Antibodies

21 pages, 3673 KiB

Open AccessEditor’s ChoiceArticle

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

Viewed by 1524

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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14 pages, 2745 KiB

Open AccessEditor’s ChoiceArticle

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

Viewed by 1305

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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18 pages, 5533 KiB

Open AccessEditor’s ChoiceArticle

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 1678

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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14 pages, 2589 KiB

Open AccessEditor’s ChoiceArticle

Identification of a Fully Human Antibody VH Domain Targeting Anaplastic Lymphoma Kinase (ALK) with Applications in ALK-Positive Solid Tumor Immunotherapy

by Chuan Chen, Zehua Sun, Zening Wang, Seungmin Shin, Abigail Berrios, John W. Mellors, Dimiter S. Dimitrov and Wei Li

Antibodies 2024, 13(2), 39; https://doi.org/10.3390/antib13020039 - 7 May 2024

Viewed by 2068

Abstract

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length [...] Read more.

The anaplastic lymphoma kinase (ALK, CD247) is a potential target for antibody-based therapy. However, no antibody-based therapeutics targeting ALK have entered clinical trials, necessitating the development of novel antibodies with unique therapeutic merits. Single-domain antibodies (sdAb) bear therapeutic advantages compared to the full-length antibody including deeper tumor penetration, cost-effective production and fast washout from normal tissues. In this study, we identified a human immunoglobulin heavy chain variable domain (VH domain) (VH20) from an in-house phage library. VH20 exhibits good developability and high specificity with no off-target binding to ~6000 human membrane proteins. VH20 efficiently bound to the glycine-rich region of ALK with an EC₅₀ of 0.4 nM and a KD of 6.54 nM. Both VH20-based bispecific T cell engager (TCE) and chimeric antigen receptor T cells (CAR Ts) exhibited potent cytolytic activity to ALK-expressing tumor cells in an ALK-dependent manner. VH20 CAR Ts specifically secreted proinflammatory cytokines including IL-2, TNFα and IFNγ after incubation with ALK-positive cells. To our knowledge, this is the first reported human single-domain antibody against ALK. Our in vitro characterization data indicate that VH20 could be a promising ALK-targeting sdAb with potential applications in ALK-expressing tumors, including neuroblastoma (NBL) and non-small cell lung cancer. 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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25 pages, 3699 KiB

Open AccessEditor’s ChoiceArticle

A Conditionally Activated Cytosol-Penetrating Antibody for TME-Dependent Intracellular Cargo Delivery

by Carolin Sophie Dombrowsky, Dominic Happel, Jan Habermann, Sarah Hofmann, Sasi Otmi, Benny Cohen and Harald Kolmar

Antibodies 2024, 13(2), 37; https://doi.org/10.3390/antib13020037 - 2 May 2024

Cited by 1 | Viewed by 3200

Abstract

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody [...] Read more.

Currently, therapeutic and diagnostic applications of antibodies are primarily limited to cell surface-exposed and extracellular proteins. However, research has been conducted on cell-penetrating peptides (CPP), as well as cytosol-penetrating antibodies, to overcome these limitations. In this context, a heparin sulfate proteoglycan (HSPG)-binding antibody was serendipitously discovered, which eventually localizes to the cytosol of target cells. Functional characterization revealed that the tested antibody has beneficial cytosol-penetrating capabilities and can deliver cargo proteins (up to 70 kDa) to the cytosol. To achieve tumor-specific cell targeting and cargo delivery through conditional activation of the cell-penetrating antibody in the tumor microenvironment, a single-chain Fc fragment (scFv) and a V_L domain were isolated as masking units. Several in vitro assays demonstrated that fusing the masking protein with a cleavable linker to the cell penetration antibody results in the inactivation of antibody cell binding and internalization. Removal of the mask via MMP-9 protease cleavage, a protease that is frequently overexpressed in the tumor microenvironment (TME), led to complete regeneration of binding and cytosol-penetrating capabilities. Masked and conditionally activated cytosol-penetrating antibodies have the potential to serve as a modular platform for delivering protein cargoes addressing intracellular targets in tumor cells. Full article

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19 pages, 4024 KiB

Open AccessEditor’s ChoiceArticle

Balancing the Affinity and Tumor Cell Binding of a Two-in-One Antibody Simultaneously Targeting EGFR and PD-L1

by Julia Harwardt, Felix Klaus Geyer, Katrin Schoenfeld, David Baumstark, Vera Molkenthin and Harald Kolmar

Antibodies 2024, 13(2), 36; https://doi.org/10.3390/antib13020036 - 2 May 2024

Viewed by 2705

Abstract

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the [...] Read more.

