Antibodies

20 pages, 2671 KiB

Open AccessArticle

Three-Dimensional Modeling of Camelus dromedarius T Cell Receptor Gamma (TRG)_Delta (TRD)/CD1D Complex Reveals Different Binding Interactions Depending on the TRD CDR3 Length

by Salvatrice Ciccarese, Marie-Paule Lefranc, Giulia C. M. Perrone, Pietro D’Addabbo and Ciro Leonardo Pierri

Antibodies 2025, 14(2), 46; https://doi.org/10.3390/antib14020046 - 29 May 2025

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Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Recombinant Binding Proteins and Genetically Engineered T-cells Targeting Intracellular Neoantigens)

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18 pages, 8713 KiB

Open AccessArticle

Protective Potential and Functional Role of Antibodies Against SARS-CoV-2 Nucleocapsid Protein

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

Antibodies 2025, 14(2), 45; https://doi.org/10.3390/antib14020045 - 28 May 2025

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Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Humoral Immunity)

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20 pages, 399 KiB

Open AccessReview

IgM Antibody Detection as a Diagnostic Marker for Acute Toxoplasmosis: Current Status of Studies and Main Limitations

by Karolina Sołowińska and Lucyna Holec-Gąsior

Antibodies 2025, 14(2), 44; https://doi.org/10.3390/antib14020044 - 21 May 2025

Viewed by 379

Abstract

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 [...] Read more.

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. Full article

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15 pages, 2551 KiB

Open AccessArticle

IgG to Galactose-Alpha-1,3-Galactose: Impact of Alpha-Gal IgE Sensitization, Blood Type, and Tick Bites

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

Antibodies 2025, 14(2), 43; https://doi.org/10.3390/antib14020043 - 16 May 2025

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Abstract

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 [...] Read more.

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. Full article

(This article belongs to the Section Humoral Immunity)

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16 pages, 1990 KiB

Open AccessArticle

Neutralization of the Pandemic Influenza A/H1N1 Virus with Lama glama Humanized Nanobodies (VHH)

by Zeila Yazmín Páez-Hernández, Jose Luis Stephano-Hornedo, Jose Alberto Bolaños-Prats, Iván Córdova-Guerrero, Mariana Macías-Alonso, Joaquín G. Marrero, Angel Pulido Capiz and Victor García González

Antibodies 2025, 14(2), 42; https://doi.org/10.3390/antib14020042 - 16 May 2025

Viewed by 356

Abstract

Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant [...] Read more.

Background/Objetives: Nanobodies (VHH) have become an excellent tool for diagnosis, therapy, and research since VHH shows a high capability of recognizing and neutralizing antigens. VHHs are highly soluble and stable at high temperatures, and in the presence of chaotropic agents, they offer significant advantages over other biological therapeutic agents. This study aimed to identify and humanize VHH fragments with neutralizing potential against the influenza A/H1N1 virus. Methods: A library of VHH antibody fragments was produced by phage display technique against an inactivated influenza A/H1N1 vaccine. Three VHH sequences were selected and humanized. Specifically, the recognition capacity of the antibodies denominated 2-C10 and 2-C10H was confirmed by ELISA and western blot (WB), as well as their microneutralization capacity in a cellular model, suggesting their potential therapeutic use in patients infected with the influenza A/H1N1 virus. Molecular docking assays were used to support the mechanism of viral inhibition. Results: The VHHs 2-C10 and 2-C10H showed specific recognition of influenza A/H1N1 antigens by ELISA and Western Blot and demonstrated neutralizing activity in vitro. The optimal VHH, 2-C10H, showed 75% neutralization capacity at a concentration of 1.56 μg/mL against the A/H1N1 viral strain, potentially through the inactivation of hemagglutinin protein, a phenomenon supported by molecular docking assays. Conclusions: This study presents a strategic approach to identify VHH candidates that may be useful for diagnosing and potentially treating patients already infected by the A/H1N1 virus, as it may reduce the severity of their symptoms. Full article

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13 pages, 1960 KiB

Open AccessArticle

Generative Deep Learning Design of Single-Domain Antibodies Against Venezuelan Equine Encephalitis Virus

by Jinny L. Liu, Gabrielle C. Bayacal, Jerome Anthony E. Alvarez, Lisa C. Shriver-Lake, Ellen R. Goldman and Scott N. Dean

Antibodies 2025, 14(2), 41; https://doi.org/10.3390/antib14020041 - 14 May 2025

Viewed by 441

Abstract

Background/Objectives: Venezuelan equine encephalitis virus (VEEV) represents a significant biothreat with no FDA-approved vaccine currently available, highlighting the need for alternative therapeutic strategies. Single-domain antibodies (sdAbs) present a potential alternative to conventional antibodies, due to their small size and ability to recognize cryptic [...] Read more.

