Antibodies

14 pages, 3179 KiB

Open AccessArticle

A Novel Tetravalent Bispecific Immune Cell Engager Activates Natural Killer Cells to Kill Cancer Cells without Mediating Fratricide

by Ge Yang, Shahryar Khoshtinat Nikkhoi, Hajar Owji, Geng Li, Mohammad Massumi, Jessica Cervelli, Venu Gopal Vandavasi and Arash Hatefi

Antibodies 2024, 13(3), 75; https://doi.org/10.3390/antib13030075 - 10 Sep 2024

Viewed by 171

Abstract

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by [...] Read more.

We previously reported the structure, affinity, and anticancer activity of a bivalent bispecific natural killer cell engager (BiKE) composed of one anti-CD16a VHH and one anti-HER2 VHH fused via a linker. In this study, we explored the engineering of a tetravalent BiKE by fusing two anti-CD16a and two anti-HER2 VHHs in tandem, using bivalent BiKE as a template. The tetravalent BiKE was genetically engineered, and its tertiary structure was predicted using in silico modeling. The antigen binding and affinity of the tetravalent BiKE were assessed using ELISA, flow cytometry, and biolayer interferometry. The ability of the BiKEs to kill cancer cells was evaluated through classical and residual antibody-dependent cellular cytotoxicity (ADCC) assays. Additionally, we investigated the potential for NK cell fratricide via CD16a-CD16a crosslinking. Our results revealed that the tetravalent BiKE exhibited at least 100-fold higher affinity toward its target antigens compared to its bivalent counterpart. The residual ADCC assay indicated that the tetravalent BiKE was more effective in killing cancer cells than the bivalent BiKE, attributable to its lower K_off value, which prolonged its binding to NK cell surfaces. Fratricide assays demonstrated that neither the bivalent nor the tetravalent BiKE mediated fratricide. Notably, our findings showed that daratumumab-induced NK fratricide was restricted to CD38-CD38 crosslinking and was not related to ADCC via CD16a-CD38 crosslinking. This study is the first in the literature to show the successful engineering of a tetravalent immune cell engager composed of tandem VHH units, which achieves high affinity and anticancer activity without mediating fratricide. Full article

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

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21 pages, 3673 KiB

Open AccessArticle

The Accurate Prediction of Antibody Deamidations by Combining High-Throughput Automated Peptide Mapping and Protein Language Model-Based Deep Learning

by Ben Niu, Benjamin Lee, Lili Wang, Wen Chen and Jeffrey Johnson

Antibodies 2024, 13(3), 74; https://doi.org/10.3390/antib13030074 - 10 Sep 2024

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Abstract

Therapeutic antibodies such as monoclonal antibodies (mAbs), bispecific and multispecific antibodies are pivotal in therapeutic protein development and have transformed disease treatments across various therapeutic areas. The integrity of therapeutic antibodies, however, is compromised by sequence liabilities, notably deamidation, where asparagine (N) and [...] Read more.

Therapeutic antibodies such as monoclonal antibodies (mAbs), bispecific and multispecific antibodies are pivotal in therapeutic protein development and have transformed disease treatments across various therapeutic areas. The integrity of therapeutic antibodies, however, is compromised by sequence liabilities, notably deamidation, where asparagine (N) and glutamine (Q) residues undergo chemical degradations. Deamidation negatively impacts the efficacy, stability, and safety of diverse classes of antibodies, thus necessitating the critical need for the early and accurate identification of vulnerable sites. In this article, a comprehensive antibody deamidation-specific dataset (n = 2285) of varied modalities was created by using high-throughput automated peptide mapping followed by supervised machine learning to predict the deamidation propensities, as well as the extents, throughout the entire antibody sequences. We propose a novel chimeric deep learning model, integrating protein language model (pLM)-derived embeddings with local sequence information for enhanced deamidation predictions. Remarkably, this model requires only sequence inputs, eliminating the need for laborious feature engineering. Our approach demonstrates state-of-the-art performance, offering a streamlined workflow for high-throughput automated peptide mapping and deamidation prediction, with the potential of broader applicability to other antibody sequence liabilities. Full article

(This article belongs to the Collection Computational Antibody and Antigen Design)

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9 pages, 914 KiB

Open AccessArticle

Therapeutic Drug Monitoring of Infliximab and Adalimumab through Concentration and Anti-Drug Antibodies Assessment; Comparison of Sanquin Diagnostics and Theradiag Assays

by Wim H. M. Vroemen, Shakira S. Agata, Joyce J. B. C. van Beers and Jan G. M. C. Damoiseaux

Antibodies 2024, 13(3), 73; https://doi.org/10.3390/antib13030073 - 5 Sep 2024

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Abstract

Background: Therapeutic drug monitoring of biological Tumor Necrosis Factor (TNF)-alpha inhibitors is of critical importance. In this study, the performance of practically advantageous chemiluminescent immunoassays of Theradiag, assessing Infliximab and Adalimumab serum concentrations and anti-drug antibodies (ADA) against these biologics, were compared to [...] Read more.

Background: Therapeutic drug monitoring of biological Tumor Necrosis Factor (TNF)-alpha inhibitors is of critical importance. In this study, the performance of practically advantageous chemiluminescent immunoassays of Theradiag, assessing Infliximab and Adalimumab serum concentrations and anti-drug antibodies (ADA) against these biologics, were compared to the Enzyme-Linked Immuno-Sorbent Assays (ELISAs) from Sanquin Diagnostics. Methods: Leftover serum samples (n = 80 for each parameter) from patients treated with Infliximab or Adalimumab were collected. Correlation and agreement analyses for serum concentration and ADAs, respectively, were performed. Both Theradiag ADA assays, an assay targeting both free and bound ADAs and an assay targeting solely free ADAs, were investigated and compared to the Sanquin Diagnostics ADA assay, targeting both free and bound ADAs. Results: Strong positive correlations were observed between the biologic concentration assessment of Infliximab (Spearman’s Rho = 0.91) and Adalimumab (Spearman’s Rho = 0.94). However, there appeared to be significant bias in the Theradiag assay when compared to Sanquin (Infliximab median (Confidence Interval (CI)) = 2.1 (1.7–2.6) µg/mL; Adalimumab median (CI) = 0.8 (0.5–0.9) µg/mL). Agreement analyses showed moderate to good agreement for the Theradiag and Sanquin Diagnostics ADA assays, when detecting both free and bound ADAs, for Infliximab (Cohen’s k = 0.717) and Adalimumab (Cohen’s k = 0.802). In contrast, the Theradiag ADA assay detecting solely free ADAs had zero to poor agreement for Infliximab (Cohen’s k = 0.458) and Adalimumab (Cohen’s k = 0.119), respectively. Conclusions: This study demonstrated strong correlations and good agreement between the Theradiag and Sanquin Diagnostics assays measuring Infliximab and Adalimumab serum concentrations and ADAs, both free and bound, against these biologics. Discordance analyses showed significantly decreased drug concentrations in the solely free assays, indicating that the combined detection of free and bound ADAs better aligns with drug levels. Full article