The optimization of the affinity of monoclonal antibodies is crucial for the development of drug candidates, as it can impact the efficacy of the drug and, thus, the dose and dosing regimen, limit adverse effects, and reduce therapy costs. Here, we present the affinity maturation of an EGFR×PD-L1 Two-in-One antibody for EGFR binding utilizing site-directed mutagenesis and yeast surface display. The isolated antibody variants target EGFR with a 60-fold-improved affinity due to the replacement of a single amino acid in the CDR3 region of the light chain. The binding properties of the Two-in-One variants were confirmed using various methods, including BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding experiments using flow cytometry as well as real-time interaction cytometry. An AlphaFold-based model predicted that the amino acid exchange of tyrosine to glutamic acid enables the formation of a salt bridge to an arginine at EGFR position 165. This easily adaptable approach provides a strategy for the affinity maturation of bispecific antibodies with respect to the binding of one of the two antigens. Full article

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

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14 pages, 8324 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Cross-Reactivity of N6AMT1 Antibodies with Aurora Kinase A: An Example of Antibody-Specific Non-Specificity

by Baiba Brūmele, Evgeniia Serova, Aleksandra Lupp, Mihkel Suija, Margit Mutso and Reet Kurg

Antibodies 2024, 13(2), 33; https://doi.org/10.3390/antib13020033 - 22 Apr 2024

Viewed by 2480

Abstract

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with [...] Read more.

Primary antibodies are one of the main tools used in molecular biology research. However, the often-occurring cross-reactivity of primary antibodies complicates accurate data analysis. Our results show that three commercial polyclonal antibodies raised against N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) strongly cross-react with endogenous and recombinant mitosis-associated protein Aurora kinase A (AURKA). The cross-reactivity was verified through immunofluorescence, immunoblot, and immunoprecipitation assays combined with mass spectrometry. N6AMT1 and AURKA are evolutionarily conserved proteins that are vital for cellular processes. Both proteins share the motif ENNPEE, which is unique to only these two proteins. We suggest that N6AMT1 antibodies recognise this motif in N6AMT1 and AURKA proteins and exhibit an example of “specific” non-specificity. This serves as an example of the importance of controls and critical data interpretation in molecular biology research. Full article

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19 pages, 771 KiB

Open AccessEditor’s ChoiceReview

A Narrative Review of the State of the Art of CCR4-Based Therapies in Cutaneous T-Cell Lymphomas: Focus on Mogamulizumab and Future Treatments

by Corrado Zengarini, Alba Guglielmo, Martina Mussi, Giovanna Motta, Claudio Agostinelli, Elena Sabattini, Bianca Maria Piraccini and Alessandro Pileri

Antibodies 2024, 13(2), 32; https://doi.org/10.3390/antib13020032 - 22 Apr 2024

Viewed by 2398

Abstract

The CCR4 receptor is a pivotal target in cutaneous T-cell lymphoma (CTCL) therapy due to its role in impairing immune responses against malignant T-cells and expression profiles. Monoclonal antibodies like mogamulizumab effectively bind to CCR4, reducing tumour burden and enhancing patient outcomes by [...] Read more.

The CCR4 receptor is a pivotal target in cutaneous T-cell lymphoma (CTCL) therapy due to its role in impairing immune responses against malignant T-cells and expression profiles. Monoclonal antibodies like mogamulizumab effectively bind to CCR4, reducing tumour burden and enhancing patient outcomes by inhibiting the receptor’s interaction with ligands, thereby hindering malignant T-cell migration and survival. Combining CCR4 antibodies with chemotherapy, radiation, and other drugs is being explored for synergistic effects. Additionally, small-molecular inhibitors, old pharmacological agents interacting with CCR4, and CAR-T therapies are under investigation. Challenges include drug resistance, off-target effects, and patient selection, addressed through ongoing trials refining protocols and identifying biomarkers. Despite advancements, real-life data for most of the emerging treatments are needed to temper expectations. In conclusion, CCR4-targeted therapies show promise for CTCL management, but challenges persist. Continued research aims to optimise treatments, enhance outcomes, and transform CTCL management. This review aims to elucidate the biological rationale and the several agents under various stages of development and clinical evaluation with the actual known data. Full article

