mariateresa Rossi

Background: Dupilumab, a monoclonal antibody targeting the interleukin-4 receptor α, is approved for moderate-to-severe atopic dermatitis (AD). However, its safety profile in patients with concomitant hematologic disorders remains unclear, as such populations were excluded from pivotal trials. Objective: To evaluate the safety and effectiveness of dupilumab in adolescents and adults with AD and underlying hematologic comorbidities. Methods: This retrospective, multicenter study included 139 patients aged ≥15 years with moderate-to-severe AD and at least one hematologic disorder, treated with dupilumab across 21 dermatology centers. Data on disease severity, laboratory markers, and hematologic outcomes were collected over a median follow-up of 52 weeks (range 4–156). Results: The most common hematologic conditions included monoclonal gammopathies, leukemias, lymphomas, myeloproliferative neoplasms, and immune cytopenias. Clinical response to dupilumab was sustained across all endpoints, with median EASI scores decreasing from 26.0 at the baseline to 1.0 at week 52. NRS pruritus and sleep scores similarly declined to 0.0 by week 52. Serum IgE levels and eosinophil counts progressively decreased. The clinical response to dupilumab was sustained across all endpoints, with significant and progressive improvements in EASI, pruritus NRS, and sleep NRS observed up to week 52, followed by long-term stability through week 156. Serum IgE levels decreased steadily at all timepoints, while eosinophil counts declined after week 4 and stabilized beyond week 52. Hematologic conditions remained stable in 82.7% of patients, resolved in 16.5%, and progressed in only one case. Twelve patients (8.6%) received a new hematologic diagnosis during follow-up; no causal relationship could be established due to the retrospective design and absence of systematic screening, and these findings should be interpreted as descriptive associations only. Conclusions: Dupilumab appears to be safe and effective in AD patients with a broad range of hematologic comorbidities, including malignancies. These findings support its use in real-world settings, though prospective studies are warranted to further assess long-term safety in this population.

Antibodies doi: 10.3390/antib14030074

Authors: Raphael Trenker Deepti Rokkam Andrew Morin Priyanka Balasubrahmanyam Verenice Paredes Ivan Cheng Rene de Waal Malefyt Martin Oft Patrick Lupardus Sandro Vivona

Background: Bispecific antibodies have emerged as a promising class of therapeutics, enabling simultaneous targeting of two distinct antigens. Single-domain antibodies (sdAbs) comprising camelid variable heavy chains (VHHs) provide a compact and adaptable platform for bispecific antibody design due to their small size and ease of linkage. Methods: Here we investigate structure-activity relationship of VHH-based cytokine surrogates by combining cell signaling and functional assays with x-ray crystallography and other biophysical techniques. Results: We describe crystal structures of four unique bispecific VHHs that engage and activate the cytokine receptor pairs IL-18Rα/IL-18Rβ and IL-2Rβ/IL-2Rγ. These bispecific VHH molecules, referred to as surrogate cytokine agonists (SCAs), create unique cytokine signals that can be tuned by linker engineering. Our structural analysis reveals multiple dimeric conformations for these bispecific SCAs, where the two VHH domains can interact to form a compact structure. We demonstrate that the dimeric conformation can be enforced via engineering of a non-native disulfide bond between the VHH subunits, thus enhancing molecular thermostability. Conclusion: Our findings have important implications for the design and engineering of bispecific VHHs or sdAbs, offering a novel strategy for tuning their activity and increasing their stability.

Antibodies doi: 10.3390/antib14030073

Authors: Felix Klaus Geyer Julian Borbeck Wiktoria Palka Xueyuan Zhou Jeffrey Takimoto Brian Rabinovich Bernd Reifenhäuser Karlheinz Friedrich Harald Kolmar

Background/Objectives: Single-domain immunoglobulins are small protein modules with specific affinities. Among them, the variable domains of heavy chains of heavy-chain-only antibodies (VHH) as the antigen-binding fragment of heavy-chain-only antibodies (also termed nanobodies) have been widely investigated for their applicability, e.g., therapeutics and immunodiagnostics. However, despite their advantageous biochemical and biophysical characteristics, protein aggregation throughout recombinant synthesis is a serious drawback in the development of nanobodies with application perspectives. Therefore, we aimed to develop a computational method to predict the aggregation propensity of VHH antibodies for the selection of promising candidates in early discovery. Methods: We employed a deep learning-based structure prediction for VHHs and derived from it likely biophysical and biochemical properties of the framework region 2 with relevance for aggregation. A total of 106 nanobody variants were produced by recombinant expression and characterized for their aggregation behavior using size exclusion chromatography (SEC). Results: Quantitative characteristics of framework region 2 patches were combined into a function that defines an aggregation score (AS) predicting the aggregation propensities of VHH variants. AS was evaluated for its capability to forecast recombinant VHH aggregation by experimentally studying VHH Fc-fusion proteins for their aggregation. We observed a clear correlation between the calculated aggregation score and the actual aggregation propensities of biochemically characterized VHHs Fc-fusion proteins. Moreover, we implemented an easily accessible pipeline of software modules to design nanobodies with desired solubility properties. Conclusions: AI-based prediction of VHH structures, followed by analysis of framework region 2 properties, can be used to predict the aggregation propensities of VHHs, providing a convenient and efficient tool for selecting stable recombinant nanobodies.

Antibodies doi: 10.3390/antib14030072

Authors: Sarah Döring Georg Tscheuschner Sabine Flemig Michael G. Weller Zoltán Konthur

Background: Monoclonal antibodies play an important role in therapeutic and analytical applications. For recombinant expression, the coding sequences of the variable regions of the heavy and light chains are required. In addition, cloning antibody sequences, including constant regions, reduces the impact of hybridoma cell loss and ensures preservation of the naturally occurring full antibody sequence. Method: We combined amplification of IgG antibody variable regions from hybridoma mRNA with an advanced method for full-length cloning of monoclonal antibodies in a simple two-step workflow. Following Sanger sequencing and evaluation of consensus sequences, the best matching variable, diversity, and joining (V-(D-)J) gene segments were identified according to identity scores from IgBLAST reference sequences. Simultaneously, the mouse IgG subclass was determined at the DNA level based on isotype-specific sequence patterns in the CH1 domain. Knowing the DNA sequence of V-(D-)J recombination responsible for the complementary determining region 3 (CDR 3), variable region-specific primers were designed and used to amplify the corresponding antibody constant regions. Results: To verify the approach, we applied it to the hybridoma clone BAM-CCMV-29-81 and obtained identical full-length antibody sequences as with RNA Illumina sequencing. Further validation at the protein level using an established MALDI-TOF MS-fingerprinting protocol showed that five out of six genetically encoded CDR domains of the monoclonal antibody BAM-CCMV-29-81 could be efficiently correlated. Conclusion: This simple, streamlined method enables the cost-effective determination of the full-length sequence of monoclonal antibodies from hybridoma cell lines, with the added benefit of obtaining the DNA sequence of the antibody ready for recombinant expression.

