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A High-Affinity Monoclonal Antibody Against the Pancreatic Ductal Adenocarcinoma Target, Anterior Gradient-2 (AGR2/PDIA17)
Metabolic Engineering of Glycofusion Bispecific Antibodies for α-Dystroglycanopathies
Limited Biomarker Potential for IgG Autoantibodies Reactive to Linear Epitopes in Systemic Lupus Erythematosus or Spondyloarthropathy
Anti-MET Antibody Therapies in Non-Small-Cell Lung Cancer: Current Progress and Future Directions
A Brief Chronicle of Antibody Research and Technological Advances

Journal Description

Antibodies

Antibodies is an international, peer-reviewed, open access journal on immunoglobulins, published quarterly online by MDPI.

Open Access— free for readers, with article processing charges (APC) paid by authors or their institutions.
High Visibility: indexed within Scopus, ESCI (Web of Science), PubMed, PMC, Embase, CAPlus / SciFinder, and other databases.
Journal Rank: CiteScore - Q2 (Drug Discovery)
Rapid Publication: manuscripts are peer-reviewed and a first decision is provided to authors approximately 22.8 days after submission; acceptance to publication is undertaken in 4.9 days (median values for papers published in this journal in the second half of 2024).
Recognition of Reviewers: reviewers who provide timely, thorough peer-review reports receive vouchers entitling them to a discount on the APC of their next publication in any MDPI journal, in appreciation of the work done.

Impact Factor: 3.0 (2023); 5-Year Impact Factor: 4.7 (2023)

Imprint Information Journal Flyer Open Access ISSN: 2073-4468

Latest Articles

23 pages, 1025 KiB

Open AccessReview

Monoclonal Antibodies in Light of Mpox Outbreak: Current Research, Therapeutic Targets, and Animal Models

by Vladimir N. Nikitin, Iuliia A. Merkuleva and Dmitriy N. Shcherbakov

Antibodies 2025, 14(1), 20; https://doi.org/10.3390/antib14010020 - 26 Feb 2025

The rapid rise in monkeypox virus infections among humans from 2022 to 2024 has captured the attention of the global healthcare community. In light of the lack of mandatory vaccination and limited data on next-generation vaccines for monkeypox prevention, the urgent development of [...] Read more.

The rapid rise in monkeypox virus infections among humans from 2022 to 2024 has captured the attention of the global healthcare community. In light of the lack of mandatory vaccination and limited data on next-generation vaccines for monkeypox prevention, the urgent development of therapeutic agents has become a priority. One promising approach involves the use of neutralizing monoclonal antibodies. This review highlights significant advancements in the search for antibodies against human pathogenic orthopoxviruses, particularly focusing on their potential application against the monkeypox virus. We also analyze viral proteins that serve as targets for identifying therapeutic antibodies capable of neutralizing a wide range of viruses. Finally, we deemed it essential to address the challenges associated with selecting an animal model that can adequately reflect the infectious process of each orthopoxvirus species in humans. Full article

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

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11 pages, 1086 KiB

Open AccessArticle

IgE-Crosslinking-Induced Luciferase Expression Test as a Sensitive Indicator of Anisakis Allergy

by Haruyo Akiyama, Masashi Niwa, Chisato Kurisaka, Yuto Hamada, Yuma Fukutomi and Reiko Teshima

Antibodies 2025, 14(1), 19; https://doi.org/10.3390/antib14010019 - 25 Feb 2025

Background: Anisakis allergy has been increasing, and the diagnosis of it is based on specific serum IgE detection. Recently, the IgE-crosslinking-induced luciferase expression (EXiLE) test has been proposed as convenient tool for detecting functionally specific IgE antibodies. Here, we investigated if the [...] Read more.

Background: Anisakis allergy has been increasing, and the diagnosis of it is based on specific serum IgE detection. Recently, the IgE-crosslinking-induced luciferase expression (EXiLE) test has been proposed as convenient tool for detecting functionally specific IgE antibodies. Here, we investigated if the EXiLE test is a useful tool in the diagnosis of Anisakis allergy. Methods: HuRa-40 cells were sensitized using six serum types from three patients with Anisakis allergy at the time of the initial test and after 6–12 months. Thereafter, various concentrations of Anisakis worm protein (AWP) were reacted to measure the degree of EXiLE. The degree of EXiLE was compared with Anisakis-specific IgE antibody levels measured by the CAP-FEIA method, and the IgE-antibody-binding protein profile was examined using IgE immunoblotting. Results: The results showed a good correlation between the CAP-FEIA values and EXiLE obtained with 5 μg/mL of AWP (R = 0.91, p < 0.01), a strong response on IgE immunoblotting in the region containing proteins weighing ≥40,000 Da. In addition, after the onset of Anisakis allergy, the degree of serum EXiLE decreased in two patients whose Anisakis-specific IgE antibody levels decreased over time but increased in one patient whose specific IgE antibodies increased after repeated antigen sensitization. Conclusions: Based on these data, the AWP-induced EXiLE test seemed to be useful and convenient for the diagnosis of Anisakis allergy, supplementing specific IgE determinants. After allergy onset, the use of this method to observe changes in specific IgE levels over time may be important for predicting the risk of recurrence. Full article

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

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17 pages, 2302 KiB

Open AccessReview

Avian Antibodies as Potential Therapeutic Tools

by Mats Eriksson and Anders Larsson

Antibodies 2025, 14(1), 18; https://doi.org/10.3390/antib14010018 - 14 Feb 2025

Immunoglobulin Y (IgY) is the primary antibody found in the eggs of chicken (Gallus domesticus), allowing for large-scale antibody production with high titers, making them cost-effective antibody producers. IgY serves as a valuable alternative to mammalian antibodies typically used in immunodiagnostics [...] Read more.

