Antibodies

20 pages, 310 KB

Open AccessReview

Post-Chemotherapy Antibody-Based Continuation and Maintenance Strategies in HER2-Positive Metastatic Breast Cancer: A Translational Narrative Review

by Katarzyna Pogoda, Karolina Lewińska, Paulina Kalman, Anna Bałata and Piotr J. Wysocki

Antibodies 2026, 15(2), 36; https://doi.org/10.3390/antib15020036 - 16 Apr 2026

Abstract

The treatment paradigm for HER2-positive metastatic breast cancer has evolved from continuous chemotherapy-based regimens to a model of finite chemotherapy induction followed by sustained antibody-driven disease control. The CLEOPATRA trial established dual HER2 blockade with trastuzumab and pertuzumab plus a taxane as the [...] Read more.

The treatment paradigm for HER2-positive metastatic breast cancer has evolved from continuous chemotherapy-based regimens to a model of finite chemotherapy induction followed by sustained antibody-driven disease control. The CLEOPATRA trial established dual HER2 blockade with trastuzumab and pertuzumab plus a taxane as the biological and clinical anchor of this approach, demonstrating that chemotherapy is administered for a defined induction period, after which antibody maintains disease suppression. An increasing body of clinical evidence indicates that antibody-based regimens can be combined with targeted agents, including CDK4/6 inhibitors or HER2 tyrosine kinase inhibitors, to achieve durable disease control without the need for continuous chemotherapy. In the PATINA trial, the addition of palbociclib to trastuzumab, pertuzumab, and endocrine therapy was associated with a clinically meaningful improvement in progression-free survival in hormone receptor-positive, HER2-positive metastatic breast cancer. At the same time, quality of life was maintained despite higher rates of hematologic toxicity. More recently, HER2-CLIMB-05 demonstrated that the addition of tucatinib to dual HER2 antibody therapy significantly prolonged progression-free survival, supporting a model of sustained, multi-agent HER2 pathway suppression. The monarcHER trial provided biological proof of concept that antibody plus CDK4/6 inhibition can achieve disease control without chemotherapy in hormone receptor-positive, HER2-positive disease. Collectively, these advances support a translational framework in which antibody therapy serves as a central component of treatment strategies, with targeted partners selected according to tumor biology and prior therapy. This review summarizes the biological basis, clinical evidence, and future perspectives of antibody-driven maintenance in HER2-positive metastatic breast cancer. Full article

(This article belongs to the Section Antibody-Based Therapeutics)

► Show Figures

Graphical abstract

59 pages, 6332 KB

Open AccessReview

IMGT^® Nomenclature of Immunoglobulins (IG) or Antibodies and T Cell Receptors (TR): A Common Language for Immunoinformatics and Artificial Intelligence (AI)

by Marie-Paule Lefranc and Gérard Lefranc

Antibodies 2026, 15(2), 35; https://doi.org/10.3390/antib15020035 - 15 Apr 2026

Abstract

The immunoglobulins (IG) or antibodies and the T cell receptors (TR) are the antigen receptors of the adaptive immune responses (AIR) of jawed vertebrates (Gnathostomata). IMGT^®, the international ImMunoGeneTics information system^®, was created in 1989 by Marie-Paule [...] Read more.

The immunoglobulins (IG) or antibodies and the T cell receptors (TR) are the antigen receptors of the adaptive immune responses (AIR) of jawed vertebrates (Gnathostomata). IMGT^®, the international ImMunoGeneTics information system^®, was created in 1989 by Marie-Paule Lefranc (Laboratoire d’ImmunoGénétique Moléculaire (LIGM), Université de Montpellier and CNRS) to deal with and to manage the huge diversity of IG or antibodies and TR. The founding of IMGT^® marked the advent of immunoinformatics, a new science which emerged at the interface between immunogenetics and bioinformatics. For the first time, the IG and TR variable (V), diversity (D), joining (J) and constant (C) genes were officially recognized as ‘genes’, as were the conventional genes. The IMGT-ONTOLOGY CLASSIFICATION axiom and the concepts of classification have generated the IMGT nomenclature and the IMGT Scientific chart rules for assigning IMGT names to IG and TR genes and alleles of Homo sapiens and of any other jawed vertebrate species. The IMGT nomenclature is used for genes in locus, in sequences (genomic or rearranged, expressed or not) and in structures enabling comparative immunology, evolutionary immunogenetics, standardized analysis and comparison of IG and TR repertoires analysis in normal or pathologic situations. IMGT nomenclature is used in basic, veterinary, and medical research, in clinical applications (mutation analysis in leukemia and lymphoma), and in therapeutic antibody design, engineering and humanization. By providing consistent and high standard biocuration for the description of the IG and TR loci, genes and alleles, and for the analysis of the IG or antibody and TR-expressed rearranged sequences and proteins and structures, the IMGT nomenclature is the common language for immunoinformatics and artificial intelligence (AI). Full article