Background/Objectives: Venezuelan equine encephalitis virus (VEEV) represents a significant biothreat with no FDA-approved vaccine currently available, highlighting the need for alternative therapeutic strategies. Single-domain antibodies (sdAbs) present a potential alternative to conventional antibodies, due to their small size and ability to recognize cryptic epitopes. Methods: This research describes the development and preliminary evaluation of VEEV-binding sdAbs generated using a generative artificial intelligence (AI) platform. Using a dataset of known alphavirus-binding sdAbs, the AI model produced sequences with predicted affinity for the E2 glycoprotein of VEEV. These candidate sdAbs were expressed in a bacterial periplasmic system and purified for initial assessment. Results: Enzyme-linked immunosorbent assays (ELISAs) indicated binding activity of the sdAbs to VEEV antigens. In vitro neutralization tests suggested inhibition of VEEV infection in cultured cells for some of the candidates. Conclusions: This study demonstrates how generative AI can expedite antiviral therapeutic development and establishes a framework for quick responses to emerging viral threats when extensive example databases are unavailable. Additional refinement and validation of AI-generated sdAbs could establish effective VEEV therapeutics. Full article

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

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14 pages, 2399 KiB

Open AccessArticle

Purification of Human Immunoglobulin G with Bathophenanthroline–Zn²⁺, –Fe²⁺, or –Cu²⁺ Complexes

by Thisara Jayawickrama Withanage, Ron Alcalay, Olga Krichevsky, Ellen Wachtel, Ohad Mazor and Guy Patchornik

Antibodies 2025, 14(2), 40; https://doi.org/10.3390/antib14020040 - 12 May 2025

Viewed by 361

Abstract

Background/Objectives: Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic-grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human [...] Read more.

Background/Objectives: Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic-grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG), which does not require any expensive ligands, chromatography columns, polymers, or membranes. Methods/Results: Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5–15 mg/mL) in the supernatant. [(Batho)₃:Zn²⁺] complexes were the most promising, resulting in hIgG with a purity of ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves the native secondary structure (by far UV circular dichroism spectroscopy, CD). The process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume, which required only proportional increases in reagents. Conclusions: Although Protein A chromatographic columns, the industry gold standard, have a limited binding capacity, are costly, and require familiarity with column maintenance, we are attempting, by our efforts, to help to produce a more efficient, simple, and economical purification platform. Full article

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

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18 pages, 4678 KiB

Open AccessArticle

Validation and Optimization of PURE Ribosome Display for Screening Synthetic Nanobody Libraries

by Bingying Liu and Daiwen Yang

Antibodies 2025, 14(2), 39; https://doi.org/10.3390/antib14020039 - 2 May 2025

Viewed by 548

Abstract

Background/Objectives: PURE (Protein synthesis Using Recombinant Elements), an ideal system for ribosome display, has been successfully used for nanobody selection. However, its limitations in nanobody selection, especially for synthetic nanobody libraries, have not been clearly elucidated, thereby restricting its utilization. Methods: The PURE [...] Read more.

Background/Objectives: PURE (Protein synthesis Using Recombinant Elements), an ideal system for ribosome display, has been successfully used for nanobody selection. However, its limitations in nanobody selection, especially for synthetic nanobody libraries, have not been clearly elucidated, thereby restricting its utilization. Methods: The PURE ribosome display selection process was closely monitored using RNA agarose gel electrophoresis to assess the presence of mRNA molecules in each fraction, including the flow-through, washing, and elution fractions. Additionally, a real-time validation method for monitoring each biopanning round was implemented, ensuring the successful enrichment of target protein-specific binders. The selection process was further optimized by introducing a target protein elution step prior to the EDTA-mediated disassembly, as well as by altering the immobilization surfaces. Finally, the efficiency of PURE ribosome display was enhanced by replacing the spacer gene. Results: The efficiency of PURE ribosome display was merely 4% with an unfavourable spacer gene. Using this spacer gene, EGFP- and human fatty acid-binding protein 4-specific nanobodies from a synthetic nanobody library were we successfully identified through optimizing the selection process. Choosing a spacer gene less prone to secondary structure formation increased significantly its efficiency in displaying synthetic nanobody libraries. Conclusions: Implementing a target protein elution step prior to EDTA-mediated disassembly and modifying the immobilization surfaces effectively increase selection efficiency. For PURE ribosome display, efficiency was further improved using a suitable spacer gene, enabling the display of large libraries. Full article

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24 pages, 4948 KiB

Open AccessArticle

A Targeted Integration-Based CHO Cell Platform for Simultaneous Antibody Display and Secretion

by Jessica P. Z. Ng, Mariati Mariati, Jiawu Bi, Matthew Wook Chang and Yuansheng Yang

Antibodies 2025, 14(2), 38; https://doi.org/10.3390/antib14020038 - 28 Apr 2025

Viewed by 564

Abstract

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes. Methods: The platform consists of a CHO master cell line with [...] Read more.