Antibodies doi: 10.3390/antib14030071

Authors: Mark A. Tornetta Brian P. Whitaker Olivia M. Cantwell Peter N. Haytko Eileen D. Pisors Fulai Zhou Mark L. Chiu

Background/Objectives: The complexity of diseases such as cancer and auto-immune disorders drives the need for unique, target-driven therapeutics. A broader arsenal to generate better biologics-based therapeutics is needed to provide more efficient and effective antibody generation technologies. The critical parameter for antibody generation is to generate as much candidate diversity to each target as possible. Method/Results: We present guidelines for having an efficient process using a fully synthetic human single-domain antibody (sdAb) phage display library. Critical milestones for success focused on library quality control (QC) assessments, evaluation of specific biopanning outputs, and construct designs that enabled efficient transition to mammalian expression. The synthetic VHO libraries produced epitope diversity better than an immunized sourced library with candidates possessing nM potencies and monodispersity > 90% via SEC. Conclusions: Synthetic human scaffold sdAb phage display libraries was constructed, biopanned, and selected candidates that could be directly transitioned for mammalian expression. The diverse VHO sets of candidates produced from many targets easily provided opportunities to make a multi-specific biological compound. Both synthetic and immunized phage selection campaign results suggested that these technologies complemented each other to generate therapeutic candidates. Finally, we demonstrated how diverse data produced from a process that used VHO synthetic libraries could accelerate drug discovery.

Antibodies doi: 10.3390/antib14030070

Authors: Olena Denysenko Anselm H. C. Horn Heinrich Sticht

Background/Objectives: Cows produce antibodies with ultralong CDRH3 segments (ulCABs) that contain a disulfide-stabilized knob domain. This domain is connected to the globular core of the antibody by a β-strand stalk. In the crystal structures, the stalk protrudes from the core in an extended conformation and presents the knob at its distal end. However, the rigidity of this topology has been questioned due to the extensive crystal packing present in most ulCAB crystal structures. To gain more insight into the dynamics of ultralong CDRH3s, we performed a comparative molecular dynamics (MD) study of 19 unique ulCABs. Methods: For all 19 systems, one-microsecond MD simulations were performed in explicit solvent. The analyses included an investigation of the systems’ conformational stability and the dynamics of the knob domain as well as an energetic analysis of the intramolecular knob interactions. Results: The simulations show that the extended stalk–knob conformation observed in the crystal structures is not preserved in solution. There are significant differences in the degree of knob dynamics, the orientations of the knobs, the number of flexible stalk residues, and the frequency of the motions. Furthermore, interactions between the knob and the light chain (LC) of the ulCABs were observed in about half of the systems. Conclusions: The study reveals that pronounced knob dynamics is a general feature of ulCABs rather than an exception. The magnitude of knob motions depends on the system, thus reflecting the high sequence diversity of the CDRH3s in ulCABs. The observed knob–LC interactions might play a role in stabilizing distinct knob orientations. The MD simulations of ulCABs could also help to identify suitable knob fragments as mini-antibodies by suggesting appropriate truncation points based on flexible sites in the stalks.

Antibodies doi: 10.3390/antib14030069

Authors: Aurelia Knispel Christian Jassoy

Background: The concentration of antigen-specific antibodies in serum is usually measured in international units/mL. Therefore, the actual concentration of virus-specific antibodies in sera is unknown. Objectives: The aim of the study was to determine conversion factors for concentrations of IgG against hepatitis B surface antigen (HBs), SARS-CoV-2 receptor binding domain (RBD) and nucleoprotein (NP) as well as tetanus toxin (Ttx) in serum and to compare antigen-specific IgG concentrations in serum samples. Methods: Absorption equivalence ELISAs were used to determine conversion factors for international units (IU) for anti-HBs, anti-SARS-CoV-2-RBD and NP and for anti-Ttx immunoglobulin G. The antigen-specific IgG concentrations in serum samples were then measured in units/mL and the ratio of IgG concentrations in the sera was determined using the conversion factors. Results: One IU of anti-HBs IgG corresponded to 24.4 BAU of anti-CoV-2 RBD IgG, 6.87 BAU of anti-CoV-2 NP and 14 mIU of anti-Ttx IgG. One BAU anti-SARS-CoV-2 NP-specific IgG is equivalent to 3.5 BAU SARS-CoV-2 RBD-specific IgG. Conversion of international units showed that median serum anti-Ttx-IgG concentrations were 50 times higher and anti-CoV-2-RBD-IgG concentrations were 390 times higher than median anti-HBs-IgG concentrations. In addition, after SARS-CoV-2 infection, the concentration of NP-specific IgG in serum was generally higher than that of RBD-specific IgG. Conclusions: The study provides conversion factors for serum concentrations of IgG against HBs, SARS-CoV-2 RBD and NP, as well as Ttx-IgG. This offers new insights into serum IgG concentrations and allows conclusions to be drawn about plasma cell pools.

Antibodies doi: 10.3390/antib14030068

Authors: József Prechl Ágnes Kovács Krisztián Papp Zoltán Hérincs Tamás Pfeil

Background: When an antigen molecule is exposed to serum, many different kinds of antibodies bind to it. The complexity of these binding events is only poorly characterized by assays that generate a single variable, generally reflecting the fractional saturation of the antigen, as the readout. Methods: We have previously devised an assay that delivers the essential biochemical variables to determine fractional saturation as the output: an equilibrium dissociation constant for affinity, the ratio of antibody concentration to the equilibrium constant and the concentration of bound antibodies under reference conditions. Here we propose a visualization method for the practical and informative display of these variables. Results: Using total antigen concentration and free and bound antibody concentration as coordinates in a three-dimensional space, a surface plot can depict the behavior of serum antibodies in the measurement range and identify the values of the key variables of binding activity. This surface display (antibody binding in 3-concentration display, Ab3cD) was used for the characterization of antibody binding to the SARS-CoV-2 spike protein in seronegative and seropositive sera. We demonstrate that this visualization scheme is suitable for presenting both individual and group differences and that epitope density changes, not commonly measured by immunoassays, are also revealed by the method. Conclusions: We recommend the use of 3D visualization whenever detailed, informative and characteristic differences in serum antibody reactivity are studied.