(This article belongs to the Section Antibody Discovery and Engineering)

► Show Figures

Graphical abstract

15 pages, 12077 KB

Open AccessArticle

Development of a Human IgG1 Monoclonal Antibody Targeting Transferrin Receptor 1 for Antitumor Drug Delivery

by Tingting Ji, Zhaoyun Zong, Ningyuan Gong, Minghui Yan and Shiyu Chen

Antibodies 2026, 15(2), 34; https://doi.org/10.3390/antib15020034 - 13 Apr 2026

Abstract

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) [...] Read more.

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 (K_D = 214 ± 1 nM), which was substantially enhanced following conversion to the IgG1 format (T8IgG1, K_D = 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. Full article

(This article belongs to the Topic Antibody-Mediated Therapy and Other Emerging Therapies in Cancer Treatment)

► Show Figures

Graphical abstract

12 pages, 992 KB

Open AccessReview

Non-COVID-19 Vaccinations and the Induction of Autoantibodies in Pemphigus Diseases: A Review of the Speculative Issue and Our Clinical-Laboratory Experience

by Maksymilian Markwitz, Natalia Welc, Klementyna Kępińska, Monika Bowszyc-Dmochowska and Marian Dmochowski

Antibodies 2026, 15(2), 33; https://doi.org/10.3390/antib15020033 - 10 Apr 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Humoral Immunity)

► Show Figures

Figure 1

12 pages, 1198 KB

Open AccessCase Report

Monoclonal Antibodies in Pregnancy of Patients with Systemic Lupus Erythematosus: Friend or Foe? A Case Report of a Patient with Multiple Pregnancies

by 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 and Ilaria Cavazzana

Antibodies 2026, 15(2), 32; https://doi.org/10.3390/antib15020032 - 8 Apr 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Topic Antibody-Mediated Therapy and Other Emerging Therapies in Cancer Treatment)

► Show Figures

Figure 1

11 pages, 389 KB

Open AccessReview

The Possible Role of Antibodies in Alopecia: A Narrative Review

by Julia Cieślawska, Mariola Pawlaczyk and Justyna Gornowicz-Porowska

Antibodies 2026, 15(2), 31; https://doi.org/10.3390/antib15020031 - 3 Apr 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody-Based Therapeutics)

► Show Figures

Figure 1

14 pages, 737 KB

Open AccessArticle

SARS-CoV-2 Infection and COVID-19 Vaccine Antibody Responses in Two Canadian Cohorts of Persons Living with HIV

by 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 and Marina B. Klein

Antibodies 2026, 15(2), 30; https://doi.org/10.3390/antib15020030 - 3 Apr 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Antiviral Antibody Immune Responses in the Context of Vaccination and Infection)

► Show Figures

Graphical abstract

36 pages, 9313 KB

Open AccessArticle

Development of Bispecific Antibody Targeting Human IL-17A and IL-6

by 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 and Olga Abramczyk

Antibodies 2026, 15(2), 29; https://doi.org/10.3390/antib15020029 - 30 Mar 2026

Abstract

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, [...] Read more.

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. Full article

(This article belongs to the Section Antibody Discovery and Engineering)

► Show Figures

Figure 1

13 pages, 529 KB

Open AccessReview

Dynamics of 1,3-β-D-Glucan in Invasive Candidiasis: A Narrative Review of Microbiological Aspects and Diagnostic Implications

by Maddalena Calvo, Marta Caccamo, Dalila Maria Cammarata and Laura Trovato

Antibodies 2026, 15(2), 28; https://doi.org/10.3390/antib15020028 - 27 Mar 2026

Abstract

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 [...] Read more.

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. Full article

► Show Figures

Figure 1

19 pages, 2063 KB

Open AccessArticle

The Effect of FcRn Binding on Ocular Disposition of Monoclonal Antibodies

by Sanika Naware, Saurav Kulkarni, Sahil Salvi, Dhvani Patel and Dhaval K. Shah

Antibodies 2026, 15(2), 27; https://doi.org/10.3390/antib15020027 - 25 Mar 2026

Abstract

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 [...] Read more.