Immunoglobulin Y (IgY) is the primary antibody found in the eggs of chicken (Gallus domesticus), allowing for large-scale antibody production with high titers, making them cost-effective antibody producers. IgY serves as a valuable alternative to mammalian antibodies typically used in immunodiagnostics and immunotherapy. Compared to mammalian antibodies, IgY offers several biochemical advantages, and its straightforward purification from egg yolk eliminates the need for invasive procedures like blood collection, reducing stress in animals. Due to the evolutionary differences between birds and mammals, chicken antibodies can bind to a broader range of epitopes on mammalian proteins than their mammalian counterparts. Studies have shown that chicken antibodies bind 3–5 times more effectively to rabbit IgG than swine antibodies, enhancing the signal in immunological assays. Additionally, IgY does not interact with rheumatoid factors or human anti-mouse IgG antibodies (HAMA), helping to minimize interference from these factors. IgY obtained from egg yolk of hens immunized against Pseudomonas aeruginosa has been used in patients suffering from cystic fibrosis and chronic pulmonary colonization with this bacterium. Furthermore, IgY has been used to counteract streptococcus mutans in the oral cavity and for the treatment of enteral infections in both humans and animals. However, the use of avian antibodies is limited to pulmonary, enteral, or topical application and should, due to immunogenicity, not be used for systemic administration. Thus, IgY expands the range of strategies available for combating pathogens in medicine, as a promising candidate both as an alternative to antibiotics and as a valuable tool in research and diagnostics. Full article

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

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22 pages, 2446 KiB

Open AccessArticle

Investigation of Antibody Pharmacokinetics in Male Reproductive System and Its Characterization Using a Translational PBPK Model

by Sree Ojili and Dhaval K. Shah

Antibodies 2025, 14(1), 17; https://doi.org/10.3390/antib14010017 - 13 Feb 2025

Objectives: To investigate the pharmacokinetics (PK) of the monoclonal antibody (mAb) in male reproductive tissues and develop a translational physiologically based pharmacokinetic (PBPK) model to characterize the PK data. Method: The PK of a non-cross-reactive antibody (trastuzumab) was investigated in human FcRn-expressing male [...] Read more.

Objectives: To investigate the pharmacokinetics (PK) of the monoclonal antibody (mAb) in male reproductive tissues and develop a translational physiologically based pharmacokinetic (PBPK) model to characterize the PK data. Method: The PK of a non-cross-reactive antibody (trastuzumab) was investigated in human FcRn-expressing male mice following a 10 mg/kg intravenous dose. The PK in plasma and male reproductive tissues (i.e., epididymis, testes, vas deferens, seminal vesicles, and prostate glands) were evaluated. The observed PK data in mice were mathematically characterized using a novel PBPK model for antibodies that contained male reproductive systems. The mouse PBPK model was scaled to rats, monkeys, and humans to predict the PK of antibodies in male reproductive organs across animal species. Results: Plasma and tissue PK data generated in mice suggest that antibody distribution in male reproductive tissues is generally lower compared to that of most of the organs. The antibody exposure in the testes was 1.70%, in the epididymis was 2.57%, in the vas deferens was 2.01%, in the seminal vesicle was 0.42%, and in the prostate gland was 0.52% of the plasma exposure. The plasma and tissue PK data were simultaneously characterized using the PBPK model, which incorporated the novel male reproductive system. All the predicted PK profiles were within two-fold of the observed data, as indicated by percentage prediction error (%PE) values. The mouse model was successfully translated to bigger animals, and the model was used to simulate the PK of antibodies in rat, monkey, and human male reproductive systems. Conclusions: The combination of the experimental data and novel PBPK model presented here provides unprecedented insights into the antibody distributions in different male reproductive tissues. The PBPK model can serve as a crucial tool for advancing the development of antibody-based therapies for treating sexually transmitted infections (STIs), cancers, and contraceptives. Full article

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

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29 pages, 6227 KiB

Open AccessReview

Immune Cell Engagers: Advancing Precision Immunotherapy for Cancer Treatment

by Hyukmin In, Minkyoung Park, Hyeonsik Lee and Kyung Ho Han

Antibodies 2025, 14(1), 16; https://doi.org/10.3390/antib14010016 - 11 Feb 2025

Immune cell engagers (ICEs) are an emerging class of immunotherapies designed to harness the immune system’s anti-tumor potential through precise targeting and activation of immune effector cells. By engaging T cells, natural killer (NK) cells, and phagocytes, ICEs overcome challenges such as immune [...] Read more.

Immune cell engagers (ICEs) are an emerging class of immunotherapies designed to harness the immune system’s anti-tumor potential through precise targeting and activation of immune effector cells. By engaging T cells, natural killer (NK) cells, and phagocytes, ICEs overcome challenges such as immune evasion and MHC downregulation, addressing critical barriers in cancer treatment. T-cell engagers (TCEs), led by bispecific T-cell engagers (BiTEs), dominate the field, with innovations such as half-life-extended BiTEs, trispecific antibodies, and checkpoint inhibitory T-cell engagers driving their application in hematologic and solid malignancies. NK cell engagers (NKCEs) and phagocyte cell engagers (PCEs) are rapidly progressing, drawing on NK cells’ innate cytotoxicity and macrophages’ phagocytic abilities to target tumors, particularly in immunosuppressive microenvironments. Since the FDA approval of Blinatumomab in 2014, ICEs have transformed the oncology landscape, with nine FDA-approved products and numerous candidates in clinical trials. Despite challenges such as toxicity, resistance, and limited efficacy in solid tumors, ongoing research into advanced platforms and combination therapies highlights the growing potential of ICEs to provide personalized, scalable, and effective cancer treatments. This review investigates the mechanisms, platforms, research trends, and clinical progress of ICEs, emphasizing their pivotal role in advancing precision immunotherapy and their promise as a cornerstone of next-generation cancer therapies. Full article

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

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16 pages, 2547 KiB

Open AccessArticle

Assessing the Influence of Selected Permeabilization Methods on Lymphocyte Single-Cell Multi-Omics

by Shifan Ding, Na Lu and Hassan Abolhassani

Antibodies 2025, 14(1), 15; https://doi.org/10.3390/antib14010015 - 10 Feb 2025

(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is [...] Read more.

