Antibodies

20 pages, 8964 KB

Open AccessArticle

A Robust, High-Titer, Semi-Automated, and In-Culture Antibody-Capturing Transient CHO Platform Technology

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

Antibodies 2025, 14(4), 87; https://doi.org/10.3390/antib14040087 (registering DOI) - 11 Oct 2025

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Abstract

Background: Recent advances in antibody discovery technologies, especially progress in de novo synthesis through machine learning, have imposed a significant production challenge for the generation of a large diversity of antibodies against nearly any target of interest. There is a demand for the [...] Read more.

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

(This article belongs to the Special Issue A Festschrift Celebrating Dr. Dimiter Stanchev Dimitrov: Antibodies, Innovation, and Impact on Infectious Disease and Cancer Research)

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17 pages, 2840 KB

Open AccessArticle

Structural and Functional Characterization of Anti-SARS-CoV-2 Spike Monoclonal Antibodies Produced via Bicistronic Expression in CHO Cells

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

Antibodies 2025, 14(4), 86; https://doi.org/10.3390/antib14040086 - 9 Oct 2025

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Abstract

Background: Recombinant monoclonal antibodies (mAbs) represent the fastest-growing sector of the biopharmaceutical industry, with their efficient expression being a key technological factor for scalability. Objectives: In this study we compared the performance of two bicistronic vectors, which alternate the positions of the light [...] Read more.

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

(This article belongs to the Special Issue A Festschrift Celebrating Dr. Dimiter Stanchev Dimitrov: Antibodies, Innovation, and Impact on Infectious Disease and Cancer Research)

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16 pages, 1228 KB

Open AccessArticle

Monoclonal Antibodies Can Aid in the Culture-Based Detection and Differentiation of Mucorales Fungi—The Flesh-Eating Pathogens Apophysomyces and Saksenaea as an Exemplar

by Christopher R. Thornton and Genna E. Davies

Antibodies 2025, 14(4), 85; https://doi.org/10.3390/antib14040085 - 7 Oct 2025

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Abstract

Background: The frequency of necrotising cutaneous and soft tissue infections caused by the Mucorales fungi Apophysomyces and Sakasenaea is increasing. The absence of sophisticated diagnostic technologies in low- and middle-income countries (LMICs) means that detection of cutaneous mucormycosis continues to rely on culture [...] Read more.

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

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

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25 pages, 5098 KB

Open AccessArticle

Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

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

Antibodies 2025, 14(4), 84; https://doi.org/10.3390/antib14040084 - 6 Oct 2025

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Abstract

Background/Objectives: The ErbB protein family plays a critical role in the progression of various solid tumors, and HER3 has been implicated in resistance mechanisms to multiple cancer therapies due to its ability to form heterodimers with other ErbB receptors, thereby activating pathways that [...] Read more.

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

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

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16 pages, 2388 KB

Open AccessArticle

Generation Using Phage-Display of pH-Dependent Antibodies Against the Tumor-Associated Antigen AXL

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

Antibodies 2025, 14(4), 83; https://doi.org/10.3390/antib14040083 - 30 Sep 2025

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Abstract

Background/Objectives: Tumor-associated antigens are not tumor-specific antigens but proteins that are overexpressed by tumor cells and also weakly expressed at the surface of healthy tissues. Therefore, some side effects are observed when targeted by therapeutic antibodies, a phenomenon named “on-target, off-tumor toxicity”. As [...] Read more.

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

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

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22 pages, 346 KB

Open AccessReview

Serum Factors in Primary Podocytopathies

by Edward John Filippone and John L. Farber

Antibodies 2025, 14(4), 82; https://doi.org/10.3390/antib14040082 - 28 Sep 2025

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Abstract

Primary podocytopathies, including minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS), are caused by a circulating factor or factors injurious to the podocyte. An immunologic origin seems likely based on responsiveness to corticosteroids or other immunosuppressive agents, including calcineurin inhibitors targeting T-cells [...] Read more.

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

(This article belongs to the Section Humoral Immunity)

14 pages, 1274 KB

Open AccessArticle

Purification and Characterization of Immunoglobulin Y (IgY) Targeting Surface Antigen 1 (SAG1) of Toxoplasma gondii

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

Antibodies 2025, 14(4), 81; https://doi.org/10.3390/antib14040081 - 26 Sep 2025

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Abstract

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite responsible for toxoplasmosis, a disease with significant health implications for humans and animals. The surface antigen 1 (SAG1) of T. gondii is a major immunodominant protein that facilitates host cell invasion, [...] Read more.

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

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

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Antibodies, Volume 14, Issue 4 (December 2025) – 7 articles

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