Objective: We developed a targeted integration-based CHO cell platform for simultaneous antibody display and secretion, enabling a streamlined transition from antibody library screening to production without requiring the re-cloning of antibody genes. Methods: The platform consists of a CHO master cell line with a single-copy landing pad, a helper vector expressing FLPe recombinase, and bi-functional targeting vectors. Recombinase-mediated cassette exchange was utilized to integrate targeting vectors into the landing pad. Bi-functional vectors were designed by incorporating a minimal furin cleavage sequence (mFCS), RRKR, and various 2A peptides between the heavy chain (HC) and a membrane anchor. Results: Incomplete cleavage at the mFCS and 2A sites facilitated the expression of both membrane-bound and secreted antibodies, while mutations in the 2A peptide produced a range of display-to-secretion ratios. However, a fraction of secreted antibodies retained 2A residues attached to the HC polypeptides. Further analysis demonstrated that modifying the first five amino acids of the 2A peptide significantly influenced furin cleavage efficiency, resulting in different display-to-secretion ratios for targeting vectors containing mFCS-2A variant combinations. To overcome this, we designed nine-amino-acid FCS variants that, when placed between the HC and membrane anchor, provided a range of display-to-secretion ratios and eliminated the issue of attached 2A residues in the secreted antibodies. Vectors with lower display levels proved more effective at distinguishing cells expressing high-affinity antibodies with closely matched binding affinities. The platform also demonstrated high sensitivity in isolating high-affinity antibody-expressing cells and supported robust antibody production. Conclusion: This targeted integration-based CHO platform enables efficient, in-format screening and production of antibodies with tunable display-to-secretion profiles. It provides a powerful and scalable tool for accelerating the development of functional, manufacturable therapeutic antibodies. Full article

(This article belongs to the Special Issue Emerging Antibody Engineering Strategies and Applications for Immunotherapy of Cancer)

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19 pages, 1878 KiB

Open AccessReview

The Role of Monoclonal Antibodies as Therapeutics in HPV-Related Head and Neck Cancers: An Updated Review

by Michael Zalin, Shaan Patel, Carter Coggins and Vikrant Rai

Antibodies 2025, 14(2), 37; https://doi.org/10.3390/antib14020037 - 24 Apr 2025

Viewed by 700

Abstract

Background/Objectives: The increasing prevalence of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has necessitated a revaluation of therapeutic strategies. HPV-driven OPSCC differs from HPV-negative OPSCC due to its distinct molecular signatures, increased radiosensitivity, and better prognoses. However, despite these differences, treatment strategies [...] Read more.

Background/Objectives: The increasing prevalence of human papillomavirus (HPV)-positive oropharyngeal squamous cell carcinoma (OPSCC) has necessitated a revaluation of therapeutic strategies. HPV-driven OPSCC differs from HPV-negative OPSCC due to its distinct molecular signatures, increased radiosensitivity, and better prognoses. However, despite these differences, treatment strategies have remained largely uniform, resulting in minimal reductions in morbidity and exposing HPV-positive patients to unnecessary toxicity. Monoclonal antibodies (mAbs) have become a promising therapeutic option due to their ability to target treatment with fewer systemic side effects. Immune checkpoint inhibitors (ICIs) such as pembrolizumab have shown efficacy in enhancing the immune response against tumors, while EGFR inhibitors like cetuximab offer an alternative modality. Current clinical trials aim to refine dosing regimens and identify combination strategies that may enhance therapeutic outcomes. Results: Despite promising evidence, several challenges hinder the widespread adoption of mAbs as a standard treatment for HPV-positive OPSCC in clinical practice. This review examines the current role of mAbs in HPV-positive OPSCC treatment, highlighting their limitations and future research directions. Conclusions: Further studies are needed to optimize patient selection, establish standardized treatment protocols, and investigate the long-term benefits of mAb-based therapies in this patient population. Full article