(1) Background: Single-cell multi-omics is a powerful method for the dissection and detection of complicated immunologic functions and synapses. However, most currently available technologies merge datasets of different omics from separate portions of the same sample to generate combined multi-omics. This process is a source of bias, mainly in the field of immunology on cells originating from pluripotent hematopoietic stem cells with high flexibility during maturation. (2) Methods: Although new multi-omics approaches have been developed to use the advantages of cellular and molecular barcoding and next-generation sequencing to solve this issue, one of the main current challenges is intracellular proteomics, which should be combined with other omics data with high importance for immune system studies. We designed this study to evaluate previously recommended minimal permeabilization and fixation methods on the quality and quantity of transcriptomics and proteomics data generated by the BD Rhapsody™ Single-Cell Analysis System. (3) Results: Our findings showed that high-throughput sequencing with advanced quality and read-out is required for the combination of multi-omics outcomes from a permeabilized single cell. Therefore, the HiseqX platform was selected for further analysis. The effect of immune stimulation was observed clearly as the separated clusters of helper and cytotoxic T cells using unsupervised clustering. Importantly, fixation and permeabilization did not affect the general expression profile of unstimulated cells. However, fixation and permeabilization were proved to negatively impact the detection of the whole transcriptome for single-cell assay. Nevertheless, about 60% of the transcriptomic signature of the stimulation was detected. If the measurement of combined surface and intracellular markers is required to be achieved, the modified fixation and permeabilization method is recommended because of a lower transcriptomic loss and more precise proteomic fingerprint detected. (4) Conclusions: The findings of this study support the potential possibility for integrating intracellular proteomics, which needs to be optimized and tested with newly designed oligonucleotide-tagged antibodies targeting intracellular proteins. Full article

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18 pages, 3060 KiB

Open AccessArticle

Clinical Scaleup of Humanized AnnA1 Antibody Yielded Unexpected High Reticuloendothelial (RES) Uptake in Mice

by Lu Lucy Xu, Satyendra Kumar Singh, Chelsea Nayback, Abdullah Metebi, Dalen Agnew, Tim Buss, Jan Schnitzer and Kurt R. Zinn

Antibodies 2025, 14(1), 14; https://doi.org/10.3390/antib14010014 - 6 Feb 2025

Background/Objectives: A mouse antibody directed against truncated Annexin A1 showed high tumor retention in pre-clinical cancer models and was approved by the National Cancer Institute Experimental Therapeutics (NExT) program for humanization and large batch cGMP production for toxicology and clinical trials. In this [...] Read more.

Background/Objectives: A mouse antibody directed against truncated Annexin A1 showed high tumor retention in pre-clinical cancer models and was approved by the National Cancer Institute Experimental Therapeutics (NExT) program for humanization and large batch cGMP production for toxicology and clinical trials. In this process, a contractor for Leidos accidentally produced a mutated version of humanized AnnA1 (hAnnA1-mut) with a single nucleotide deletion in the terminal Fc coding region that increased the translated size by eight amino acids with random alterations in the final twenty-four amino acids. We investigated the tissue distribution of hAnnA1-mut, hAnnA1, mAnnA1, and isotope-matched human IgG1 under various injection and conjugation conditions with C57BL/6, FVB, and BALB/c nude mice strains. Methods: Biodistribution studies were performed 24 h after injection of Tc-99m-HYNIC radiolabeled antibodies (purity > 98%). Non-reducing gel electrophoresis studies were conducted with IR680 labeled antibodies incubated with various mouse sera. Results: Our results showed that Tc-99m-HYNIC-hAnnA1 had low spleen and liver retention not statistically different from Tc-99m-HYNIC-IgG1 and Tc-99m-HYNIC-mAnnA1, with corresponding higher blood levels; however, Tc-99m-HYNIC-hAnnA1-mut had high levels in the spleen and liver with differences identified among the mouse strains, radiolabeling conditions, and injection routes. Histopathology showed no morphological change in the liver or spleen from any conditions. Gel electrophoresis showed an upward shift of hAnnA1-mut, consistent with the binding of blood serum protein. Conclusions: The changes in the Fc region of hAnnA1-mut led to higher liver and spleen uptake, suggesting the antibody’s recognition by the innate immune system (likely complement protein binding) and subsequent clearance. Future clinical translation using hAnnA1 and other antibodies needs to limit protein modifications that could drastically reduce blood clearance. Full article

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11 pages, 3358 KiB

Open AccessArticle

Evaluation of SARS-CoV-2 Antibody Response Between Paired Fingerprick (HemaPEN^®) and Venepuncture Collected Samples in Children and Adults

by Nadia Mazarakis, Zheng Quan Toh, Jill Nguyen, Rachel A. Higgins, James Rudge, Belinda Whittle, Nicholas J. Woudberg, Justin Devine, Andrew Gooley, Florian Lapierre, Nigel W. Crawford, Shidan Tosif and Paul V. Licciardi

Antibodies 2025, 14(1), 13; https://doi.org/10.3390/antib14010013 - 5 Feb 2025

Serological surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is important to monitor population COVID-19 immunity. Dried blood spots (DBS) are a valuable method for serosurveys, particularly in remote settings and in children. We compared the measurement of SARS-CoV-2 spike-specific IgG [...] Read more.