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14 pages, 2257 KiB

Open AccessArticle

Long-Term Immunity against SARS-CoV-2 Wild-Type and Omicron XBB.1.5 in Indonesian Residents after Vaccination and Infection

by Karismananda, Ammar Abdurrahman Hasyim, Akihiko Sakamoto, Kyouhei Yamagata, Kartika Hardianti Zainal, Desi Dwirosalia Ningsih Suparman, Ika Yustisia, Marhaen Hardjo, Syahrijuita Kadir, Mitsuhiro Iyori, Shigeto Yoshida and Yenni Yusuf

Antibodies 2024, 13(3), 72; https://doi.org/10.3390/antib13030072 - 2 Sep 2024

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Abstract

In the post-pandemic era, evaluating long-term immunity against COVID-19 has become increasingly critical, particularly in light of continuous SARS-CoV-2 mutations. This study aimed to assess the long-term humoral immune response in sera collected in Makassar. We measured anti-RBD IgG levels and neutralization capacity [...] Read more.

In the post-pandemic era, evaluating long-term immunity against COVID-19 has become increasingly critical, particularly in light of continuous SARS-CoV-2 mutations. This study aimed to assess the long-term humoral immune response in sera collected in Makassar. We measured anti-RBD IgG levels and neutralization capacity (NC) against both the Wild-Type (WT) Wuhan-Hu and Omicron XBB.1.5 variants across groups of COVID-19-vaccinated individuals with no booster (NB), single booster (SB), and double booster (DB). The mean durations since the last vaccination were 25.11 months, 19.24 months, and 16.9 months for the NB, SB, and DB group, respectively. Additionally, we evaluated the effect of breakthrough infection (BTI) history, with a mean duration since the last confirmed infection of 21.72 months. Our findings indicate fair long-term WT antibody (Ab) titers, with the DB group showing a significantly higher level than the other groups. Similarly, the DB group demonstrated the highest anti-Omicron XBB.1.5 Ab titer, yet it was insignificantly different from the other groups. Although the level of anti-WT Ab titers was moderate, we observed near-complete (96–97%) long-term neutralization against the WT pseudo-virus for all groups. There was a slight decrease in NC against Omicron XBB.1.5 compared to the WT among all groups, as DB group, SB group, and NB group showed 80.71 ± 3.9%, 74.29 ± 6.7%, and 67.2 ± 6.3% neutralization activity, respectively. A breakdown analysis based on infection and vaccine status showed that booster doses increase the NC against XBB.1.5, particularly in individuals without BTI. Individuals with BTI demonstrate a better NC compared to their counterpart uninfected individuals with the same number of booster doses. Our findings suggest that long-term immunity against SARS-CoV-2 persists and is effective against the mutant variant. Booster doses enhance the NC, especially among uninfected individuals. Full article

(This article belongs to the Special Issue Review Collection on Humoral Immunity)

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14 pages, 2745 KiB

Open AccessArticle

Functional Activity of Cytokine-Induced Killer Cells Enhanced by CAR-CD19 Modification or by Soluble Bispecific Antibody Blinatumomab

by Silvia Zaninelli, Silvia Panna, Sarah Tettamanti, Giusi Melita, Andrea Doni, Francesca D’Autilia, Rut Valgardsdottir, Elisa Gotti, Alessandro Rambaldi, Josée Golay and Martino Introna

Antibodies 2024, 13(3), 71; https://doi.org/10.3390/antib13030071 - 30 Aug 2024

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Abstract

Strategies to increase the anti-tumor efficacy of cytokine-induced killer cells (CIKs) include genetic modification with chimeric antigen receptors (CARs) or the addition of soluble T-cell engaging bispecific antibodies (BsAbs). Here, CIKs were modified using a transposon system integrating two distinct anti-CD19 CARs (CAR-MNZ [...] Read more.

Strategies to increase the anti-tumor efficacy of cytokine-induced killer cells (CIKs) include genetic modification with chimeric antigen receptors (CARs) or the addition of soluble T-cell engaging bispecific antibodies (BsAbs). Here, CIKs were modified using a transposon system integrating two distinct anti-CD19 CARs (CAR-MNZ and CAR-BG2) or combined with soluble CD3xCD19 BsAb blinatumomab (CIK + Blina). CAR-MNZ bearing the CD28-OX40-CD3ζ signaling modules, and CAR-BG2, designed on the Tisagenlecleucel CAR sequence (Kymriah^®), carrying the 4-1BB and CD3ζ signaling elements, were employed. After transfection and CIK expansion, cells expressed CAR-CD19 to a similar extent (35.9% CAR-MNZ and 17.7% CAR-BG2). In vitro evaluations demonstrated robust proliferation and cytotoxicity (~50% cytotoxicity) of CARCIK-MNZ, CARCIK-BG2, and CIK + Blina against CD19⁺ target cells, suggesting similar efficacy. All effectors formed an increased number of synapses, activated NFAT and NFkB, and secreted IL-2 and IFN-ɣ upon encountering targets. CIK + Blina displayed strongest NFAT and IFN-ɣ induction, whereas CARCIK-BG2 demonstrated superior synapse formation. All the effectors have shown therapeutic activity in vivo against the CD19⁺ Daudi tumor model, with CARCIK cells showing a more durable response compared to CIK + Blina, likely due to the short half-life of Blina in this model. Full article

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12 pages, 1099 KiB

Open AccessArticle

A Physiologically Based Pharmacokinetic Model Relates the Subcutaneous Bioavailability of Monoclonal Antibodies to the Saturation of FcRn-Mediated Recycling in Injection-Site-Draining Lymph Nodes

by Felix Stader, Cong Liu, Abdallah Derbalah, Hiroshi Momiji, Xian Pan, Iain Gardner, Masoud Jamei and Armin Sepp

Antibodies 2024, 13(3), 70; https://doi.org/10.3390/antib13030070 - 15 Aug 2024

Viewed by 875

Abstract

The bioavailability of a monoclonal antibody (mAb) or another therapeutic protein after subcutaneous (SC) dosing is challenging to predict from first principles, even if the impact of injection site physiology and drug properties on mAb bioavailability is generally understood. We used a physiologically [...] Read more.