Antibodies doi: 10.3390/antib14030067

Authors: Hiroyuki Suzuki Tomokazu Ohishi Takuro Nakamura Miyuki Yanaka Saori Handa Tomohiro Tanaka Mika K. Kaneko Yukinari Kato

Background: Podocalyxin (PODXL) has been identified as a promising therapeutic target and a potential diagnostic biomarker in various tumors. Despite the therapeutic potential of anti-PODXL monoclonal antibodies (mAbs), their further development has been limited by concerns regarding potential on-target off-tumor toxicities. To minimize adverse effects on normal tissues, developing a cancer-specific mAb (CasMab) against PODXL is essential. Methods: Our group established a cancer-specific anti-PODXL mAb, PcMab-60 (IgM, κ), through the screening of over one hundred hybridoma clones. In this study, PcMab-60 was engineered into a humanized IgG1-type mAb (humPcMab-60), and its antitumor activity was examined using mouse xenograft models of pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer. Results: HumPcMab-60 retains cancer-specific reactivity; humPcMab-60 reacted to PDAC cell lines (PK-45H and MIA PaCa-2) and the colorectal cancer cell line (Caco-2), but not to a normal lymphatic endothelial cell line in flow cytometry. Furthermore, humPcMab-60 exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against PODXL-expressing cell lines and showed antitumor effects against the tumor xenografts. Conclusions: A humanized anti-PODXL CasMab, humPcMab-60, could be a promising mAb-based tumor therapy.

Antibodies doi: 10.3390/antib14030066

Authors: Maciej Marek Spałek Magdalena Jałowska Natalia Welc Monika Bowszyc-Dmochowska Takashi Hashimoto Justyna Gornowicz-Porowska Marian Dmochowski

Background/Objectives: Anti-p200 pemphigoid is a rare and likely underdiagnosed autoimmune blistering disorder. Laminin γ1 and laminin β4 have been implicated as potential target antigens in its pathogenesis. Recently, a novel indirect immunofluorescence assay targeting anti-laminin β4 antibodies has been developed, demonstrating high sensitivity and specificity, and offering a valuable tool for improved diagnosis. Methods: Of the 451 patients, 21 were selected for further laboratory analysis based on medical records. Sera from 10 patients, which showed a positive direct immunofluorescence (DIF) result and negative results in multiplex enzyme-linked immunosorbent assays (ELISAs) and/or mosaic six-parameter indirect immunofluorescence (IIF) for various autoimmune bullous diseases, were tested for the presence of anti-laminin β4 antibodies. Additionally, sera from 11 patients with positive DIF and positive ELISA for antibodies against BP180 and/or BP230 were analyzed. Results: Among the 10 patients with positive DIF and negative ELISA and/or mosaic six-parameter IIF, 6 sera were positive for anti-laminin β4 antibodies. These patients presented with atypical clinical features. In contrast, all 11 sera from patients with both positive DIF and positive ELISA for BP180 and/or BP230 were negative for anti-laminin β4 antibodies. Conclusions: In patients with a positive DIF result but negative ELISA and/or mosaic six-parameter IIF findings, testing for anti-laminin β4 antibodies should be considered. Furthermore, in cases presenting with atypical clinical features—such as acral distribution of lesions, intense pruritus, or erythematous–edematous plaques—the possibility of anti-p200 pemphigoid should be included in the differential diagnosis.

Antibodies doi: 10.3390/antib14030065

Authors: Michael Sandhu Irina Murakhovskaya

Antibody-mediated autoimmune diseases are common, can involve any organ system, and pose a large burden for patients and healthcare systems. Most antibody-mediated diseases are mediated by IgG antibodies. Selective targeting of pathogenic antibodies is an attractive treatment option which has already proven to be effective in antibody-positive generalized myasthenia gravis, maternal-fetal alloimmune cytopenias, and immune thrombocytopenic purpura. Warm autoimmune hemolytic anemia (wAIHA) is an autoimmune disorder mediated by pathogenic antibodies mainly of the IgG class with no approved therapy. Current treatment includes non-specific immunosuppression with corticosteroids, rituximab, and other immunosuppressive agents. With most therapies, time to response can be delayed and transfusions may be needed. Neonatal Fc receptor (FcRN) therapies provide rapid and sustained reduction of pathogenic IgG levels providing potential for fast, effective therapy in antibody-mediated autoimmune diseases including warm autoimmune hemolytic anemia. This review focuses on the emerging role of FcRn inhibition in autoimmune hematologic diseases, and their therapeutic potential in wAIHA.

Antibodies doi: 10.3390/antib14030064

Authors: Evgeniy V. Petrotchenko Andreas Hahn Christoph H. Borchers

Pompe disease is a rare autosomal-recessive neuromuscular disorder caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA), leading to the pathological accumulation of glycogen and impaired autophagy. Enzyme replacement therapy (ERT) with recombinant human alpha-glucosidase (rhGAA) has been available since 2006, but may lead to the formation of anti-drug antibodies (ADAs) against the recombinant human enzyme, which, in turn, may adversely affect the response to ERT. Knowledge of the antigenic determinants of rhGAA involved in interaction with ADAs may facilitate the development of strategies to attenuate the anti-drug immune response in patients. Here, we determined the rhGAA ADA epitopes in the plasma of Pompe disease patients using a series of affinity purifications combined with epitope extraction and label free quantitation LC-MS.