Serological surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies is important to monitor population COVID-19 immunity. Dried blood spots (DBS) are a valuable method for serosurveys, particularly in remote settings and in children. We compared the measurement of SARS-CoV-2 spike-specific IgG in paired blood samples collected using standard venepuncture (serum) and the hemaPEN^® microsampling DBS device from children and adults. A total of 83 participants (10 months to 65 years of age), comprising COVID-positive and -negative participants, were recruited. Paired serum and DBS samples were assayed for SARS-CoV-2 receptor-binding domain (RBD) and Spike (S1) antibodies using an established in-house ELISA. RBD and S1 IgG concentrations of paired hemaPEN DBS eluates and serum samples were compared using a non-parametric Wilcoxon matched-pairs signed ranked test. A Pearson’s correlation was used for RBD and S1 IgG concentrations and the level of agreement between the hemaPEN DBS eluates and serum samples was assessed by Bland–Altman analysis. A total of N = 41 adults (36 COVID-positive and 5 COVID-negative), and N = 42 children (37 COVID-positive, and 5 COVID-negative) have paired serum and DBS assayed. We found moderate to strong correlations between paired hemaPEN DBS eluates and serum SARS-CoV-2 IgG antibodies for RBD (r = 0.9472, p < 0.0001) and S1 proteins (r = 0.6892, p < 0.0001). Similar results were observed in both adult and paediatric populations. No significant differences in S1-specific IgG levels were observed in hemaPEN DBS samples stored for up to 35 weeks at room temperature. Eluted hemaPEN samples showed high specificity and sensitivity (100% and 89.89%, respectively) compared with serum. The use of the microsampling hemaPEN device for DBS sample collection is a feasible approach for assessing SARS-CoV-2 antibodies for serosurveillance studies, particularly in remote settings and in children. Full article

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

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19 pages, 2289 KiB

Open AccessArticle

Efficient Identification of Monoclonal Antibodies Against Rift Valley Fever Virus Using High-Throughput Single Lymphocyte Transcriptomics of Immunized Mice

by Ronit Rosenfeld, Ron Alcalay, Yfat Yahalom-Ronen, Sharon Melamed, Avital Sarusi-Portuguez, Tal Noy-Porat, Ofir Israeli, Adi Beth-Din, Ronnie Blecher-Gonen, Theodor Chitlaru, Erez Bar-Haim, Tomer Israely, Anat Zvi and Efi Makdasi

Antibodies 2025, 14(1), 12; https://doi.org/10.3390/antib14010012 - 4 Feb 2025

Background: Rift Valley fever virus (RVFV) is a zoonotic virus that poses a significant threat to both livestock and human health and has caused outbreaks in endemic regions. In humans, most patients experience a febrile illness; however, in some patients, RVF disease [...] Read more.

Background: Rift Valley fever virus (RVFV) is a zoonotic virus that poses a significant threat to both livestock and human health and has caused outbreaks in endemic regions. In humans, most patients experience a febrile illness; however, in some patients, RVF disease may result in hemorrhagic fever, retinitis, or encephalitis. While several veterinary vaccines are being utilized in endemic countries, currently, there are no licensed RVF vaccines or therapeutics for human use. Neutralizing antibodies specifically targeting vulnerable pathogen epitopes are promising candidates for prophylactic and therapeutic interventions. In the case of RVFV, the surface glycoproteins Gc and Gn, which harbor neutralizing epitopes, represent the primary targets for vaccine and neutralizing antibody development. Methods: We report the implementation of advanced 10x Genomics technology, enabling high-throughput single-cell analysis for the identification of rare and potent antibodies against RVFV. Following the immunization of mice with live attenuated rMP-12-GFP virus and successive Gc/Gn boosts, memory B cell populations (both general and antigen-specific) were sorted from splenocytes by flow cytometry. Deep sequencing of the antibody repertoire at a single-cell resolution, together with bioinformatic analyses, was applied for BCR pair selection based on their abundance and specificity. Results: Twenty-three recombinant monoclonal antibodies (mAbs) were selected and expressed, and their antigen-binding capacities were characterized. About half of them demonstrated specific binding to their cognate antigen with relatively high binding affinities. Conclusions: These antibodies could be used for the future development of efficacious therapeutics, as well as for studying virus-neutralizing mechanisms. The current study, in which the single-cell sequencing approach was implemented for the development of antibodies targeting the RVFV surface proteins Gc and Gn, demonstrates the effective applicability of this technique for antibody discovery purposes. Full article

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

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15 pages, 1785 KiB

Open AccessArticle

Purification of a Fc-Fusion Protein with [Bathophenathroline:metal] Complexes

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

Antibodies 2025, 14(1), 11; https://doi.org/10.3390/antib14010011 - 31 Jan 2025

In this study, we assess an alternative Fc-fusion protein purification method that does not rely on chromatographic media or ligands. Recombinant human acetylcholinesterase, fused to the Fc domain of human IgG1 (henceforth, AChE-Fc), was purified with precipitated aromatic complexes composed of the bathophenanthroline [...] Read more.