The bioavailability of a monoclonal antibody (mAb) or another therapeutic protein after subcutaneous (SC) dosing is challenging to predict from first principles, even if the impact of injection site physiology and drug properties on mAb bioavailability is generally understood. We used a physiologically based pharmacokinetic model to predict pre-systemic clearance after SC administration mechanistically by incorporating the FcRn salvage pathway in antigen-presenting cells (APCs) in peripheral lymph nodes, draining the injection site. Clinically observed data of the removal rate of IgG from the arm as well as its plasma concentration after SC dosing were mostly predicted within the 95% confidence interval. The bioavailability of IgG was predicted to be 70%, which mechanistically relates to macropinocytosis in the draining lymph nodes and transient local dose-dependent partial saturation of the FcRn receptor in the APCs, resulting in higher catabolism and consequently less drug reaching the systemic circulation. The predicted free FcRn concentration was reduced to 40–45%, reaching the minimum 1–2 days after the SC administration of IgG, and returned to baseline after 8–12 days, depending on the site of injection. The model predicted the uptake into APCs, the binding affinity to FcRn, and the dose to be important factors impacting the bioavailability of a mAb. Full article

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18 pages, 1915 KiB

Open AccessArticle

Dynamics of IgM and IgG Antibody Response Profile against Linear B-Cell Epitopes from Exoerythrocytic (CelTOS and TRAP) and Erythrocytic (CyRPA) Phases of Plasmodium vivax: Follow-Up Study

by Cinthia Magalhães Rodolphi, Isabela Ferreira Soares, Ada da Silva Matos, Rodrigo Nunes Rodrigues-da-Silva, Marcelo Urbano Ferreira, Lilian Rose Pratt-Riccio, Paulo Renato Rivas Totino, Kézia Katiani Gorza Scopel and Josué da Costa Lima-Junior

Antibodies 2024, 13(3), 69; https://doi.org/10.3390/antib13030069 - 15 Aug 2024

Viewed by 644

Abstract

Malaria is a serious health problem worldwide affecting mainly children and socially vulnerable people. The biological particularities of P. vivax, such as the ability to generate dormant liver stages, the rapid maturation of gametocytes, and the emergence of drug resistance, have contributed [...] Read more.

Malaria is a serious health problem worldwide affecting mainly children and socially vulnerable people. The biological particularities of P. vivax, such as the ability to generate dormant liver stages, the rapid maturation of gametocytes, and the emergence of drug resistance, have contributed to difficulties in disease control. In this context, developing an effective vaccine has been considered a fundamental tool for the efficient control and/or elimination of vivax malaria. Although recombinant proteins have been the main strategy used in designing vaccine prototypes, synthetic immunogenic peptides have emerged as a viable alternative for this purpose. Considering, therefore, that in the Brazilian endemic population, little is known about the profile of the humoral immune response directed to synthetic peptides that represent different P. vivax proteins, the present work aimed to map the epitope-specific antibodies’ profiles to synthetic peptides representing the linear portions of the ookinete and sporozoite cell passage protein (CelTOS), thrombospondin-related adhesive protein (TRAP), and cysteine-rich protective antigen (CyRPA) proteins in the acute (AC) and convalescent phases (Conv30 and Conv180 after infection) of vivax malaria. The results showed that the studied subjects responded to all proteins for at least six months following infection. For IgM, a few individuals (3–21%) were positive during the acute phase of the disease; the highest frequencies were observed for IgG (28–57%). Regarding the subclasses, IgG2 and IgG3 stood out as the most prevalent for all peptides. During the follow-up, the stability of IgG was observed for all peptides. Only one significant positive correlation was observed between IgM and exposure time. We conclude that for all the peptides, the immunodominant epitopes are recognized in the exposed population, with similar frequency and magnitude. However, if the antibodies detected in this study are potential protectors, this needs to be investigated. Full article

(This article belongs to the Section Humoral Immunity)

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16 pages, 5274 KiB

Open AccessArticle

Novel Monoclonal Antibody Specific toward Amyloid-β Binds to a Unique Epitope within the N-Terminal Region

by Giavanna Paterno, Brenda D. Moore, Brach M. Bell, Kimberly-Marie M. Gorion, Yong Ran, Stefan Prokop, Todd E. Golde and Benoit I. Giasson

Antibodies 2024, 13(3), 68; https://doi.org/10.3390/antib13030068 - 9 Aug 2024

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Abstract

Amyloid-β (Aβ) deposition throughout the neuroaxis is a classical hallmark of several neurodegenerative diseases, most notably Alzheimer’s disease (AD). Aβ peptides of varied length and diverse structural conformations are deposited within the parenchyma and vasculature in the brains of individuals with AD. Neuropathologically, [...] Read more.

Amyloid-β (Aβ) deposition throughout the neuroaxis is a classical hallmark of several neurodegenerative diseases, most notably Alzheimer’s disease (AD). Aβ peptides of varied length and diverse structural conformations are deposited within the parenchyma and vasculature in the brains of individuals with AD. Neuropathologically, Aβ pathology can be assessed using antibodies to label and characterize their features, which in turn leads to a more extensive understanding of the pathological process. In the present study, we generated a novel monoclonal antibody, which we found to be specific for the N-terminal region of Aβ. This antibody reacted to amyloid precursor protein expressed in cultured cells and labels Aβ plaques and cerebral amyloid angiopathy in brain tissue from a mouse model of amyloidosis as well as post-mortem brain tissue from patients diagnosed with AD. This highly specific novel antibody will serve as a unique tool for future studies investigating Aβ deposition in novel mouse models and cross-sectional studies using post-mortem human tissue. Full article

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16 pages, 3270 KiB

Open AccessArticle

Identification of Conserved Linear Epitopes on Viral Protein 2 of Foot-and-Mouth Disease Virus Serotype O by Monoclonal Antibodies 6F4.D11.B6 and 8D6.B9.C3

by Wantanee Tommeurd, Kanyarat Thueng-in, Sirin Theerawatanasirikul, Nongnaput Tuyapala, Sukontip Poonsuk, Nantawan Petcharat, Nattarat Thangthamniyom and Porntippa Lekcharoensuk

Antibodies 2024, 13(3), 67; https://doi.org/10.3390/antib13030067 - 7 Aug 2024

Viewed by 793

Abstract

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide [...] Read more.