Antibodies doi: 10.3390/antib14030063

Authors: Maurizio Benucci Riccardo Terenzi Francesca Li Gobbi Emanuele Antonio Maria Cassarà Tommaso Picchioni Edda Russo Barbara Lari Mariangela Manfredi Maria Infantino

Background/Objectives: Immune-mediated necrotizing myopathy (IMNM) is a severe inflammatory myopathy marked by proximal muscle weakness, elevated creatine kinase (CK), and the presence of anti-HMGCR antibodies. Statin exposure is a recognized trigger for anti-HMGCR-positive IMNM, which may persist despite statin withdrawal. This pilot case series explores, for the first time, the use of bempedoic acid—a liver-specific lipid-lowering agent with minimal muscle toxicity—as an alternative to statins in these patients. Methods: We report 10 anti-HMGCR-antibody-positive IMNM patients (6 females, 4 males) previously on statins for primary prevention (8 on atorvastatin, 2 on simvastatin) without prior cardiovascular events. Statins were discontinued at myositis onset. All patients received prednisone and immunosuppressants (methotrexate in 7, mycophenolate in 3), plus bempedoic acid. Anti-HMGCR antibodies were measured using a chemiluminescence method. Results: Their mean anti-HMGCR antibody levels decreased significantly from 390.93 ± 275.22 to 220.89 ± 113.37 CU/L (p = 0.027) after 6 months of treatment. Their CK levels dropped from 1278.9 ± 769.39 to 315.1 ± 157.72 IU/L (p = 0.001), and aldolase dropped from 11.63 ± 2.18 to 6.61 ± 1.22 U/L (p = 0.0001). The mean LDL-C value was 96.1 ± 8.16 mg/dL. No disease recurrence was observed. Autoimmune panels were negative for other myositis-associated and/or -specific antibodies. Conclusions: Bempedoic acid appears to be a safe, effective, and cost-efficient lipid-lowering alternative in statin-intolerant IMNM patients. Larger studies are warranted to confirm its efficacy across different subgroups and to optimize dyslipidemia management in this setting.

Antibodies doi: 10.3390/antib14030062

Authors: Kevin M. Budge Ross Blankenship Patricia Brown-Augsburger Lukasz K. Chlewicki

Background/Objectives: Anti-drug antibody (ADA) formation can impact the safety, pharmacokinetics, and/or efficacy of biotherapeutics, including monoclonal antibodies (mAbs). Current strategies for ADA/immunogenicity risk prediction of mAbs include in silico algorithms, T cell proliferation assays, MHC-associated peptide proteomics assays (MAPPs), and dendritic cell internalization assays. However, B cell-mediated responses are not assessed in these assays. B cells are professional antigen-presenting cells (APCs) and secrete antibodies toward immunogenic mAbs. Therefore, methods to determine B cell responses would be beneficial for immunogenicity risk prediction and may provide a more comprehensive assessment of risk. Methods: We used a PBMC culture method with the addition of IL-4, IL-21, B cell activating factor (BAFF), and an anti-CD40 agonist mAb to support B cell survival and activation. Results: B cells in this assay format become activated, proliferate, and secrete IgG. A panel of 51 antibodies with varying clinical immunogenicity rates were screened in this assay with IgG secretion used as a readout for immunogenicity risk. IgG secretion differed among test articles but did not correlate with the clinical immunogenicity rating. Conclusions: This dataset highlights the challenges of developing a B cell assay for immunogenicity risk prediction and provides a framework for further refinement of a B cell-based assay for immunogenicity risk prediction of mAbs.

Antibodies doi: 10.3390/antib14030061

Authors: Matteo Bonato Francesca Savoia Enrico Orzes Elisabetta Favero Gianenrico Senna Micaela Romagnoli

Background: Mepolizumab is an effective treatment for severe eosinophilic asthma, leading to a depletion of blood eosinophil levels, the clinical relevance of which remains unclear. Objective: The aim of this study was to assess the relationship between mepolizumab-induced blood eosinophil reduction and clinical outcome in patients with severe eosinophilic asthma, in particular, whether the magnitude of blood eosinophil reduction was associated with clinical remission. Methods: We conducted a real-world retrospective analysis of 58 adult patients with severe eosinophilic asthma treated with mepolizumab. Clinical and respiratory functional parameters were evaluated at the start of mepolizumab treatment (T0) and after two years of treatment (T2; mean follow-up: 22.8 ± 7.5 months). Blood eosinophil counts were recorded at T0 and during the first year of treatment (T1; mean follow-up: 7.7 ± 4.1 months). Results: After two years of mepolizumab treatment, 58 severe asthmatic patients showed significant improvements in ACT score, FVC, and FEV1 and a reduction in acute exacerbations and the use of maintenance therapies. Clinical remission was achieved in 55.1% of patients. Lower blood eosinophil counts during the first year (T1) were associated with greater improvements in lung function and fewer exacerbations. A greater relative decrease in eosinophils from baseline to T1 (ΔEOS%) was significantly associated with remission, reductions in exacerbations, and no maintenance OCS use. ΔEOS% was the only independent predictor of remission in the multivariate analysis. A ≥90% reduction predicted remission with 80% specificity (AUC = 0.726). Conclusions: Monitoring blood eosinophils after mepolizumab initiation could be a useful tool for predicting long-term response to treatment. In particular, a reduction by over 90% of peripheral blood eosinophils during the first year of mepolizumab treatment predicts clinical remission with a specificity of 80%. Considering the accessibility and the low cost of this biomarker, it may help to optimize long-term asthma management.

Antibodies doi: 10.3390/antib14030060

Authors: Eligia González-Rodríguez Marta Rodero J. Alberto Montoya-Alonso Kevin M. Santana-Hernández Myriam R. Ventura Carmen Cuéllar Eligia Rodríguez-Ponce

Food-borne zoonoses, particularly anisakiosis caused by Anisakis spp., are an increasing public health concern due to the rising consumption of raw fish. Anisakiosis results from the ingestion of third-stage larvae of Anisakidae nematodes, with the genus Anisakis re-sponsible for approximately 97% of human cases. While regulatory protocols exist to minimize infection risk in commercial settings, domestic food preparation often lacks such safeguards, creating a gap in public health protection. In the Canary Islands, a major Spanish aquaculture region, farmed fish exhibit a low Anisakis prevalence, suggesting minimal risk from aquaculture products. In contrast, wild-caught fish demonstrate varia-ble parasitism, with recent studies reporting a 25% prevalence among commercial species. Methods: This study assessed Anisakis exposure in the Canary Islands by measuring specific IgG and IgE antibodies in 1043 serum samples collected from all seven islands between March 2014 and October 2015. ELISA assays detected anti-Anisakis antibodies, and the results were analyzed by age, sex, island, and isoclimatic zone. Results: Overall, 16.9% of samples were IgG-positive and 6.8% were IgE-positive. Seroprevalence was significantly higher in indi-viduals aged 60 years and above. Geographic heterogeneity was notable: La Palma had the highest IgG seroprevalence (35.3%), while El Hierro showed the highest IgE prevalence (16.3%). Temperate isoclimatic zones exhibited higher antibody prevalence than dry zones. These findings indicate variable Anisakis exposure across the Canary Islands, likely influenced by environmental and behavioral factors. Conclusions: The results highlight the need for targeted public health interventions to reduce the anisakiosis risk, particularly in regions and populations with elevated exposure.