In this study, we assess an alternative Fc-fusion protein purification method that does not rely on chromatographic media or ligands. Recombinant human acetylcholinesterase, fused to the Fc domain of human IgG1 (henceforth, AChE-Fc), was purified with precipitated aromatic complexes composed of the bathophenanthroline (henceforth, batho) chelator with either Zn²⁺ or Cu²⁺ ions (i.e., [(batho)₃:Zn²⁺] or [(batho)₂:Cu²⁺]) in the presence of polyethylene glycol 6000 (PEG-6000). In a three-step purification process conducted at pH 7, AChE-Fc was captured by the aromatic complexes (Step 1); unbound or weakly bound protein impurities were removed with 20 mM NaCl (Step 2); and AChE-Fc was then extracted at pH 7 (Step 3) using 100 mM Na citrate buffer in 250 mM NaCl. Purified AChE-Fc was not aggregated (as determined by dynamic light scattering (DLS) and Native PAGE). However, full enzymatic activity was only preserved with the [(batho)₃:Zn²⁺] complex. Interaction between AChE-Fc and [(batho)₃:Zn²⁺] led to ~83–88% overall protein yield. Thirty-fold process upscaling by volume required only proportional increase in the amounts of [(batho)₃:Zn²⁺] and PEG-6000. Efficient (95–97%) chelator recycling was achieved by recrystallization. Chelator leaching into purified AchE-Fc was estimated to be ~0.3% relative to the total amount used. Taken together, this novel procedure has the potential to provide an economical and practical avenue for the industrial purification of Fc-fusion proteins. Full article

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

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28 pages, 5117 KiB

Open AccessArticle

Exploring Anticitrullinated Antibodies (ACPAs) and Serum-Derived Exosomes Cargoes

by Mohammed A. Alghamdi, Sami M. Bahlas, Sultan Abdulmughni Alamry, Ehab H. Mattar and Elrashdy M. Redwan

Antibodies 2025, 14(1), 10; https://doi.org/10.3390/antib14010010 - 26 Jan 2025

Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein autoantibodies (ACPAs) are useful tools for rheumatoid arthritis (RA). The presence of ACPAs against citrullinated proteins (CPs), especially citrullinated fibrinogen (cFBG), seems to be a useful serological marker for diagnosing RA. RA patients’ [...] Read more.

Background: Autoantibodies such as rheumatoid factor (RF) and anticitrullinated protein autoantibodies (ACPAs) are useful tools for rheumatoid arthritis (RA). The presence of ACPAs against citrullinated proteins (CPs), especially citrullinated fibrinogen (cFBG), seems to be a useful serological marker for diagnosing RA. RA patients’ sera were found to be enriched in exosomes that can transmit many proteins. Exosomes have been found to express citrullinated protein such as cFBG. Objective: We conducted this study in two stages. In the first phase, we aimed to evaluate the association between autoantibodies and risk factors. In the next step, ACPA-positive serum samples from the first phase were subjected to exosomal studies to explore the presence of cFBG, which is a frequent target for ACPAs. Methods: We investigated the autoantibodies in one hundred and sixteen Saudi RA patients and correlated with host-related risk factors. Exosomes were extracted from patients’ sera and examined for the presence of cFBG using monoclonal antibodies. Results: The study reported a high female-to-male ratio of 8:1, and seropositive RA (SPRA) was more frequent among included RA patients. The frequency and the levels of ACPAs were similar in both genders. Autoantibodies incidences have a direct correlations with patient age, while the average titers decreased as the age increased. Further, the highest incidence and levels of autoantibodies were reported in patients with RA duration between 5 and 10 years. Smoking and family history have no impact on autoantibody, except for ACPAs titers among smokers’ RA. Our analysis of serum exosomes revealed that about 50% of SPRA patients expressed cFBG. Conclusions: The female-to-male ratio is 8:1, which is higher than the global ratio. We can conclude that patients’ age and disease duration contribute to the autoantibodies, particularly RF and anti-MCV, whereas smoking and family history had no effects on autoantibodies. We detected cFBG in all exosomes from SPRA patients; thus, we suggest that the precise mechanism of exosomes in RA pathogenesis can be investigated to develop effective treatment strategies. Full article

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21 pages, 4090 KiB

Open AccessArticle

Influence of High Eimeria tenella Immunization Dosages on Total Oocyst Output and Specific Antibodies Recognition Response in Hybrid Pullets (Gallus gallus)—A Pilot Study

by Marco A. Juarez-Estrada, Guillermo Tellez-Isaias, Víctor M. Petrone-Garcia, Amanda Gayosso-Vazquez, Xochitl Hernandez-Velasco and Rogelio A. Alonso-Morales

Antibodies 2025, 14(1), 9; https://doi.org/10.3390/antib14010009 - 26 Jan 2025

Background: Two high primary-immunization doses of a wild-type E. tenella strain were assessed in healthy pullets (5K versus 10K sporulated oocysts/bird) to understand the effects of coccidia infection. Methods: Acquired immunity was evaluated following primary immunization and two booster doses with the homologous [...] Read more.

Background: Two high primary-immunization doses of a wild-type E. tenella strain were assessed in healthy pullets (5K versus 10K sporulated oocysts/bird) to understand the effects of coccidia infection. Methods: Acquired immunity was evaluated following primary immunization and two booster doses with the homologous strain. Total oocyst shedding, clinical signs, and viability of every bird/group after each immunization/booster were recorded. Indirect ELISA measured the time course of humoral responses from each immunization group against sporozoite and second-generation merozoite of E. tenella. Antigen pattern recognition on these two asexual zoite stages of E. tenella was analyzed using Western blotting with antibodies from each immunization program. Afterwards, antigen recognition of specific life-cycle stages was performed using individual pullet serums from the best immunization program. Results: A primary-immunization dose of 1 × 10⁴ oocysts/bird reduced the oocyst output; however, all pullets exhibited severe clinical signs and low specific antibodies titers, with decreased polypeptide recognition on both E. tenella asexual zoite stages. In contrast, immunization with 5 × 10³ oocysts/bird yielded the best outcomes regarding increased oocyst collection and early development of sterilizing immunity. After the first booster dosage, this group’s antisera revealed a strong pattern of specific antigen recognition on the two assayed E. tenella life-cycle stages. Conclusions: The E. tenella-specific antibodies from the 5 × 10³ oocysts/bird immunization program can aid in passive immunization trials and further research to identify B-cell immunoprotective antigens, which could help in the development of a genetically modified anticoccidial vaccine. Full article