Foot-and-mouth disease (FMD) is a highly infectious disease of cloven-hoofed animals with a significant economic impact. Early diagnosis and effective prevention and control could reduce the spread of the disease which could possibly minimize economic losses. Epitope characterization based on monoclonal antibodies provide essential information for developing diagnostic assays and vaccine designs. In this study, monoclonal antibodies raised against FMD virus (FMDV) were produced. Sixty-six monoclonal antibodies demonstrated strong reactivity and specificity to FMDV. The purified monoclonal antibodies were further used for bio-panning to select phage expressing specific epitopes from phage-displayed 12 mer-peptide library. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. Two hybridoma clones secreted monoclonal antibodies recognizing linear epitopes on VP2 of FMDV serotype O. The non-neutralizing monoclonal antibody 6F4.D11.B6 recognized the residues 67–78 on antigenic site 2 resinding in VP2, while the neutralizing monoclonal antibody 8D6.B9.C3 recognized a novel linear epitope encompassing residues 115–126 on VP2. This information and the FMDV-specific monoclonal antibodies provide valuable sources for further study and application in diagnosis, therapeutics and vaccine designs to strengthen the disease prevention and control measures. Full article

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16 pages, 3881 KiB

Open AccessArticle

Enhanced N-Glycan Profiling of Therapeutic Monoclonal Antibodies through the Application of Upper-Hinge Middle-Up Level LC-HRMS Analysis

by Natalia Mesonzhnik, Anton Belushenko, Polina Novikova, Alexey Kukharenko and Mikhail Afonin

Antibodies 2024, 13(3), 66; https://doi.org/10.3390/antib13030066 - 6 Aug 2024

Viewed by 528

Abstract

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up [...] Read more.

Therapeutic monoclonal antibodies (mAbs) are crucial in modern medicine due to their effectiveness in treating various diseases. However, the structural complexity of mAbs, particularly their glycosylation patterns, presents challenges for quality control and biosimilarity assessment. This study explores the use of upper-hinge middle-up (UHMU)-level ultra-high-performance liquid chromatography–high-resolution mass spectrometry (LC-HRMS) analysis to improve N-glycan profiling of mAbs. Two specific enzymes, known as IgG degradation enzymes (IGDEs), were used to selectively cleave therapeutic mAbs above the hinge region to separate antibody subunits for further Fc glycan analysis by means of the UHMU/LC-HRMS workflow. The complexity of the mass spectra of IGDEs-digested mAbs was significantly reduced compared to the intact MS level, enabling reliable assignment and relative quantitation of paired Fc glycoforms. The results of the UHMU/LC-HRMS analysis of nine approved therapeutics highlight the significance of this approach for in-depth glycoform profiling. Full article

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

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12 pages, 6357 KiB

Open AccessArticle

The Seraph 100^® Microbind Affinity Blood Filter Does Not Alter Levels of Circulating or Mucosal Antibodies in Critical COVID-19 Patients

by Tonia L. Conner, Pooja Vir, Eric D. Laing, Ian J. Stewart, Edward Mitre and Kathleen P. Pratt

Antibodies 2024, 13(3), 65; https://doi.org/10.3390/antib13030065 - 6 Aug 2024

Viewed by 604

Abstract

PURIFY-OBS-1 is an observational study evaluating the safety and efficacy of Seraph 100^® Microbind Affinity Blood Filter (Seraph 100) use for COVID-19 patients with respiratory failure admitted to the intensive care unit (ICU). The Seraph 100 is a hemoperfusion device containing heparin-coated [...] Read more.

PURIFY-OBS-1 is an observational study evaluating the safety and efficacy of Seraph 100^® Microbind Affinity Blood Filter (Seraph 100) use for COVID-19 patients with respiratory failure admitted to the intensive care unit (ICU). The Seraph 100 is a hemoperfusion device containing heparin-coated beads that can bind to, and reduce levels of, some circulating pathogens and inflammatory molecules. This study evaluated whether treatment with the Seraph 100 affected circulating and mucosal antibody levels in critically ill COVID-19 subjects. SARS-CoV-2 anti-spike and anti-nucleocapsid IgG and IgA levels in serum were evaluated at enrollment and on days 1, 4, 7, and 28 after Seraph 100 application, while anti-spike and nucleocapsid IgG, IgA, and secretory IgA levels in tracheal aspirates were evaluated at enrollment and on days 1, 2, 3, 7, and 28. Serum samples were also collected from the pre- and post-filter lines at 1 and 4 h following Seraph 100 application to evaluate the direct impact of the filter on circulating antibody levels. Treatment with the Seraph 100 did not alter the levels of circulating or mucosal antibodies in critically ill COVID-19 subjects admitted to the ICU. Full article

(This article belongs to the Section Humoral Immunity)

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24 pages, 12834 KiB

Open AccessArticle

Prevention of Blood Incompatibility Related Hemagglutination: Blocking of Antigen A on Red Blood Cells Using In Silico Designed Recombinant Anti-A scFv

by Saleha Hafeez and Najam Us Sahar Sadaf Zaidi

Antibodies 2024, 13(3), 64; https://doi.org/10.3390/antib13030064 - 1 Aug 2024

Viewed by 1022

Abstract

Critical blood shortages plague healthcare systems, particularly in lower-income and middle-income countries. This affects patients requiring regular transfusions and creates challenges during emergencies where universal blood is vital. To address these shortages and support blood banks during emergencies, this study reports a method [...] Read more.

Critical blood shortages plague healthcare systems, particularly in lower-income and middle-income countries. This affects patients requiring regular transfusions and creates challenges during emergencies where universal blood is vital. To address these shortages and support blood banks during emergencies, this study reports a method for increasing the compatibility of blood group A red blood cells (RBCs) by blocking surface antigen-A using anti-A single chain fragment variable (scFv). To enhance stability, the scFv was first modified with the addition of interdomain disulfide bonds. The most effective location for this modification was found to be H44-L232 of mutant-1a scFv. ScFv was then produced from E.coli BL21(DE3) and purified using a three-step process. Purified scFvs were then used to block maximum number of antigens-A on RBCs, and it was found that only monomers were functional, while dimers formed through incorrect domain-swapping were non-functional. These antigen-blocked RBCs displayed no clumping in hemagglutination testing with incompatible blood plasma. The dissociation constant K_D was found to be 0.724 μM. Antigen-blocked RBCs have the potential to be given to other blood groups during emergencies. This innovative approach could significantly increase the pool of usable blood, potentially saving countless lives. Full article

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

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10 pages, 380 KiB

Open AccessCommunication

Knowledge of the Serological Response to the Third BNT162b2 Vaccination May Influence Compliance of Healthcare Workers to Booster Dose

by Avi Magid, Khetam Hussein, Halima Dabaja-Younis, Moran Szwarcwort-Cohen, Ronit Almog, Michal Mekel, Avi Weissman, Gila Hyams, Vardit Gepstein, Netanel A. Horowitz, Hagar Cohen Saban, Jalal Tarabeia, Michael Halberthal and Yael Shachor-Meyouhas

Antibodies 2024, 13(3), 63; https://doi.org/10.3390/antib13030063 - 1 Aug 2024

Viewed by 552

Abstract

Background: Previous studies showed that the fourth SARS-CoV-2 vaccine dose has a protective effect against infection, as well as against severe disease and death. This study aimed to examine whether knowledge of a high-level antibody after the third dose may reduce compliance to [...] Read more.