Antibodies doi: 10.3390/antib14030059

Authors: Yalcin Pisil Tomoyuki Miura Kiyoki Ito Yoshihiro Watanabe

Objectives: The durability and breadth of neutralizing antibodies following SARS-CoV-2 mRNA vaccination remain incompletely understood. This study aimed to investigate how longitudinal changes in antibody isotype composition impact neutralization against structurally diverse SARS-CoV-2 variants. Methods: After screening a broader cohort of mRNA-vaccinated sera, time-matched samples collected one month (1 mpv) and three months post-vaccination (3 mpv) were selected for detailed analysis. Neutralization assays against live virus variants, enzyme-linked immunosorbent assays (ELISA), and immunogold electron microscopy were performed to assess antibody titers, isotype levels, and virion morphology. Results: Neutralization titers declined markedly at 3 mpv, particularly against immune-evasive variants. Notably, the Lambda variant showed disproportionately high sensitivity to early-phase sera despite its divergence from the vaccine strain. Antibody isotyping showed that IgA and IgM decreased over time, while IgG levels were relatively more sustained. Electron microscopy revealed broader virion size heterogeneity in Lambda (50–200 nm) compared to Wuhan (80–120 nm), potentially enhancing multivalent antibody engagement. Consistently, ELISA under reduced spike density conditions showed that IgA and IgM retained stronger binding than IgG. Conclusions: These findings indicate that the decline of IgA and IgM compromises neutralization breadth, especially against structurally divergent variants such as Lambda. Sustaining dynamic multivalent isotype responses that adapt to diverse spike morphologies may be critical for broad cross-variant immunity.

Antibodies doi: 10.3390/antib14030058

Authors: Supriya Peshin Faizan Bashir Naga Anvesh Kodali Adit Dharia Sajida Zaiter Sakshi Singal Nagaishwarya Moka

Immunotherapy has emerged as a transformative approach in gastrointestinal (GI) cancers, addressing historically poor survival rates in advanced-stage disease. Immune checkpoint inhibitors (ICIs) targeting the PD-1/PD-L1 axis demonstrate remarkable efficacy in colorectal cancer with deficient mismatch repair (dMMR) or high microsatellite instability (MSI-H), exemplified by trials like NICHE-2 achieving exceptional pathological response rates. However, significant limitations persist, including resistance in some dMMR/MSI-H tumors, minimal efficacy in proficient mismatch repair (pMMR) tumors, and low overall response rates across most GI malignancies due to tumor heterogeneity and immune evasion mechanisms. Predictive biomarkers such as tumor mutational burden (TMB) and PD-L1 expression are crucial for optimizing patient selection, while hypermutated pMMR tumors with POLE mutations represent emerging therapeutic opportunities. In pancreatic adenocarcinoma, where survival remains dismal, combination strategies with chemotherapy and novel approaches like cancer vaccines show promise but lack transformative breakthroughs. Esophagogastric cancers benefit from ICIs combined with chemotherapy, particularly in MSI-H and HER2-positive tumors, while hepatocellular carcinoma has achieved significant progress with combinations like atezolizumab–bevacizumab and durvalumab–tremelimumab surpassing traditional therapies. Biliary tract cancers show modest improvements with durvalumab–chemotherapy combinations. Despite these advances, immunotherapy faces substantial challenges including immune-related adverse events, acquired resistance through cancer immunoediting, and the need for biomarker-driven approaches to overcome tumor microenvironment barriers. This review discusses key clinical trials, therapeutic progress, and emerging modalities including CAR T-cell therapies and combination strategies, emphasizing the critical need to address resistance mechanisms and refine precision medicine approaches to fully realize immunotherapy’s potential in GI malignancies.

Antibodies doi: 10.3390/antib14030057

Authors: Kazuhisa Aoki Rikio Yabe Sayaka Ono Mayumi Saeki Yuri Tanno Hidetaka Tanno

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae, and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Conclusions: Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics.

Antibodies doi: 10.3390/antib14030056

Authors: Abeline Kapuczinski Dorian Parisis Nour Kassab Julie Smet Muhammad Soyfoo

Connective tissue diseases (CTDs) comprise a heterogeneous group of autoimmune conditions characterized by diverse clinical manifestations and autoantibody profiles, posing significant diagnostic challenges. This systematic review and meta-analysis evaluated the diagnostic performance of automated connective tissue disease screening assays, commonly known as CTD screens, in diagnosing systemic rheumatic diseases. Eleven studies, including cohort and case–control designs, involving a total of 2384 CTD-positive patients, 8972 controls without CTD, and 679 healthy blood donors, were analyzed. The results demonstrated a pooled sensitivity of 79.36% and specificity of 90.79% for Elia® CTD-screen, and a sensitivity of 87.23% and specificity of 83.56% for QuantaFlash® CTD-screen. These tests exhibited varied sensitivity across individual CTDs, with excellent specificity for distinguishing CTD patients from healthy controls. Despite their utility, CTD screens should not be solely relied upon for diagnosis due to limitations in positive predictive value, particularly in low-prevalence populations. Clinical context and expert rheumatological evaluation remain indispensable. Optimizing the use of CTD screens can enhance diagnostic efficiency, reduce unnecessary testing, and mitigate patient anxiety and healthcare costs. Further research focusing on integrating these assays with clinical evaluation is recommended.