(This article belongs to the Special Issue Unravelling Effector Functions of B cells in Infectious Diseases and Cancer)

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30 pages, 24558 KiB

Open AccessArticle

In Vitro Functional Validation of an Anti-FREM2 Nanobody for Glioblastoma Cell Targeting

by Gloria Krapež, Neja Šamec, Alja Zottel, Mojca Katrašnik, Ana Kump, Jernej Šribar, Igor Križaj, Jurij Stojan, Rok Romih, Gregor Bajc, Matej Butala, Serge Muyldermans and Ivana Jovčevska

Antibodies 2025, 14(1), 8; https://doi.org/10.3390/antib14010008 - 24 Jan 2025

Background/Objectives: Glioblastomas are the most common brain malignancies. Despite the implementation of multimodal therapy, patient life expectancy after diagnosis is barely 12 to 18 months. Glioblastomas are highly heterogeneous at the genetic and epigenetic level and comprise multiple different cell subpopulations. Therefore, [...] Read more.

Background/Objectives: Glioblastomas are the most common brain malignancies. Despite the implementation of multimodal therapy, patient life expectancy after diagnosis is barely 12 to 18 months. Glioblastomas are highly heterogeneous at the genetic and epigenetic level and comprise multiple different cell subpopulations. Therefore, small molecules such as nanobodies, able to target membrane proteins specific to glioblastoma cells or specific cell types within the tumor are being investigated as novel tools to treat glioblastomas. Methods: Here, we describe the identification of such a nanobody and its in silico and in vitro validation. NB3F18, as we named it, is directed against the membrane-associated protein FREM2, overexpressed in glioblastoma stem cells. Results: Three dimensional in silico modeling indicated that NB3F18 and FREM2 form a stable complex. Surface plasmon resonance confirmed their interaction with moderate affinity. As we demonstrated by flow cytometry, NB3F18 binds to glioblastoma stem cells to a greater extent than to differentiated glioblastoma cells and astrocytes. Immunocytochemistry revealed surface localization of NB3F18 on glioblastoma stem cells, whereas cytoplasmic localization of NB3F18 was observed in other cell lines. NB3F18 was detected by transmission electron microscopy on the plasma membrane and in various compartments of the endocytic pathway, from endocytic vesicles to multivesicular bodies (endosomes) and lysosomes. Interestingly, NB3F18 was cytotoxic to glioblastoma stem cells. Conclusions: Collectively, NB3F18 has been qualified as an interesting tool to target glioblastoma cells and as a potential vehicle to deliver biological or pharmaceutical agents to these cells. Full article

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

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18 pages, 2550 KiB

Open AccessArticle

A Strategy for Simultaneous Engineering of Interspecies Cross-Reactivity, Thermostability, and Expression of a Bispecific 5T4 x CD3 DART^® Molecule for Treatment of Solid Tumors

by Renhua R. Huang, Michael Spliedt, Tom Kaufman, Sergey Gorlatov, Bhaswati Barat, Kalpana Shah, Jeffrey Gill, Kurt Stahl, Jennifer DiChiara, Qian Wang, Jonathan C. Li, Ralph Alderson, Paul A. Moore, Jennifer G. Brown, James Tamura, Xiaoyu Zhang, Ezio Bonvini and Gundo Diedrich

Antibodies 2025, 14(1), 7; https://doi.org/10.3390/antib14010007 - 17 Jan 2025

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties [...] Read more.

Background: Bispecific antibodies represent a promising class of biologics for cancer treatment. However, their dual specificity and complex structure pose challenges in the engineering process, often resulting in molecules with good functional but poor physicochemical properties. Method: To overcome limitations in the properties of an anti-5T4 x anti-CD3 (α5T4 x αCD3) DART molecule, a phage-display method was developed, which succeeded in simultaneously engineering cross-reactivity to the cynomolgus 5T4 ortholog, improving thermostability and the elevating expression level. Results: This approach generated multiple DART molecules that exhibited significant improvements in all three properties. The lead DART molecule demonstrated potent in vitro and in vivo anti-tumor activity. Although its clearance in human FcRn-transgenic mice was comparable to that of the parental molecule, faster clearance was observed in cynomolgus monkeys. The lead α5T4 x αCD3 DART molecule displayed no evidence of off-target binding or polyspecificity, suggesting that the increased affinity for the target may account for its accelerated clearance in cynomolgus monkeys. Conclusions: This may reflect target-mediated drug disposition (TMDD), a potential limitation of targeting 5T4, despite its limited expression in healthy tissues. Full article

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

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23 pages, 3442 KiB

Open AccessArticle

Phenomenological Modeling of Antibody Response from Vaccine Strain Composition

by Victor Ovchinnikov and Martin Karplus

Antibodies 2025, 14(1), 6; https://doi.org/10.3390/antib14010006 - 16 Jan 2025

The elicitation of broadly neutralizing antibodies (bnAbs) is a major goal of vaccine design for highly mutable pathogens, such as influenza, HIV, and coronavirus. Although many rational vaccine design strategies for eliciting bnAbs have been devised, their efficacies need to be evaluated in [...] Read more.