Background: Previous studies showed that the fourth SARS-CoV-2 vaccine dose has a protective effect against infection, as well as against severe disease and death. This study aimed to examine whether knowledge of a high-level antibody after the third dose may reduce compliance to the fourth booster dose among healthcare workers (HCWs). Methods: We conducted a prospective cohort study among HCWs vaccinated with the first three doses at Rambam Healthcare Campus, a tertiary hospital in northern Israel. Participants underwent a serological test before the fourth booster vaccine was offered to all of them, with results provided to participants. The population was divided into two groups, namely those with antibodies below 955 AU/mL and those with 955 AU/mL and higher, a cutoff found protective in a previous study. Multiple logistic regression was carried out to compare the compliance to the fourth booster between the two groups, adjusted for demographic and clinical variables. Results: After adjusting for the confounding variables, the compliance was higher in those with antibody levels below 955 AU/mL (OR = 1.41, p = 0.05, 95% CI 1.10–1.96). In addition, male sex and age of 60 years and above were also associated with higher vaccination rates (OR = 2.28, p < 0.001, 95% CI 1.64–3.17), (OR = 1.14, p = 0.043, 95% CI 1.06–1.75), respectively. Conclusions: Knowledge of the antibody status may affect compliance with the booster dose. Considering waning immunity over time, reduced compliance may affect the protection of HCWs who declined the fourth dose. Full article

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16 pages, 1684 KiB

Open AccessArticle

Novel Flow Cytometry Method Detecting Complement C1q Bound to Blood Type A/B IgG Antibody for Preventing Severe Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation

by Tsutomu Ishizuka, Kazuhiro Iwadoh, Hiroshi Kataoka, Junichi Hoshino, Kosaku Nitta and Hideki Ishida

Antibodies 2024, 13(3), 62; https://doi.org/10.3390/antib13030062 - 1 Aug 2024

Viewed by 711

Abstract

We aimed to develop a novel method for measuring the complement-binding ability of anti-blood type antibodies (ab-Abs), the flow cytometry method for the complement C1q test (FCM-C1q) for detecting antibody-mediated rejection (AMR) caused by ab-Abs in ABO-incompatible kidney transplantation (ABOI-KTx). FCM-C1q distribution was [...] Read more.

We aimed to develop a novel method for measuring the complement-binding ability of anti-blood type antibodies (ab-Abs), the flow cytometry method for the complement C1q test (FCM-C1q) for detecting antibody-mediated rejection (AMR) caused by ab-Abs in ABO-incompatible kidney transplantation (ABOI-KTx). FCM-C1q distribution was surveyed in 44 healthy participants and 43 dialysis patients (Cohort A). The relationship between AMR and FCM-C1q levels was examined along with ab-Ab titers by the flow cytometry method for the IgG test (FCM-IgG) in 62 ABOI-KTx patients (Cohort B). FCM-IgG and C1q levels were significantly higher in type O participants than in A/B participants in Cohort A. There were minimal differences in the distribution of FCM-IgG and C1q between dialysis and healthy participants. Sixteen cases were suspected of acute rejections (ARs) in Cohort B, of whom nine had AR clinically. One patient with severe AMR was highly suspected of hyperacute rejection along with another patient with severe AMR. Their postoperative FCM-C1q and FCM-IgG levels were elevated. Another two patients showed high FCM-IgG and C1q levels before KTx, and these levels remained low after KTx with no or mild rejection. In conclusion, our results suggest that a high positivity rate for FCM-C1q may predict moderate to severe AMR caused by ab-Abs and poor prognosis in ABOI-KTx. Full article

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

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15 pages, 3024 KiB

Open AccessReview

Allogeneic HLA Humoral Immunogenicity and the Prediction of Donor-Specific HLA Antibody Development

by Vadim Jucaud

Antibodies 2024, 13(3), 61; https://doi.org/10.3390/antib13030061 - 24 Jul 2024

Viewed by 538

Abstract

The development of de novo donor-specific HLA antibodies (dnDSAs) following solid organ transplantation is considered a major risk factor for poor long-term allograft outcomes. The prediction of dnDSA development is a boon to transplant recipients, yet the assessment of allo-HLA immunogenicity remains imprecise. [...] Read more.

The development of de novo donor-specific HLA antibodies (dnDSAs) following solid organ transplantation is considered a major risk factor for poor long-term allograft outcomes. The prediction of dnDSA development is a boon to transplant recipients, yet the assessment of allo-HLA immunogenicity remains imprecise. Despite the recent technological advances, a comprehensive evaluation of allo-HLA immunogenicity, which includes both B and T cell allorecognition, is still warranted. Recent studies have proposed using mismatched HLA epitopes (antibody and T cell) as a prognostic biomarker for humoral alloimmunity. However, the identification of immunogenic HLA mismatches has not progressed despite significant improvements in the identification of permissible mismatches. Certainly, the prediction of dnDSA development may benefit permissible HLA mismatched organ transplantations, personalized immunosuppression, and clinical trial design. However, characteristics that go beyond the listing of mismatched HLA antibody epitopes and T cell epitopes, such as the generation of HLA T cell epitope repertoires, recipient’s HLA class II phenotype, and immunosuppressive regiments, are required for the precise assessment of allo-HLA immunogenicity. Full article

(This article belongs to the Special Issue Review Collection on Humoral Immunity)

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17 pages, 1927 KiB

Open AccessReview

Application of Monoclonal Antibodies against Naturally Occurring Bioactive Ingredients

by Shunsuke Fujii, Takuhiro Uto, Hiroaki Hayashi, Waraporn Putalun, Seiichi Sakamoto, Hiroyuki Tanaka and Yukihiro Shoyama

Antibodies 2024, 13(3), 60; https://doi.org/10.3390/antib13030060 - 24 Jul 2024

Viewed by 678

Abstract

Monoclonal antibodies (Mabs) are widely used in a variety of fields, including protein identification, life sciences, medicine, and natural product chemistry. This review focuses on Mabs against naturally occurring active compounds. The preparation of Mabs against various active compounds began in the 1980s, [...] Read more.