Antibodies doi: 10.3390/antib14030055

Authors: Yuan Zong Shuang Qiu Mingming Yang Jing Zhang Yaru Zou Yuxin Jing Kyoko Ohno-Matsui Koju Kamoi

Thyroid eye disease (TED) is a complex autoimmune disorder characterized by orbital inflammation and tissue remodeling. Teprotumumab, a fully human monoclonal antibody targeting insulin-like growth factor-1 receptor (IGF-1R), represents a significant breakthrough in TED treatment. This review comprehensively analyzes the therapeutic role of teprotumumab in TED management. Mechanistically, teprotumumab inhibits the IGF-1R/TSHR signaling complex, thereby reducing orbital fibroblast differentiation and inflammatory responses. Phase II and III clinical trials have demonstrated its remarkable efficacy in reducing proptosis and improving clinical activity scores, with the benefits extending to both active and chronic TED cases. Real-world studies have validated these findings further and expanded its potential applications to various clinical scenarios, including dysthyroid optic neuropathy and steroid-resistant cases. However, several challenges remain. These include treatment-related adverse effects such as hyperglycemia and hearing impairment, with emerging evidence suggesting ethnic variations in susceptibility. The high cost of treatment poses significant accessibility barriers, while limited long-term follow-up data and potential disease recurrence necessitate further investigation. This review synthesizes the current evidence to inform clinical decision-making and highlights areas requiring additional research to optimize teprotumumab’s therapeutic application in TED management.

Antibodies doi: 10.3390/antib14030054

Authors: Zhaklin Apostolova Tanya Shivacheva Tsvetoslav Georgiev

Objectives: The present study aimed to evaluate the long-term survival of patients with rheumatoid arthritis (RA) receiving biologic or targeted synthetic disease-modifying antirheumatic drugs (b/tsDMARDs) in a real-world setting, and to identify key prognostic factors influencing mortality within this cohort. Methods: This retrospective, observational cohort study analyzed 165 patients with confirmed RA who were on b/tsDMARD treatment for at least six months as of June 2017. Patient data, including demographics, disease duration, prior therapeutic regimens, and global functional status were extracted from medical records to collect data covering a seven-year follow-up period, extending from June 2017 to December 2024. Corticosteroid use was defined as continuous systemic intake during the RA activity analysis period. Survival outcomes were analyzed using Kaplan-Meier methods and multivariate Cox proportional hazards models to identify independent predictors of mortality. Results: Over a mean follow-up of 9.4 years, the mortality rate was 13.5 deaths per 1000 treatment-years, with an overall survival rate of 87.3%. Advanced functional disability and prolonged corticosteroid use were independently associated with higher mortality risk. In subgroup analyses, chronic kidney disease significantly increased mortality among patients on TNF inhibitors. In contrast, patients who remained on their initial anti-IL6 therapy had lower mortality, though this may reflect survivor bias. Conclusions: This study highlights the importance of long-term b/tsDMARD intervention in RA patients, with observed low mortality and high survival rates. Subgroup findings suggest the importance of comorbidity management in TNFi users and therapeutic stability in anti-IL6 users.

Antibodies doi: 10.3390/antib14030053

Authors: Alexander Veber Dennis Lenau Polyniki Gkragkopoulou David Kornblüh Bauer Ingo Focken Wulf Dirk Leuschner Christian Beil Sandra Weil Ercole Rao Thomas Langer

Recombinantly produced monoclonal antibodies (mabs) belong to the fastest growing class of biotherapeutics. In humans, antibodies are classified into five different classes: IgA, IgD, IgE, IgG and IgM. Most of the therapeutic mabs used in the clinic belong to the IgG class, albeit other antibody classes, e.g., IgM, have been evaluated in clinical stages. Antibodies are composed of heavy chains paired with a light chain. In IgM and IgA, an additional chain, the J-chain, is present. Two types of light chains exist in humans: the κ-light chain and the λ-light chain. The κ-light chain predominates in humans and is used in the vast majority of therapeutic IgG. The reason for the preference of the κ-light chain in humans is not known. Our study investigates whether light-chain selection influences the productivity of the clinically validated mabs adalimumab and trastuzumab. Both mabs were expressed as IgG and IgM with a κ- or a λ-light chain in HEK293 cells. Besides comparing the expression levels of the different mabs, we also evaluated whether the passage number of the cell line has an impact on product yield. In addition, the expressions of adalimumab, trastuzumab, an anti-CD38 and an anti-PD-L1-antibody were analyzed in HEK293 and CHO cells when both the κ- and λ-light chains are present. In summary, IgG outperformed IgM variants in expression efficacy, while light-chain selection had minimal impact on the overall expression levels. The yields of all mab variants were higher in fresh cells, despite cell cultures with a high cell passage number having higher cell densities and cell numbers at the time of harvest. The incorporation of a particular light chain occurred at similar rates in HEK293 and CHO cells.

Antibodies doi: 10.3390/antib14030052

Authors: Ge Yang Mohammad Massumi

Since the advent of recombinant DNA technologies and leading up to the clinical approval of T cell engager blinatumomab, the modular design of therapeutic antibodies has enabled the fusion of antibody fragments with proteins of various functionalities. This has resulted in an expansive array of possible mechanisms of action and has given birth to fragment-based antibodies (fbAbs) with immune cell engager modalities. In searchable databases, the preclinical development of these antibodies has shown promise; however, clinical outcomes and restructuring efforts involving these agents have produced mixed results and uncertainties. Amid budgetary cuts in both academia and industry, critical planning and evaluation of drug R&D would be more essential than ever before. While many reviews have provided outstanding summaries of preclinical phase fbAbs and cataloged relevant clinical trials, to date, very few of the articles in searchable databases have comprehensively reviewed the details of clinical outcomes along with the underlying reasons or potential explanations for the success and failures of these fbAb drug products. To fill the gap, in this review, we seek to provide the readers with clinically driven insights, accompanied by translational and mechanistic studies, on the current landscape of fragment-based immune cell engager antibodies in treating cancer, infectious, and autoimmune diseases.

Antibodies doi: 10.3390/antib14020051

Authors: Domenico De Falco Francesca Iaquinta Doriana Pedone Alberta Lucchese Dario Di Stasio Massimo Petruzzi

Oral Lichen Planus (OLP) is a chronic autoimmune disease with potential overlap with Pemphigus Vulgaris (PV), particularly in erosive forms. Desmoglein 1 and 3 are transmembrane glycoproteins of desmosomes, typically involved in PV. This scoping review aims to evaluate the presence and potential pathogenetic role of anti-desmoglein 1 (Dsg1) and anti-desmoglein 3 (Dsg3) antibodies in OLP. A literature search was conducted on MEDLINE/PubMed, Ovid, and Scopus up to April 2025. Human studies reporting OLP patients with anti-Dsg1 and/or anti-Dsg3 antibodies were included. Data from 11 studies were analyzed by diagnosis, age/sex, oral site involvement, immunofluorescence, and ELISA testing. Erosive OLP was most frequently associated with anti-Dsg1/Dsg3 positivity, mainly in women aged 40–60. Immunofluorescence was positive in some cases, while the ELISA test almost consistently detected anti-Dsg1 and Dsg3 antibodies. However, in many instances, antibody titers did not reach the threshold value, despite the presence being detectable. This finding suggests that anti-Dsg1/Dsg3 antibodies may represent epiphenomena of chronic inflammation in erosive OLP, indicating an immune-serological overlap with PV but lacking direct pathogenicity. Furthermore, the role of Dsg3 in oral squamous cell carcinoma, by promoting enzymes that degrade the extracellular matrix and enhance tumor invasiveness, highlights the complex functions of desmogleins beyond autoimmunity.