The elicitation of broadly neutralizing antibodies (bnAbs) is a major goal of vaccine design for highly mutable pathogens, such as influenza, HIV, and coronavirus. Although many rational vaccine design strategies for eliciting bnAbs have been devised, their efficacies need to be evaluated in preclinical animal models and in clinical trials. To improve outcomes for such vaccines, it would be useful to develop methods that can predict vaccine efficacies against arbitrary pathogen variants. As a step in this direction, here, we describe a simple biologically motivated model of antibody reactivity elicited by nanoparticle-based vaccines using only antigen amino acid sequences, parametrized with a small sample of experimental antibody binding data from influenza or SARS-CoV-2 nanoparticle vaccinations. Results: The model is able to recapitulate the experimental data to within experimental uncertainty, is relatively insensitive to the choice of the parametrization/training set, and provides qualitative predictions about the antigenic epitopes exploited by the vaccine, which are testable by experiment. For the mosaic nanoparticle vaccines considered here, model results suggest indirectly that the sera obtained from vaccinated mice contain bnAbs, rather than simply different strain-specific Abs. Although the present model was motivated by nanoparticle vaccines, we also apply it to a mutlivalent mRNA flu vaccination study, and demonstrate good recapitulation of experimental results. This suggests that the model formalism is, in principle, sufficiently flexible to accommodate different vaccination strategies. Finally, we show how the model could be used to rank the efficacies of vaccines with different antigen compositions. Conclusions: Overall, this study suggests that simple models of vaccine efficacy parametrized with modest amounts of experimental data could be used to compare the effectiveness of designed vaccines. Full article

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11 pages, 3139 KiB

Open AccessArticle

Targeting CD44 and EpCAM with Antibody Dye Conjugates for the Photoimmunotherapy of Prostate Cancer

by Isis Wolf, Susanne Schultze-Seemann, Christian Gratzke and Philipp Wolf

Antibodies 2025, 14(1), 5; https://doi.org/10.3390/antib14010005 - 9 Jan 2025

Background/Objectives: Photoimmunotherapy (PIT) is an innovative approach for the targeted therapy of cancer. In PIT, photosensitizer dyes are conjugated to tumor-specific antibodies for targeted delivery into cancer cells. Upon irradiation with visible light, the photosensitizer dye is activated and induces cancer-specific cell death. [...] Read more.

Background/Objectives: Photoimmunotherapy (PIT) is an innovative approach for the targeted therapy of cancer. In PIT, photosensitizer dyes are conjugated to tumor-specific antibodies for targeted delivery into cancer cells. Upon irradiation with visible light, the photosensitizer dye is activated and induces cancer-specific cell death. In the present article, we describe the PIT of prostate cancer (PC) as a therapeutic option for the targeted treatment of localized prostate cancer. Methods: We conjugated the silicon phthalocyanine dye WB692-CB2 to recombinant cysteine-modified anti-CD44 and anti-EpCAM antibodies via a maleimide linker and tested the antibody dye conjugates for PIT on PC cells and prostate cancer stem cell (PCSC)-like cells. Results: The anti-CD44 and anti-EpCAM antibody dye conjugates showed specific binding and high cytotoxicity against PC and PCSC-like cells following irradiation with red light. Combined treatment with both conjugates led to enhanced cytotoxic effects. Conclusions: PIT with our dye WB692-CB2 can serve as an effective focal therapy against prostate cancer, preserving the prostate gland and minimizing side effects. It can be employed during radical prostatectomy (RP) to treat residual tumor cells or lymph node metastases in areas where further surgical intervention is not feasible. Utilizing multiple conjugates against antigens expressed on differentiated PC and PCSC-like cells, such as CD44 and EpCAM, could be an effective method to eradicate residual cancer cells in heterogeneous tumors. This approach could reduce the risk of local recurrence after RP and thus increase the therapeutic outcome of PC patients. Full article

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

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28 pages, 1043 KiB

Open AccessReview

How Broadly Neutralising Antibodies Are Redefining Immunity to Influenza

by Rebecca Steventon, Lucas Stolle and Craig Peter Thompson

Antibodies 2025, 14(1), 4; https://doi.org/10.3390/antib14010004 - 7 Jan 2025

Recent avian influenza outbreaks have heightened global concern over viral threats with the potential to significantly impact human health. Influenza is particularly alarming due to its history of causing pandemics and zoonotic reservoirs. In response, significant progress has been made toward the development [...] Read more.

Recent avian influenza outbreaks have heightened global concern over viral threats with the potential to significantly impact human health. Influenza is particularly alarming due to its history of causing pandemics and zoonotic reservoirs. In response, significant progress has been made toward the development of universal influenza vaccines, largely driven by the discovery of broadly neutralising antibodies (bnAbs), which have the potential to neutralise a broad range of influenza viruses, extending beyond the traditional strain-specific response. This could lead to longer-lasting immunity, reducing the need for seasonal vaccinations, and improve preparedness for future pandemics. This review offers a comprehensive analysis of these antibodies, their application in clinical studies, and both their potential and possible shortcomings in managing future influenza outbreaks. Full article

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

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26 pages, 1514 KiB

Open AccessReview

Challenges and Insights in Absolute Quantification of Recombinant Therapeutic Antibodies by Mass Spectrometry: An Introductory Review

by Sarah Döring, Michael G. Weller, Yvonne Reinders, Zoltán Konthur and Carsten Jaeger

Antibodies 2025, 14(1), 3; https://doi.org/10.3390/antib14010003 - 7 Jan 2025

This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer and inflammatory, infectious, and autoimmune diseases. We trace their development from [...] Read more.