Monoclonal antibodies (Mabs) are widely used in a variety of fields, including protein identification, life sciences, medicine, and natural product chemistry. This review focuses on Mabs against naturally occurring active compounds. The preparation of Mabs against various active compounds began in the 1980s, and now there are fewer than 50 types. Eastern blotting, which was developed as an antibody staining method for low-molecular-weight compounds, is useful for its ability to visually represent specific components. In this method, a mixture of lower-molecular-weight compounds, particularly glycosides, are separated by thin-layer chromatography (TLC). The compounds are then transferred to a membrane by heating, followed by treatment with potassium periodate (KIO₄) to open the sugar moiety of the glycoside on the membrane to form an aldehyde group. Proteins are then added to form Schiff base bonds to enable adsorption on the membrane. A Mab is bound to the glycoside moiety on the membrane and reacts with a secondary antibody to produce color. Double Eastern blotting, which enables the simultaneous coloration of two glycosides, can be used to evaluate quality and estimate pharmacological effects. An example of staining by Eastern blotting and a component search based on the results will also be presented. A Mab-associated affinity column is a method for isolating antigen molecules in a single step. However, the usefulness of the wash fractions that are not bound to the affinity column is unknown. Therefore, we designated the wash fraction the “knockout extract”. Comparing the nitric oxide (NO) production of a glycyrrhizin (GL)-knockout extract of licorice with a licorice extract revealed that the licorice extract is stronger. Therefore, the addition of GL to the GL-knockout extract of licorice increased NO production. This indicates that GL has synergic activity with the knockout extract. The GL-knockout extract of licorice inhibited high-glucose-induced epithelial–mesenchymal transition in NRK-52E cells, primarily by suppressing the Notch2 pathway. The real active constituent in licorice may be constituents other than GL, which is the causative agent of pseudohyperaldosteronism. This suggests that a GL-knockout extract of licorice may be useful for the treatment of diabetic nephritis. Full article

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10 pages, 213 KiB

Open AccessArticle

Adverse Events of PD-1, PD-L1, CTLA-4, and LAG-3 Immune Checkpoint Inhibitors: An Analysis of the FDA Adverse Events Database

by Connor Frey and Mahyar Etminan

Antibodies 2024, 13(3), 59; https://doi.org/10.3390/antib13030059 - 17 Jul 2024

Viewed by 964

Abstract

This study aimed to identify the 25 most prevalent adverse events (AEs) associated with FDA-approved immune checkpoint inhibitors (ICIs)—specifically, PD-1, PD-L1, CTLA-4, and LAG-3 inhibitors—using data from the FDA Adverse Events Reporting System (FAERS), a publicly available repository of reported drug adverse events, [...] Read more.

This study aimed to identify the 25 most prevalent adverse events (AEs) associated with FDA-approved immune checkpoint inhibitors (ICIs)—specifically, PD-1, PD-L1, CTLA-4, and LAG-3 inhibitors—using data from the FDA Adverse Events Reporting System (FAERS), a publicly available repository of reported drug adverse events, and AERSMine, an open-access pharmacovigilance tool, to investigate these adverse events. For PD-1 inhibitors, the most common AEs were diarrhea, fatigue, and pyrexia, with notable instances of neutropenia and hypothyroidism, particularly with toripalimab and dostarlimab. PD-L1 inhibitors also frequently caused pyrexia, diarrhea, and fatigue, with interstitial lung disease and hypothyroidism showing a class effect, and drug-specific AEs such as hepatotoxicity and chills. CTLA-4 inhibitors predominantly resulted in diarrhea and colitis, with ipilimumab frequently causing pyrexia and rash, while tremelimumab exhibited unique AEs such as biliary tract infection. The LAG-3 inhibitor relatlimab reported fewer AEs, including pyrexia and pneumonia. Rare but significant AEs across all inhibitors included myocarditis and myasthenia gravis. This study provides a detailed overview of the 25 most common AEs associated with ICIs, offering valuable insights for clinical decision-making and AE management. Further research is necessary to elucidate the mechanisms underlying these AEs and to develop targeted interventions to enhance the safety and efficacy of ICI therapy in patients with cancer. Full article

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

13 pages, 1159 KiB

Open AccessReview

A Comparison of Natural and Therapeutic Anti-IgE Antibodies

by Monique Vogel and Paul Engeroff

Antibodies 2024, 13(3), 58; https://doi.org/10.3390/antib13030058 - 16 Jul 2024

Viewed by 1075

Abstract

Immunoglobulin E (IgE) plays a critical role for the immune system, fighting against parasites, toxins, and cancer. However, when it reacts to allergens without proper regulation, it can cause allergic reactions, including anaphylaxis, through a process initiated by effector cells such as basophils [...] Read more.

Immunoglobulin E (IgE) plays a critical role for the immune system, fighting against parasites, toxins, and cancer. However, when it reacts to allergens without proper regulation, it can cause allergic reactions, including anaphylaxis, through a process initiated by effector cells such as basophils and mast cells. These cells display IgE on their surface, bound to the high-affinity IgE receptor FcεRI. A cross-linking antigen then triggers degranulation and the release of inflammatory mediators from the cells. Therapeutic monoclonal anti-IgE antibodies such as omalizumab, disrupt this process and are used to manage IgE-related conditions such as severe allergic asthma and chronic spontaneous urticaria. Interestingly, naturally occurring anti-IgE autoantibodies circulate at surprisingly high levels in healthy humans and mice and may thus be instrumental in regulating IgE activity. Although many open questions remain, recent studies have shed new light on their role as IgE regulators and their mechanism of action. Here, we summarize the latest insights on natural anti-IgE autoantibodies, and we compare their functional features to therapeutic monoclonal anti-IgE autoantibodies. Full article

(This article belongs to the Special Issue Review Collection on Humoral Immunity)

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18 pages, 5533 KiB

Open AccessArticle

Humanization of Pan-HLA-DR mAb 44H10 Hinges on Critical Residues in the Antibody Framework

by Audrey Kassardjian, Danton Ivanochko, Brian Barber, Arif Jetha and Jean-Philippe Julien

Antibodies 2024, 13(3), 57; https://doi.org/10.3390/antib13030057 - 16 Jul 2024

Viewed by 860

Abstract

Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions [...] Read more.

Reducing the immunogenicity of animal-derived monoclonal antibodies (mAbs) for use in humans is critical to maximize therapeutic effectiveness and preclude potential adverse events. While traditional humanization methods have primarily focused on grafting antibody Complementarity-Determining Regions (CDRs) on homologous human antibody scaffolds, framework regions can also play essential roles in antigen binding. Here, we describe the humanization of the pan-HLA-DR mAb 44H10, a murine antibody displaying significant involvement of the framework region in antigen binding. Using a structure-guided approach, we identify and restore framework residues that directly interact with the antigen or indirectly modulate antigen binding by shaping the antibody paratope and engineer a humanized antibody with affinity, biophysical profile, and molecular binding basis comparable to that of the parental 44H10 mAb. As a humanized molecule, this antibody holds promise as a scaffold for the development of MHC class II-targeting therapeutics and vaccines. Full article

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11 pages, 422 KiB

Open AccessArticle

by Connor Frey and Mahyar Etminan

Antibodies 2024, 13(3), 56; https://doi.org/10.3390/antib13030056 - 15 Jul 2024

Viewed by 883

Abstract

The advancement of immuno-oncology has brought about a significant shift in cancer treatment methods, with antibody-based immune checkpoint inhibitors like atezolizumab leading the way in this regard. However, the use of this checkpoint blockade can result in immune-related adverse events due to increased [...] Read more.