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15 pages, 2241 KiB

Open AccessArticle

Potentiating Antibody-Dependent Cellular Cytotoxicity in Triple-Negative Breast Cancer via the Humanized Anti-CD147 Antibody

by Kanyarat Thongheang, Thanathat Pamonsupornwichit, Kanokporn Sornsuwan, On-anong Juntit, Tawan Chokepaichitkool, Weeraya Thongkum, Umpa Yasamut and Chatchai Tayapiwatana

Antibodies 2025, 14(2), 36; https://doi.org/10.3390/antib14020036 - 11 Apr 2025

Viewed by 560

Abstract

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with high metastatic potential, poor prognosis, and the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). The lack of these receptors limits the standard treatments, such as hormone [...] Read more.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with high metastatic potential, poor prognosis, and the absence of estrogen receptors, progesterone receptors, and human epidermal growth factor receptor 2 (HER2). The lack of these receptors limits the standard treatments, such as hormone therapies and HER2-targeted antibodies like trastuzumab. These challenges highlight the critical need for novel therapeutic strategies. CD147, a transmembrane glycoprotein overexpressed in TNBC, promotes tumor progression, metastasis, and chemoresistance, making it a promising therapeutic target. This study evaluates the antibody-dependent cellular cytotoxicity (ADCC) of HuM6-1B9, a humanized anti-CD147 antibody, against MDA-MB-231 cells, a TNBC model. Methods: CFSE-labelled MDA-MB-231 cells were co-cultured with PBMCs as effector cells (E:T ratio 80:1) in the presence of HuM6-1B9 and incubated for 4 h. Cells were then collected and stained with PI, and CFSE+/PI+ dead target cells were analyzed by flow cytometry. Results: Co-culturing MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) in the presence of HuM6-1B9 demonstrated effective ADCC induction without direct cytotoxicity. HuM6-1B9 induced 54.01% cancer cell death via ADCC, significantly outperforming trastuzumab (26.14%) while sparing PBMCs. Conclusion: These findings support HuM6-1B9 as a prospective TNBC therapeutic and warrant further investigation into its clinical potential. Full article

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25 pages, 3552 KiB

Open AccessReview

A Comprehensive Review About the Use of Monoclonal Antibodies in Cancer Therapy

by Angel Justiz-Vaillant, Bijay Raj Pandit, Chandrashekhar Unakal, Sehlule Vuma and Patrick Eberechi Akpaka

Antibodies 2025, 14(2), 35; https://doi.org/10.3390/antib14020035 - 11 Apr 2025

Viewed by 2303

Abstract

Monoclonal antibodies (mAbs) targeting various pathways in cancer therapy play crucial roles in enhancing the immune system’s ability to recognise and eliminate tumour cells. These therapies are designed to either block inhibitory immune checkpoint pathways or to target specific tumour cell markers for [...] Read more.

Monoclonal antibodies (mAbs) targeting various pathways in cancer therapy play crucial roles in enhancing the immune system’s ability to recognise and eliminate tumour cells. These therapies are designed to either block inhibitory immune checkpoint pathways or to target specific tumour cell markers for direct destruction. Additionally, mAbs can modulate the tumour microenvironment, enhance antibody-dependent cellular cytotoxicity, and inhibit angiogenesis, further amplifying their therapeutic impact. Below is a summary of monoclonal antibodies targeting key pathways, along with their indications and mechanisms of action, which are reviewed based on therapeutic mechanisms. Full article

(This article belongs to the Special Issue Emerging Antibody Engineering Strategies and Applications for Immunotherapy of Cancer)

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7 pages, 6376 KiB

Open AccessCase Report

An Exceedingly Rare Case of Mechanobullous Epidermolysis Bullosa Acquisita in a Prepubertal Child: A Review of the Clinical and Laboratory Considerations

by Aleksandra Wiktoria Bratborska, Maciej Spałek, Monika Bowszyc-Dmochowska and Marian Dmochowski

Antibodies 2025, 14(2), 34; https://doi.org/10.3390/antib14020034 - 11 Apr 2025

Viewed by 366

Abstract

Introduction: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune disease causing subepithelial blistering due to autoantibodies against type VII collagen. While mechanobullous EBA predominantly affects adults, our report presents an exceedingly rare case in an 11-year-old football player. Case Report: The patient reported [...] Read more.