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 (ABC_0–168h: 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. Full article

(This article belongs to the Section Antibody-Based Therapeutics)

► Show Figures

Graphical abstract

20 pages, 1930 KB

Open AccessArticle

The Multi-Attribute Method (MAM), An Advanced LC-MS Approach for Protein A Resin Performance and Lifecycle Evaluation

by Jingming Zhang, Matthew Larsen, Timothy Blanc, Babita S. Parekh and Ming-Ching Hsieh

Antibodies 2026, 15(2), 26; https://doi.org/10.3390/antib15020026 - 23 Mar 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody-Based Therapeutics)

► Show Figures

Graphical abstract

15 pages, 3136 KB

Open AccessArticle

Immunogenetic Architecture of Chronic Lymphocytic Leukemia at Early Stage: Insights from the O-CLL1 Cohort

by 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 and Franco Fais

Antibodies 2026, 15(2), 25; https://doi.org/10.3390/antib15020025 - 18 Mar 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody-Based Diagnostics)

► Show Figures

Graphical abstract

12 pages, 341 KB

Open AccessReview

Dermatomyositis with Anti-MDA5 Autoantibodies After SARS-CoV-2 mRNA Vaccination Treated with Tofacitinib: Integrating Literature Evidence and a Novel Observation

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

Antibodies 2026, 15(2), 24; https://doi.org/10.3390/antib15020024 - 9 Mar 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Humoral Immunity)

► Show Figures

Figure 1

11 pages, 736 KB

Open AccessArticle

Evaluation of Anti-dsDNA Antibodies in Laboratory Practice: Management of Different Analytical Methods and Correlation with HEp-2 Immunofluorescence Patterns

by Massimo Papale, Carmela Paolillo, Loredana Iafelice, Tiziana Trivisano, Giuseppe Stefano Netti, Elena Ranieri and Gaetano Corso

Antibodies 2026, 15(2), 23; https://doi.org/10.3390/antib15020023 - 5 Mar 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody-Based Diagnostics)

► Show Figures

Figure 1

21 pages, 4034 KB

Open AccessArticle

Developability Evaluation of Single-Domain Antibody-Chelator Conjugates for Diagnostic Radiotracers

by Philipp D. Kaiser, Simon Straß, Sandra Maier, Evgenia Herbold, Bjoern Traenkle and Anne Zeck

Antibodies 2026, 15(2), 22; https://doi.org/10.3390/antib15020022 - 3 Mar 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Therapeutic Antibodies: New Trends in Discovery, Developability and Characterization)

► Show Figures

Graphical abstract

18 pages, 2066 KB

Open AccessArticle

Two Highly Specific Mouse Monoclonal Antibodies to the Putative C-Telopeptide of Human Collagen XIα1, a Cancer Biomarker

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

Antibodies 2026, 15(2), 21; https://doi.org/10.3390/antib15020021 - 25 Feb 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody Discovery and Engineering)

► Show Figures

Graphical abstract

13 pages, 1338 KB

Open AccessArticle

Prevalence of Viral Hepatitis Antibodies Among Alcoholics in Croatia: A Single Center’s Results

by 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 and Tatjana Vilibić-Čavlek

Antibodies 2026, 15(2), 20; https://doi.org/10.3390/antib15020020 - 25 Feb 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Humoral Immunity)

► Show Figures

Figure 1

13 pages, 1539 KB

Open AccessArticle

Camelid-Derived Nanobodies Targeting Human Epidermal Growth Factor Receptor: Screening, Expression, and Functional Validation

by Yunfeng Liu, Qiting Huang, Dongna Zhang, Yingjun Wang, Shuaiying Zhao, Jianchuan Wen, Yingying Kong and Jianfeng Xu

Antibodies 2026, 15(2), 19; https://doi.org/10.3390/antib15020019 - 24 Feb 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Topic Antibody-Mediated Therapy and Other Emerging Therapies in Cancer Treatment)

► Show Figures

Figure 1

16 pages, 5250 KB

Open AccessArticle

Discovery of Anti-SARS-CoV-2 XBB.1.5 and JN.1 Variant-Specific Monoclonal Single-Domain Antibodies from a Synthetic Library

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

Antibodies 2026, 15(2), 18; https://doi.org/10.3390/antib15020018 - 24 Feb 2026

Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Antibody Discovery and Engineering)

► Show Figures

Graphical abstract

Journal Description

Antibodies

Latest Articles

Journal Menu

Journal Browser

Highly Accessed Articles

Latest Books

E-Mail Alert

News

Topics

Conferences

Special Issues

Topical Collections

Further Information

Guidelines

MDPI Initiatives

Follow MDPI