The advancement of immuno-oncology has brought about a significant shift in cancer treatment methods, with antibody-based immune checkpoint inhibitors like atezolizumab leading the way in this regard. However, the use of this checkpoint blockade can result in immune-related adverse events due to increased T-cell activity. The full spectrum of these events is not yet completely understood. In this study, the United States FDA Adverse Event Reporting System (FAERS) was utilized to investigate immune-related adverse events linked with the use of atezolizumab. The study identified forty-nine immune-related adverse events that affected multiple organ systems, including cardiovascular, respiratory, hematologic, hepatic, renal, gastrointestinal, neurologic, musculoskeletal, dermatologic, endocrine, and systemic disorders. The strongest signals for relative risk occurred for immune-mediated encephalitis (RR = 93.443), autoimmune myocarditis (RR = 56.641), immune-mediated hepatitis (RR = 49.062), immune-mediated nephritis (RR = 40.947), and autoimmune arthritis (RR = 39.382). Despite the morbidity associated with these adverse events, emerging evidence suggests potential associations with improved survival outcomes. Overall, this report sheds light on the widespread immune-related adverse events that cause significant morbidity and mortality in patients with cancer being treated with atezolizumab and brings attention to them for the clinicians treating these patients. Full article

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

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25 pages, 4197 KiB

Open AccessArticle

NK Cytotoxicity Mediated by NK-92 Cell Lines Expressing Combinations of Two Allelic Variants for FCGR3

by Marta Freitas Monteiro, Maria Papaserafeim, Matteo Andreani, Aline Réal, Athanasios Kouklas, Daniela Reis Galvão, Jörg D. Seebach and Gisella L. Puga Yung

Antibodies 2024, 13(3), 55; https://doi.org/10.3390/antib13030055 - 12 Jul 2024

Viewed by 826

Abstract

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by [...] Read more.

Natural killer (NK) cells play an important role in the surveillance of viral infections and cancer. NK cell antibody-dependent cellular cytotoxicity (ADCC) and direct cytotoxicity are mediated by the recognition of antibody-coated target cells through the Fc gamma receptor IIIA (FcγRIIIa/CD16) and by ligands of activating/inhibitory NK receptors, respectively. Allelic variants of the FCGR3A gene include the high-affinity single-nucleotide polymorphism (SNP) rs396991 (V176F), which is associated with the efficacy of monoclonal antibody (mAb) therapies, and the SNP rs10127939 (L66H/R). The contribution of FCGR3A SNPs to NK cell effector functions remains controversial; therefore, we generated a panel of eight NK-92 cell lines expressing specific combinations of these SNPs and tested their cytotoxicities. NK-92 cells were stably transfected with plasmids containing different combinations of FCGR3A SNPs. Messenger RNA and FcγRIIIa/CD16 cell surface expressions were detected using new generation sequencing (NGS) and flow cytometry, respectively. All FcγRIIIa/CD16-transfected NK-92 cell lines exhibited robust ADCC against three different target cell lines with minor differences. In addition, enhanced direct NK cytotoxicity against K562 target cells was observed, suggesting a mechanistic role of FcγRIIIa/CD16 in direct NK cytotoxicity. In conclusion, we generated eight FcγRIIIa/CD16-transfected NK-92 cell lines carrying different combinations of two of the most studied FCGR3A SNPs, representing the major genotypes described in the European population. The functional characterization of these cell lines revealed differences in ADCC and direct NK cytotoxicity that may have implications for the design of adoptive cancer immunotherapies using NK cells and tumor antigen-directed mAbs. Full article

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

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25 pages, 7363 KiB

Open AccessArticle

Inter-Antibody Variability in the Clinical Pharmacokinetics of Monoclonal Antibodies Characterized Using Population Physiologically Based Pharmacokinetic Modeling

by Mokshada Kumar, Sravani Lanke, Alka Yadav, Mfonabasi Ette, Donald E. Mager and Dhaval K. Shah

Antibodies 2024, 13(3), 54; https://doi.org/10.3390/antib13030054 - 9 Jul 2024

Viewed by 2137

Abstract

The objective of this work was to develop a population physiologically based pharmacokinetic (popPBPK) model to characterize the variability in the clinical PK of monoclonal antibodies (mAbs) following intravenous (IV) and subcutaneous (SC) administration. An extensive literature search was conducted and clinical PK [...] Read more.

The objective of this work was to develop a population physiologically based pharmacokinetic (popPBPK) model to characterize the variability in the clinical PK of monoclonal antibodies (mAbs) following intravenous (IV) and subcutaneous (SC) administration. An extensive literature search was conducted and clinical PK data for FDA-approved as well as non-approved mAbs were collected. Training and validation datasets of 44 and 9 mAbs exhibiting linear pharmacokinetics were used for model development. The variability in antibody PK was captured by accounting for different rate constants of pinocytosis (CL_up) and intracellular degradation (k_deg) for different mAbs. Typical values for CL_up and k_deg and their respective inter-antibody variabilities (

ω_{C l u p}

,

ω_{K d e g})

were estimated to be 0.32 L/h/L and 26.1

h^{- 1}

(73% and 46%). Varied absorption profiles following SC dosing were characterized by incorporating inter-antibody variability in local degradation (k_SC) and rate of lymphatic uptake (S_Lu) of mAbs. Estimates for typical k_SC and S_Lu values, and

ω_{K s c}, ω_{S_L u},

were found to be 0.0015

h^{- 1}

and 0.54 (193%, and 49%). FDA-approved mAbs showed less local degradation (0.0014

h^{- 1}

vs. 0.0038

h^{- 1}

) compared with other clinically tested mAbs, whereas no substantial differences in physiological processes involved in disposition were observed. To evaluate the generalizability of estimated PK parameters and model validation, the final popPBPK model was used to simulate the range of expected PK for mAbs following SC administration of nine different mAbs that were not used for model-building purposes. The predicted PK of all nine mAbs was within the expected range specified a priori. Thus, the popPBPK model presented here may serve as a tool to predict the clinical PK of mAbs with linear disposition before administering them to humans. The model may also support preclinical-to-clinical translation and ‘first-in-human’ dose determination for mAbs. Full article

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

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8 pages, 769 KiB

Open AccessCommunication

A Bead-Based Nonradioactive Immunoassay for Autoantibody Testing in a Mouse Model of Myasthenia Gravis

by Afrin Bahauddin, Kyra Curtis, Jutatip Guptarak and Ruksana Huda

Antibodies 2024, 13(3), 53; https://doi.org/10.3390/antib13030053 - 1 Jul 2024

Viewed by 787

Abstract

Serological testing for anti-acetylcholine receptor (AChR) autoantibodies is not only crucial for the diagnosing, disease monitoring, and treatment management of patients with myasthenia gravis (MG) but also for preclinical studies utilizing MG disease models. However, there are no specific guidelines on which methods [...] Read more.