This review describes mass spectrometry (MS)-based approaches for the absolute quantification of therapeutic monoclonal antibodies (mAbs), focusing on technical challenges in sample treatment and calibration. Therapeutic mAbs are crucial for treating cancer and inflammatory, infectious, and autoimmune diseases. We trace their development from hybridoma technology and the first murine mAbs in 1975 to today’s chimeric and fully human mAbs. With increasing commercial relevance, the absolute quantification of mAbs, traceable to an international standard system of units (SI units), has attracted attention from science, industry, and national metrology institutes (NMIs). Quantification of proteotypic peptides after enzymatic digestion using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) has emerged as the most viable strategy, though methods targeting intact mAbs are still being explored. We review peptide-based quantification, focusing on critical experimental steps like denaturation, reduction, alkylation, choice of digestion enzyme, and selection of signature peptides. Challenges in amino acid analysis (AAA) for quantifying pure mAbs and peptide calibrators, along with software tools for targeted MS data analysis, are also discussed. Short explanations within each chapter provide newcomers with an overview of the field’s challenges. We conclude that, despite recent progress, further efforts are needed to overcome the many technical hurdles along the quantification workflow and discuss the prospects of developing standardized protocols and certified reference materials (CRMs) for this goal. We also suggest future applications of newer technologies for absolute mAb quantification. Full article

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19 pages, 1149 KiB

Open AccessArticle

Rat as a Predictive Model for Human Clearance and Bioavailability of Monoclonal Antibodies

by Jason D. Robarge, Kevin M. Budge, Lucy Her, Andrea M. Patterson and Patricia Brown-Augsburger

Antibodies 2025, 14(1), 2; https://doi.org/10.3390/antib14010002 - 24 Dec 2024

Background: The prediction of human clearance (CL) and subcutaneous (SC) bioavailability is a critical aspect of monoclonal antibody (mAb) selection for clinical development. While monkeys are a well-accepted model for predicting human CL, other preclinical species have been less-thoroughly explored. Unlike CL, predicting [...] Read more.

Background: The prediction of human clearance (CL) and subcutaneous (SC) bioavailability is a critical aspect of monoclonal antibody (mAb) selection for clinical development. While monkeys are a well-accepted model for predicting human CL, other preclinical species have been less-thoroughly explored. Unlike CL, predicting the bioavailability of SC administered mAbs in humans remains challenging as contributing factors are not well understood, and preclinical models have not been systematically evaluated. Methods: Non-clinical and clinical pharmacokinetic (PK) parameters were mined from public and internal sources for rats, cynomolgus monkeys, and humans. Intravenous (IV) and SC PK was determined in Sprague Dawley rats for fourteen mAbs without existing PK data. Together, we obtained cross-species data for 25 mAbs to evaluate CL and SC bioavailability relationships among rats, monkeys, and humans. Results: Rat and monkey CL significantly correlated with human CL and supported the use of species-specific exponents for body-weight-based allometric scaling. Notably, rat SC bioavailability significantly correlated with human SC bioavailability, while monkey SC bioavailability did not. Bioavailability also correlated with clearance. Conclusions: The rat model enables an early assessment of mAb PK properties, allowing discrimination among molecules in the discovery pipeline and prediction of human PK. Importantly, rat SC bioavailability significantly correlated with human SC bioavailability, which has not been observed with other species. Rats are cost-effective and efficient relative to monkeys and provide a valuable tool for pharmacokinetic predictions in therapeutic antibody discovery. Full article

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13 pages, 1765 KiB

Open AccessArticle

Autoantibodies, Oxidative Stress, and Nutritional State in Anorexia Nervosa

by Andrea Amerio, Eleonora Martino, Antonella Strangio, Andrea Aguglia, Andrea Escelsior, Benedetta Conio, Samir Giuseppe Sukkar and Daniele Saverino

Antibodies 2025, 14(1), 1; https://doi.org/10.3390/antib14010001 - 24 Dec 2024

Background/Objectives: Anorexia nervosa (AN) is a complex psychiatric disorder characterized by an extreme fear of gaining weight, leading to severe calorie restriction and weight loss. Beyond its psychiatric challenges, AN has significant physical consequences affecting multiple organ systems. Recent research has increasingly [...] Read more.

Background/Objectives: Anorexia nervosa (AN) is a complex psychiatric disorder characterized by an extreme fear of gaining weight, leading to severe calorie restriction and weight loss. Beyond its psychiatric challenges, AN has significant physical consequences affecting multiple organ systems. Recent research has increasingly focused on the interplay between autoantibodies, oxidative stress, and nutritional state in this condition. Methods: Ninety-six subjects were evaluated: forty-eight with AN and forty-eight normal-weight control subjects. The serum levels of IgG reactive to hypothalamic antigens, uric acid, and total antioxidant capacity were evaluated by laboratory assays. Results: Anti-hypothalamic autoantibodies were found in AN patients. Furthermore, increased levels of oxidative stress were reported, as measured by decreased serum uric acid and total antioxidant capacity (TAC), and they reduced with the disease duration and the restoration of body mass index (BMI). Finally, a decrease in both autoantibodies and oxidative stress was observed as patients’ clinical condition improved, as measured by time since diagnosis and BMI recovery. Conclusions: The clinical improvement of AN patients seems to be associated with a decrease in the autoimmune response to hypothalamic cellular antigens and a reduction in oxidative stress. Dysregulation of the immune system and oxidative stress appear to be interconnected in various diseases, including autoimmune and psychiatric disorders. These findings, although preliminary, may offer potential avenues for the treatment of this challenging condition. Full article

(This article belongs to the Section Humoral Immunity)

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