Antibodies doi: 10.3390/antib14020050

Authors: Lin Sun Roman Palt Georg Schütz Esther Föderl-Höbenreich Laura Brod Antonia Hermle Anja Lux Herta Steinkellner Somanath Kallolimath

Therapeutic antibodies with lambda light chains (λ-Abs) are underrepresented compared to kappa light chains (κ-Abs). Here, we evaluated two SARS-CoV-2-specific monoclonal antibodies (mAbs) that exhibit high (P5C3) and low (H4) antigen binding as κ and λ variants. mAbs expressed in glycoengineered Nicotiana benthamiana did not show differences in expression levels, glycosylation, and antigen binding, while κ-Abs exhibited slightly increased thermodynamic stability over λ-Abs. SARS-CoV-2 neutralization and IgG-FcγR immune complex studies revealed increased activities of H4 IgG1κ compared to H4 IgG1λ, with no differences observed between P5C3 variants. Our results indicate that constant light chain variability and Ab specificity contribute to Ab features, a fact that should be considered in engineering therapeutics.

Antibodies doi: 10.3390/antib14020049

Authors: Ahmad Matarneh Meet Patel Kinna Parikh Amanda Karasinski Gurwant Kaur Vaqar Shah Nasrollah Ghahramani Naman Trivedi

The long-term use of immunosuppressive drugs following kidney transplantation increases the risk of life-threatening infections, malignancies, and, paradoxically, eventual allograft rejection. Therefore, achieving a balance between over-immunosuppression and under-immunosuppression is critical to optimizing patient outcomes. One promising approach is immune cell-based therapy using suppressor immune cells to modulate the immune response more precisely. Among these, regulatory T cells (Tregs) are the most extensively studied and have shown significant potential in the post-transplant setting. Tregs are broadly categorized into thymus-derived and peripherally derived subsets. Physiologically, they play key roles in maintaining immune tolerance, including in autoimmune diseases and within the tumor microenvironment. Their immunosuppressive functions are mediated through both contact-dependent and contact-independent mechanisms. Studies investigating the use of Tregs following kidney transplantation have shown encouraging results. This review summarizes the biology of Tregs and highlights current evidence supporting their role in transplant immunotherapy.

Antibodies doi: 10.3390/antib14020048

Authors: Yating Li Kexuan Cheng Jiazheng Guo Yujia Jiang Qinglin Kang Rong Wang Peng Du Chen Gao Yunzhou Yu Zhixin Yang Wei Wang Jiansheng Lu

Background: Tetanus toxin, produced by Clostridium tetani, is the second deadliest known toxin. Antibodies capable of neutralizing tetanus toxin (TeNT) are vital for preventing and treating tetanus disease. Methods: Herein, we screened thirty-six single variable domains on a heavy chain (VHHs) binding to the light chain (L) and the translocation domain (HN) (L-HN) fragment of TeNT from a phage-display library. Then, the L-HN-specific clones were identified, humanized, and fused with a human fragment crystallizable region (hFc) to form humanized VHH-hFc fusion proteins. Results: The humanized VHH-hFc fusion proteins TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc possessed potent efficacy with high binding affinity, specificity, and neutralizing activity. Only 0.3125 μg was required for TL-16-h1-hFc or TL-25-h1-hFc, and 0.625 μg was required for TL-34-h1-hFc to provide full protection against 10 × Lethal Dose 50 (LD50) TeNT. In the prophylactic setting, 125 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc provided full protection even when they were injected 12 days before exposure to 10 × LD50 TeNT, while TL-34-h1-hFc was less effective. In the therapeutic setting, 25 μg/kg of TL-16-h1-hFc or TL-25-h1-hFc could provide complete protection when administered 24 h after exposure to 5 × LD50 TeNT, while TL-34-h1-hFc required 50 μg/kg. Conclusion: Our results suggest that TL-16-h1-hFc, TL-25-h1-hFc, and TL-34-h1-hFc provide a bright future for the development of anti-TeNT preventive or therapeutic drugs.

Antibodies doi: 10.3390/antib14020047

Authors: Martina Canichella Paolo de Fabritiis

Post-transplant lymphoproliferative disorders (PTLD) represent a life-threatening complication following solid organ transplantation (SOT) and allogeneic hematopoietic stem cell transplantation (allo-HSCT), particularly in patients with relapsed or refractory (R/R) disease, where therapeutic options are limited and prognosis is poor. Among emerging strategies, adoptive cellular immunotherapy—specifically Epstein–Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs)—significantly improved outcomes in this challenging patient population. EBV-CTLs restore virus-specific immunity and induce sustained remissions with minimal toxicity, even in heavily pretreated individuals. The most promising cellular product to date is tabelecleucel, an off-the-shelf, allogeneic EBV-specific T-cell therapy, which is currently the only cellular therapy approved by the European Medicines Agency (EMA) for the treatment of R/R EBV-positive PTLD following SOT or allo-HSCT. This review aims to provide an overview of PTLD treatment with a specific focus on adoptive cellular immunotherapy. We highlight the most robust clinical outcomes reported with EBV-CTLs, particularly those achieved with tabelecleucel, and explore emerging cellular approaches such as CAR T-cell therapy, which may further broaden therapeutic strategies in the near future.