Serological testing for anti-acetylcholine receptor (AChR) autoantibodies is not only crucial for the diagnosing, disease monitoring, and treatment management of patients with myasthenia gravis (MG) but also for preclinical studies utilizing MG disease models. However, there are no specific guidelines on which methods to use in clinical diagnostic or research laboratories to detect or quantify any MG-specific autoantibodies. Conventional autoantibody assays, particularly those for anti-AChR antibodies, are varied and mostly laboratory-specific. Here, we report our new nonradioactive immunoprecipitation–immunoblotting method for assessing autoantibodies (anti-AChR antibodies) in a mouse model of MG. This simple, efficient, reproducible, and cost-effective assay appears superior to the enzyme-linked immunosorbent assay but comparable to the radioimmunoprecipitation or cell-based assay in specificity and sensitivity. Thus, the newly developed assay can serve as a valuable alternative to classical assays and is suitable for routine testing of AChR-specific autoantibodies in preclinical studies. The further optimization of our assay may facilitate its application in the diagnosis and therapeutic management of patients with MG. Full article

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

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21 pages, 3608 KiB

Open AccessArticle

Characterization of the Charge Heterogeneity of a Monoclonal Antibody That Binds to Both Cation Exchange and Anion Exchange Columns under the Same Binding Conditions

by Ming-Ching Hsieh, Jingming Zhang, Liangjie Tang, Cheng-Yen Huang, Yang Shen, Alice Matathia, Jun Qian and Babita Saxena Parekh

Antibodies 2024, 13(3), 52; https://doi.org/10.3390/antib13030052 - 30 Jun 2024

Viewed by 1058

Abstract

Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A [...] Read more.

Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody’s surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography. Full article

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15 pages, 8868 KiB

Open AccessArticle

¹⁷⁷Lu Anti-Angiogenic Radioimmunotherapy Targeting ATP Synthase in Gastric Cancer Model

by Bok-Nam Park, Young-Sil An, Su-Min Kim, Su-Jin Lee, Yong-Jin Park and Joon-Kee Yoon

Antibodies 2024, 13(3), 51; https://doi.org/10.3390/antib13030051 - 27 Jun 2024

Viewed by 699

Abstract

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope ¹⁷⁷Lu using DOTA as a chelating agent. ¹⁷⁷Lu-DOTA-ATPS mAb demonstrated high labeling [...] Read more.

This study investigated a novel radioimmunotherapy strategy for targeting tumor angiogenesis. We developed a radiopharmaceutical complex by labeling an anti-adenosine triphosphate synthase (ATPS) monoclonal antibody (mAb) with the radioisotope ¹⁷⁷Lu using DOTA as a chelating agent. ¹⁷⁷Lu-DOTA-ATPS mAb demonstrated high labeling efficiency (99.0%) and stability in serum. MKN-45 cancer cells exhibited the highest cellular uptake, which could be specifically blocked by unlabeled ATPS mAb. In mice, ¹⁷⁷Lu-DOTA-ATPS mAb accumulated significantly in tumors, with a tumor uptake of 16.0 ± 1.5%ID/g on day 7. ¹⁷⁷Lu-DOTA-ATPS mAb treatment significantly reduced the viability of MKN-45 cells in a dose-dependent manner. In a xenograft tumor model, this radioimmunotherapy strategy led to substantial tumor growth inhibition (82.8%). Furthermore, combining ¹⁷⁷Lu-DOTA-ATPS mAb with sunitinib, an anti-angiogenic drug, enhanced the therapeutic efficacy of sunitinib in the mouse model. Our study successfully developed ¹⁷⁷Lu-DOTA-ATPS mAb, a radioimmunotherapy agent targeting tumor blood vessels. This approach demonstrates significant promise for inhibiting tumor growth, both as a single therapy and in combination with other anti-cancer drugs. Full article

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

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20 pages, 2248 KiB

Open AccessArticle

Development, Optimization and Evaluation of a Sensitive Enzyme-Linked Immunosorbent Assay (ELISA) Prototype for Detection of Chicken-Based IgY Polyclonal Antibodies against Toxins of D. polylepis Venom

by Stephen Wilson Kpordze, Gideon Mutie Kikuvi, James Hungo Kimotho and Victor Atunga Mobegi

Antibodies 2024, 13(3), 50; https://doi.org/10.3390/antib13030050 - 21 Jun 2024

Cited by 1 | Viewed by 871

Abstract

Life-threatening medical issues can result from snakebite, and hence this is a public health concern. In many tropical and subtropical nations such as Kenya, where a wide variety of poisonous snakes are prevalent, diagnosis of snakebite in health facilities is imperative. Different antivenoms [...] Read more.

Life-threatening medical issues can result from snakebite, and hence this is a public health concern. In many tropical and subtropical nations such as Kenya, where a wide variety of poisonous snakes are prevalent, diagnosis of snakebite in health facilities is imperative. Different antivenoms are needed to treat the venom of different snake species. Nonetheless, it might be difficult for medical professionals to identify the exact snake species that envenomated a patient due to the similarities of several snake envenomations’ clinical symptoms. Therefore, the necessity for an assay or technique for identifying venomous species is critical. The current study sought to develop a sensitive ELISA prototype for the detection of D. polylepis venom in Kenya using generated chicken-based IgY polyclonal antibodies. Serum samples containing specific chicken-based IgY antibodies previously raised against D. polylepis venom toxins were used in the assay development. ELISA parameters were optimized, and the developed assay was assessed for applicability. The limit of detection (LoD) of the ELISA for neurotoxic venoms was determined to be 0.01 µg/mL. Successful discrimination between neurotoxic and cytotoxic venoms was achieved by the ensuing inhibition ELISA assay. The developed assay showed the capability of identifying venoms in blood samples (from spiked and venom-challenged blood samples) of BALB/c mice, providing compelling evidence of the strategy’s usefulness. This assay could help physicians diagnose and manage victims of snakebites through the evaluation of clinical samples. Full article

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

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Antibodies, Volume 13, Issue 3 (September 2024) – 26 articles

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