Introduction: Epidermolysis bullosa acquisita (EBA) is a rare autoimmune disease causing subepithelial blistering due to autoantibodies against type VII collagen. While mechanobullous EBA predominantly affects adults, our report presents an exceedingly rare case in an 11-year-old football player. Case Report: The patient reported a one-year history of blistering and scarring on the knees and scrotum. The diagnosis was established with direct immunofluorescence (DIF), mosaic indirect immunofluorescence (IIF) showing IgG antibodies reacting with the dermal side of salt-split primate skin, and multiplex ELISA revealing an elevated level of IgG antibodies against type VII collagen. Treatment with a superpotent topical glucocorticosteroid and activity modifications improved his condition. Review: This case highlights the importance of considering EBA in differential diagnoses of pediatric blistering diseases and suggests that conservative management may be effective in mild cases. We also review clinical and laboratory considerations on the topic of childhood EBA. Conclusions: Further studies are essential to develop evidence-based guidelines for pediatric EBA. Full article

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

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17 pages, 1942 KiB

Open AccessArticle

Effect of Acute Lung Injury (ALI) Induced by Lipopolysaccharide (LPS) on the Pulmonary Pharmacokinetics of an Antibody

by Shweta Jogi and Dhaval K. Shah

Antibodies 2025, 14(2), 33; https://doi.org/10.3390/antib14020033 - 6 Apr 2025

Viewed by 672

Abstract

Objective: To investigate the effect of Lipopolysaccharide (LPS)-induced acute lung injury (ALI) on the pulmonary pharmacokinetics (PK) of a systemically administered antibody in mice. Method: The PK of a non-target-binding antibody was evaluated in healthy mice and mice with intratracheal instillation of 5 [...] Read more.

Objective: To investigate the effect of Lipopolysaccharide (LPS)-induced acute lung injury (ALI) on the pulmonary pharmacokinetics (PK) of a systemically administered antibody in mice. Method: The PK of a non-target-binding antibody was evaluated in healthy mice and mice with intratracheal instillation of 5 mg/kg LPS. The plasma, bronchoalveolar lavage (BAL), trachea, bronchi, and lung homogenate PK of the antibody were measured following intravenous administration of 5 mg/kg antibody dose. Noncompartmental analysis was performed to determine AUC values. Antibody concentrations in all biological matrices were quantified using qualified ELISA. The effect of ALI on BAL albumin and total protein concentrations was also determined. BAL protein concentrations were corrected for dilution using plasma urea concentrations. Results: Intratracheal instillation of LPS and the resultant ALI led to ~2–4-fold higher concentrations of albumin and proteins in the BAL. LPS-induced ALI also notably altered the pulmonary PK of the antibody. The effect of ALI on the antibody PK was time and tissue dependent. The trachea and bronchi showed ~1.7-fold and ~1.4-fold lower antibody exposure compared with the control group, but the BAL fluid exhibited ~4-fold increase in antibody exposure following LPS treatment. Most noticeable changes in antibody PK occurred 24 h after LPS administration, and the effect was temporary for the bronchi and trachea. However, the changes in lung homogenate and, more notably, in BAL persisted until the end of the experiment. Thus, our investigation suggests that due to the acute nature of ALI-induced pathophysiology and the changing severity of the disease, the dose and timing of antibody administration following ALI may need to be optimized based on the target site of action (e.g., bronchi, trachea, BAL, lung parenchyma, etc.) to maximize the therapeutic effect of the antibody. Conclusions: ALI may significantly affect pulmonary PK of systemically administered antibodies. Changes caused by ALI are time and tissue dependent, and hence, the timing and dose of antibody following ALI may need to be optimized to maximize the therapeutic effect of the antibody at the site of action. Full article

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11 pages, 1239 KiB

Open AccessArticle

A Nationwide Seroprevalence Study for Measles in Individuals of Fertile Age in Romania

by Aurora Stanescu, Simona Maria Ruta, Mihaela Leustean, Ionel Iosif, Camelia Sultana, Anca Maria Panaitescu, Florentina Ligia Furtunescu, Costin Cernescu and Adriana Pistol

Antibodies 2025, 14(2), 32; https://doi.org/10.3390/antib14020032 - 2 Apr 2025

Viewed by 589

Abstract

Background/Objectives: Romania remains endemic for measles due to suboptimal vaccine coverage rates. During the last three epidemics, the highest incidence of measles was recorded in children younger than 1 year, who should have been partially protected by maternal antibodies. A nationwide cross-sectional seroprevalence [...] Read more.