Antibodies doi: 10.3390/antib14020046

Authors: Salvatrice Ciccarese Marie-Paule Lefranc Giulia C. M. Perrone Pietro D’Addabbo Ciro Leonardo Pierri

Background: In the adaptive immune response of the dromedary (Camelus dromedarius, Camdro), the T cell receptor (TR) repertoire of the gamma–delta (γδ) T cells is unusually diversified both by somatic hypermutation in rearranged TR gamma (TRG) and delta (TRD) genes and by the diversity in sequence and length of the third complementarity-determining region (CDR3) of the TRD chain. Methods: The purpose was to investigate, in the absence of 3D structures, the role of Camdro γδ T cells, focusing on the binding interactions at the interface between the V-gamma and V-delta domains, and in complex with the CD1D, a major histocompatibily class I (MH1)-like glycoprotein presenting lipid antigen in association with B2M. A combination of hypermutated TRG dromedary cDNA clones was paired with TRD clones bearing very long, long, or short CDR3s, all isolated from the spleen of a single animal. Results: The 3D models of the Camdro TRG_TRD/CD1D_B2M complexes were inferred using the Homo sapiens 3D structure and the ImMunoGeneTics (IMGT) numbering for V, C, and G domains, and investigated for binding interactions at the interface of the paired V-gamma_V-delta and at the interface with CD1D. Our results suggest that transcripts with long CDR3s may derive from a population of CD1D-restricted γδ T cells. Both the CD1D G-alpha1-like and G-alpha-2 like domain helices were contacted by both the V-gamma and V-delta CDR-IMGT loops. Conclusions: Our findings further emphasize the similarity between the γδ T cells population we analyzed in Camelus dromedarius and the CD1D-restricted γδ NKT cells in Homo sapiens.

Antibodies doi: 10.3390/antib14020045

Authors: Alexandra Rak Ekaterina Bazhenova Polina Prokopenko Victoria Matyushenko Yana Orshanskaya Konstantin V. Sivak Arina Kostromitina Larisa Rudenko Irina Isakova-Sivak

Cases of new COVID-19 infection, which manifested in 2019 and caused a global socioeconomic crisis, still continue to be registered worldwide. The high mutational activity of SARS-CoV-2 leads to the emergence of new antigenic variants of the virus, which significantly reduces the effectiveness of COVID-19 vaccines, as well as the sensitivity of diagnostic test systems based on variable viral antigens. These problems may be solved by focusing on highly conserved coronavirus antigens, for example nucleocapsid (N) protein, which is actively expressed by coronavirus-infected cells and serves as a target for the production of virus-specific antibodies and T cell responses. It is known that anti-N antibodies are non-neutralizing, but their protective potential and functional activity are not sufficiently studied. Here, the protective effect of anti-N antibodies was studied in Syrian hamsters passively immunized with polyclonal sera raised to N(B.1) recombinant protein. The animals were infected with 105 or 104 TCID50 of SARS-CoV-2 (B.1, Wuhan or BA.2.86.1.1.18, Omicron) 6 h after serum passive transfer, and protection was assessed by weight loss, clinical manifestation of disease, viral titers in the respiratory tract, as well as by the histopathological evaluation of lung tissues. The functional activity of anti-N(B.1) antibodies was evaluated by complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC) assays. The protection of anti-N antibodies was evident only against a lower dose of SARS-CoV-2 (B.1) challenge, whereas almost no protection was revealed against BA.2.86.1.1.18 variant. Anti-N(B.1) monoclonal antibodies were able to stimulate both CDC and ADCC. Thus, anti-N(B.1) antibodies possess protective activity against homologous challenge infection, which is possibly mediated by innate Fc-mediated immune reactions. These data may be informative for the development of N-based broadly protective COVID-19 vaccines.

Antibodies doi: 10.3390/antib14020044

Authors: Karolina Sołowińska Lucyna Holec-Gąsior

Accurate dating of Toxoplasma gondii infection is essential for effective clinical management, particularly in pregnant women and immunocompromised individuals, where distinguishing acute from chronic infection informs treatment decisions. Serological detection of IgM antibodies is a key tool in diagnosing recent toxoplasmosis; however, its reliability is compromised by persistent IgM responses, cross-reactivity, and assay variability. While IgM lacks sufficient specificity to serve as a standalone marker of acute infection, it remains an important component of serological panels. This review summarizes current IgM detection methods and explores advancements aimed at improving diagnostic accuracy with a focus on recombinant antigens, which have emerged as promising alternatives to traditional Toxoplasma lysate antigen-based immunoassays. This paper also explores alternative methods of differentiating chronic and acute toxoplasmosis and outlines key areas for future research.

Antibodies doi: 10.3390/antib14020043

Authors: Samuel M. Ailsworth Matthew MacCallum Nathan E. Richards Lisa J. Workman Pamela Schoppee Bortz Thomas Makin Thomas A. E. Platts-Mills Jeffrey M. Wilson

Background: Antibodies to galactose-alpha-1,3-galactose (alpha-gal), particularly the IgM and IgG isotypes, are abundant in human sera. These antibodies are known to be an important xenotransplantation barrier, but the full implications of these antibodies to health and disease remain incompletely understood. By contrast, IgE to alpha-gal is uncommon in the population but has been associated with tick bites and causally linked with mammalian meat allergy, often now known as alpha-gal syndrome (AGS). To date, there have been few population-based studies that have investigated alpha-gal IgG levels in relation to demographic factors, diet, tick bites, and mammalian meat allergy. Methods: Adults, predominantly healthcare workers, were recruited for a COVID-19 vaccine study. At least one serum sample was collected, and subjects completed questionnaires to provide demographic, diet, and tick exposure data. Alpha-gal IgG, IgE, and total IgG were measured using the ImmunoCAP platform, and blood group was assessed via reverse typing using stored serum. We also assessed alpha-gal IgG levels among subjects with AGS, recruited from an allergy clinic. Results: The median age of the 267 subjects in the vaccine cohort was 42 years, and median alpha-gal IgG levels were 3.0 μg/mL. Alpha-gal IgG levels were higher among the 43 (16.1%) subjects who had alpha-gal IgE sensitization (≥0.1 IU/mL) and among subjects lacking the B blood group antigen (blood groups A and O). Alpha-gal IgG levels did not differ between the subjects who had asymptomatic alpha-gal IgE sensitization and those who had meat allergy. However, both groups had higher alpha-gal IgG levels than subjects who lacked alpha-gal IgE sensitization. Subjects who reported prior tick or chigger bites had higher alpha-gal IgG levels than those without a bite history, regardless of alpha-gal IgE sensitization status. Conclusions: In a population-based cohort, alpha-gal IgG antibodies were found to be prevalent, and levels were increased in subjects with blood groups A and O, subjects who were alpha-gal IgE sensitized, and those who reported a history of tick bites.