Background/Objectives: Romania remains endemic for measles due to suboptimal vaccine coverage rates. During the last three epidemics, the highest incidence of measles was recorded in children younger than 1 year, who should have been partially protected by maternal antibodies. A nationwide cross-sectional seroprevalence study was conducted on persons of fertile age, to evaluate potential immunity gaps in the population. Methods: Between June and October 2020, 959 serum samples were collected from individuals aged 25–44 years (46.5% females) from all the geographic regions in Romania. Measles IgG antibodies were assessed using an enzyme-linked immune assay (DIA.PRO-Diagnostic Bioprobes Srl, Italy). Statistical analysis was performed in IBM SPSS Statistics 27.0, using Fisher’s exact and chi-squared tests to test for associations between seropositivity and demographic factors, with p < 0.05 considered statistically significant. Results: The overall measles seroprevalence was 77%, without gender- or geographic region-related differences. Both the seropositivity rate and the measles antibodies titers increased with age, with the highest difference between the oldest and the youngest age group (p = 0.057), suggesting persistent immunity after natural infection in older individuals or anamnestic responses in vaccinated persons, caused by repeated exposures to the circulating virus. An additional confirmatory pilot study on 444 pregnant women confirmed the low level of measles seroprevalence (68.4%), with a significant upward trend in older ages (75% in those aged >40 years old vs. 65% in those aged 25–29 years, p = 0.018 and mean reactivity of measles antibodies 3.05 ± 1.75 in those aged >40 years vs. 2.28 ± 1.39 in those aged 25–29 years, p = 0.037). Conclusions: This study signals critical immunity gaps in the population that contribute to the accumulation of susceptible individuals and recurrent measles outbreaks. The absence of measles antibodies in women of childbearing age increases the newborn’s susceptibility to infection, with potentially severe complications. Full article

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22 pages, 4237 KiB

Open AccessArticle

Impact of Monoclonal Antibody Aggregates on Effector Function Characterization

by Wendy J. Walton, Shousong Jason Zhang, Joseph J. Wilson, Briana N. Harvey, Matthew Clemens and Yingmei Gu

Antibodies 2025, 14(2), 31; https://doi.org/10.3390/antib14020031 - 2 Apr 2025

Viewed by 982

Abstract

Background/Objectives: Monoclonal antibodies have successfully been used for a variety of indications. Many therapeutic antibodies are IgG1 and elicit effector functions as part of their mechanism of action. It is well known that aggregate levels should be controlled for therapeutic antibodies. Although there [...] Read more.

Background/Objectives: Monoclonal antibodies have successfully been used for a variety of indications. Many therapeutic antibodies are IgG1 and elicit effector functions as part of their mechanism of action. It is well known that aggregate levels should be controlled for therapeutic antibodies. Although there are several reports describing the impact of antibody aggregates on FcγR binding, most of these have been performed with surface plasmon resonance in an avidity-based format. What is less well known is which Fcγ receptor is most impacted by antibody aggregation and how antibody aggregates impact binding to Fcγ receptors in solution-based formats and in cell-based assays. Methods: An effector-competent IgG1 (mAb1) was forcibly degraded and fractionated by size exclusion chromatography to enrich for aggregates. The fractions were examined for FcγR binding by SPR with different formats and in solution. The fractions were also analyzed with cell-based FcγR reporter assays. Results: All Fcγ receptors displayed increased binding to enriched mAb1 aggregates in the avidity-based SPR methods and in solution, with FcγRIIa impacted the most. When examined with an antibody-down SPR format that is not usually susceptible to avidity, FcγRIIa did not show increased binding with mAb1 aggregation. Although activity for mAb1 aggregates increased slightly in an FcγRIIa cell-based reporter assay, it decreased in the FcγRIIIa reporter assay (most likely due to differences in fucosylation from the reference standard). Conclusions: Monoclonal antibody aggregation can impact FcγR binding for avidity-based binding formats. Even at low levels of antibody aggregation, FcγRII binding increases substantially. Full article

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

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14 pages, 1893 KiB

Open AccessArticle

T330M Substitution in the Sodium-Dependent Phosphate Transporter NaPi2b Abolishes the Efficacy of Monoclonal Antibodies Against MX35 Epitope

by Leisan F. Bulatova, Vera S. Skripova, Aisylu R. Sagdeeva, Ramilia A. Vlasenkova, Tatiana A. Bugaenko, Rezeda R. Galimova, Alfiya I. Nesterova, Yuliya V. Filina and Ramziya G. Kiyamova

Antibodies 2025, 14(2), 30; https://doi.org/10.3390/antib14020030 - 1 Apr 2025

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Abstract

Background: Monoclonal antibodies against the sodium-dependent phosphate transporter NaPi2b (SLC34A2) represent a promising approach in the treatment of ovarian and lung cancer. Of particular interest is the potential cancer-specific MX35 epitope of NaPi2b, as it serves as a target for monoclonal [...] Read more.

Background: Monoclonal antibodies against the sodium-dependent phosphate transporter NaPi2b (SLC34A2) represent a promising approach in the treatment of ovarian and lung cancer. Of particular interest is the potential cancer-specific MX35 epitope of NaPi2b, as it serves as a target for monoclonal antibodies studied at various stages of preclinical and clinical trials. However, variations in the NaPi2b protein structure may limit the efficacy of therapeutic antibodies by affecting the accessibility of the MX35 epitope. Methods: An in silico analysis was performed using data from 101,562 tumor samples. Genomic DNA sequencing was conducted on blood samples from patients with ovarian carcinoma, breast cancer, and renal carcinoma to access the frequency of germline mutations in the SLC34A2 gene region encoding the MX35 epitope. To assess the impact of the selected mutation, we generated a model cell line through site-directed mutagenesis carrying the mutant NaPi2b variant. Results: Using in silico analysis, we identified 17 unique variants in the SLC34A2 gene leading to amino acid substitutions within the MX35 epitope of the NaPi2b. Among these, the most prevalent mutation, c.989C>T, resulting in p.T330M substitution, was detected in 5 out of 64 patients through genomic DNA sequencing. Using site-directed mutagenesis, we created the OVCAR-8/NaPi2b^p.T330M model cell line. L3 (28/1) monoclonal antibodies specific to the MX35 epitope failed to recognize the mutant NaPi2b^p.T330M variant compared to the wild-type of the NaPi2b in both Western blot and confocal microscopy experiments. Conclusions: The obtained data may serve as a basis for predicting the efficacy of monoclonal antibody-based targeted therapy binding to the MX35 epitope of NaPi2b in the treatment of oncological diseases. Full article

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11 pages, 2759 KiB

Open AccessEditor’s ChoiceArticle

A Novel Method for Preparing Uniform Micro-Sized Dry Powder Formulations, Including Aggregation-Controlled VHH

by Tatsuru Moritani, Hidekazu Masaki, Ryo Yonehara, Takeru Suzuki, Hidenao Arai, Masayuki Tsuchiya and Naoto Nemoto

Antibodies 2025, 14(2), 29; https://doi.org/10.3390/antib14020029 - 31 Mar 2025

Viewed by 677

Abstract

Background: The preparation of antibodies in powder form without changing their physicochemical properties may enable their use in new drug delivery system therapies or non-refrigerated storage. The variable domain of heavy-chain antibodies (VHHs) is more suited for this purpose than that of conventional [...] Read more.

Background: The preparation of antibodies in powder form without changing their physicochemical properties may enable their use in new drug delivery system therapies or non-refrigerated storage. The variable domain of heavy-chain antibodies (VHHs) is more suited for this purpose than that of conventional antibodies because of VHHs’ high thermal stability and ability to refold. Methods: In this report, the fine droplet drying (FDD) process was selected as the powderization technique because of its favorable features, such as mild drying conditions and the generation of uniform particle sizes. The aggregation, binding, particle, and in vitro inhalation properties of the prepared VHH powders (VHHps) were evaluated. Results: The amount of aggregated VHHs present in the VHHps depended on the flow temperature during the FDD process, with higher temperatures yielding a higher aggregation ratio. In contrast, no significant difference in binding activity was observed between each VHHp preparation and the native VHHs. However, this process degraded VHHs or inactivated their function, and ultimately, only about 30% of the original VHHs were functional, whereas the remaining VHHs that were not degraded showed little loss of functionality, even after storage at room temperature for more than two years. Analysis of the VHHp samples revealed that the particles were uniformly spherical with a single-micron size. The VHHps showed fine inhalation properties in the inhalation property test. Conclusions: These findings suggest that the FDD process affords various VHH powder formulations, including pharmaceutical formulations. Full article

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15 pages, 3599 KiB

Open AccessArticle

Discovery of Antibodies Against Endemic Coronaviruses with NGS-Based Human Fab Phage Display Platform

by Oscar Chi-Chien Pan, Sean Miller, Ruchin Patel, Shreya Mukhopadhyay, Giancarlo Sarullo, Gwenny Go, Jennifer Galli, Jamie Hessels, Barbara Schlingmann-Molina, Emmanuel Ndashimye, Zhiyun Wen, Christopher Warren, Eberhard Durr, Lan Zhang, Kalpit A. Vora, Arthur Fridman and Zhifeng Chen

Antibodies 2025, 14(2), 28; https://doi.org/10.3390/antib14020028 - 27 Mar 2025

Cited by 1 | Viewed by 716

Abstract

Background: There is an unmet medical need to develop a vaccine targeting endemic coronaviruses. Antigen-specific monoclonal antibodies (mAbs) are crucial for many assays to support vaccine development. Objective: In this study, we used the HuCal Fab phage display library with a diversity of [...] Read more.

Background: There is an unmet medical need to develop a vaccine targeting endemic coronaviruses. Antigen-specific monoclonal antibodies (mAbs) are crucial for many assays to support vaccine development. Objective: In this study, we used the HuCal Fab phage display library with a diversity of 4.5 × 10¹⁰ to identify antibodies specific to the spike proteins of the four endemic coronaviruses: OC43, NL63, 229E, and HKU1. Methods: As proof of concept, we established a newly designed platform using a long-read NGS workflow for antibody discovery and compared the results against the traditional workflow using Sanger sequencing consisting of lengthy and laborious benchwork. Results: The long-read NGS workflow identified most of the antibodies seen from the Sanger sequencing workflow, and many more additional antigen-specific antibodies against the endemic coronaviruses. Overall efficiency improved up to three times, comparing the traditional workflow with the NGS workflow. Of the 113 NGS-derived mAbs isolated to bind the four endemic coronavirus spike proteins, 107/113 (94.7%) had potent ELISA binding affinities (EC50 < 150 ng/mL, or <1 nM), and 61/113 (54%) had extremely potent ELISA binding affinities (EC50 of <15 ng/mL, or <0.1 nM). Conclusions: We successfully developed and incorporated the long-read NGS workflow to generate target-specific antibodies with many antibodies at sub-nanomolar affinities that are likely missed by a traditional workflow. We identified strong neutralizing antibodies, proving that our endemic spike proteins are capable of generating antibodies that could offer protection against the endemic HCoVs. Full article

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13 pages, 633 KiB

Open AccessArticle

Low Serological Agreement of Hepatitis E in Immunocompromised Cancer Patients: A Comparative Study of Three Anti-HEV Assays

by Isabel-Elena Haller, Mark Reinwald, Janine Kah, Franz A. M. Eggert, Sandra Schwarzlose-Schwarck, Kristoph Jahnke, Stefan Lüth and Werner Dammermann

Antibodies 2025, 14(2), 27; https://doi.org/10.3390/antib14020027 - 24 Mar 2025

Viewed by 562

Abstract

Background/Objectives: Hepatitis E virus (HEV) is one of the leading causes of acute hepatitis, with immunosuppressed individuals, such as oncology patients, being particularly vulnerable to chronic infections that may progress to liver disease or fatal outcomes. Assay variability complicates HEV prevalence assessment in [...] Read more.

Background/Objectives: Hepatitis E virus (HEV) is one of the leading causes of acute hepatitis, with immunosuppressed individuals, such as oncology patients, being particularly vulnerable to chronic infections that may progress to liver disease or fatal outcomes. Assay variability complicates HEV prevalence assessment in at-risk groups. This study aimed to compare the reliability and concordance of three HEV antibody assays—Wantai, Euroimmun, and Elecsys^®—in immunosuppressed oncology patients. Methods: In this prospective pilot study, serum samples were obtained from oncology patients between September 2020 and October 2021. Samples were collected both at baseline (treatment-naive) and during ongoing treatment. A healthy control group was retrospectively included for comparative analysis. Anti-HEV IgM and IgG antibodies were tested in all samples using enzyme-linked immunosorbent assays (Wantai, Euroimmun) and an electrochemiluminescence immunoassay (Elecsys^®). Demographic and clinical data, along with information on HEV risk factors, were extracted from medical records and patient questionnaires. Results: HEV IgM prevalence ranged from 0% (Wantai) to 6% (Elecsys^®), while IgG prevalence was 12% (Euroimmun), 38% (Wantai), and 53% (Elecsys^®). Concordance was poor, with Cohen’s Kappa values indicating slight to moderate agreement (κ = 0.000–0.553). Patients with hematological malignancies exhibited the highest IgG seroprevalence. Risk factor analysis revealed the highest association between HEV exposure and the consumption of undercooked pork or crop-based agriculture. Conclusions: Significant variability among HEV serological assays highlights the challenges of reliable HEV diagnostics in immunosuppressed oncology patients. Assay selection and improved testing strategies are critical for this high-risk group. Full article

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Antibodies, Volume 14, Issue 2 (June 2025) – 20 articles

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