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17 pages, 3047 KiB

Open AccessEditor’s ChoiceArticle

Fcγ-Receptor-Independent Controlled Activation of CD40 Canonical Signaling by Novel Therapeutic Antibodies for Cancer Therapy

by Karsten Beckmann, Carmen Reitinger, Xianglei Yan, Anna Carle, Eva Blümle, Nicole Jurkschat, Claudia Paulmann, Sandra Prassl, Linda V. Kazandjian, Karin Loré, Falk Nimmerjahn and Stephan Fischer

Antibodies 2024, 13(2), 31; https://doi.org/10.3390/antib13020031 - 18 Apr 2024

Cited by 1 | Viewed by 2335

Abstract

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have [...] Read more.

The activation of CD40-mediated signaling in antigen-presenting cells is a promising therapeutic strategy to promote immune responses against tumors. Most agonistic anti-CD40 antibodies currently in development require the Fcγ-receptor (FcγR)-mediated crosslinking of CD40 molecules for a meaningful activation of CD40 signaling but have limitations due to dose-limiting toxicities. Here we describe the identification of CD40 antibodies which strongly stimulate antigen-presenting cells in an entirely FcγR-independent manner. These Fc-silenced anti-CD40 antibodies induce an efficient upregulation of costimulatory receptors and cytokine release by dendritic cells. Finally, the most active identified anti-CD40 antibody shows activity in humanized mice. More importantly, there are no signs of obvious toxicities. These studies thus demonstrate the potent activation of antigen-presenting cells with anti-CD40 antibodies lacking FcγR-binding activity and open the possibility for an efficacious and safe combination therapy for cancer patients. Full article

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

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18 pages, 2417 KiB

Open AccessEditor’s ChoiceArticle

Enhanced Characterization of Lysine-Linked Antibody Drug Conjugates Enabled by Middle-Down Mass Spectrometry and Higher-Energy Collisional Dissociation-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation

by Eleanor Watts, Aarti Bashyal, Sean D. Dunham, Christopher M. Crittenden and Jennifer S. Brodbelt

Antibodies 2024, 13(2), 30; https://doi.org/10.3390/antib13020030 - 17 Apr 2024

Cited by 1 | Viewed by 2143

Abstract

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow [...] Read more.

As the development of new biotherapeutics advances, increasingly sophisticated tandem mass spectrometry methods are needed to characterize the most complex molecules, including antibody drug conjugates (ADCs). Lysine-linked ADCs, such as trastuzumab-emtansine (T-DM1), are among the most heterogeneous biotherapeutics. Here, we implement a workflow that combines limited proteolysis with HCD-triggered EThcD and UVPD mass spectrometry for the characterization of the resulting middle-down large-sized peptides of T-DM1. Fifty-three payload-containing peptides were identified, ranging in mass from 1.8 to 16.9 kDa, and leading to the unambiguous identification of 46 out of 92 possible conjugation sites. In addition, seven peptides were identified containing multiple payloads. The characterization of these types of heterogeneous peptides represents an important step in unraveling the combinatorial nature of lysine-conjugated ADCs. Full article

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

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17 pages, 1931 KiB

Open AccessEditor’s ChoiceReview

Beyond bNAbs: Uses, Risks, and Opportunities for Therapeutic Application of Non-Neutralising Antibodies in Viral Infection

by Kahlio Mader and Lynn B. Dustin

Antibodies 2024, 13(2), 28; https://doi.org/10.3390/antib13020028 - 3 Apr 2024

Cited by 1 | Viewed by 2363

Abstract

The vast majority of antibodies generated against a virus will be non-neutralising. However, this does not denote an absence of protective capacity. Yet, within the field, there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, NAbs) of [...] Read more.

The vast majority of antibodies generated against a virus will be non-neutralising. However, this does not denote an absence of protective capacity. Yet, within the field, there is typically a large focus on antibodies capable of directly blocking infection (neutralising antibodies, NAbs) of either specific viral strains or multiple viral strains (broadly-neutralising antibodies, bNAbs). More recently, a focus on non-neutralising antibodies (nNAbs), or neutralisation-independent effects of NAbs, has emerged. These can have additive effects on protection or, in some cases, be a major correlate of protection. As their name suggests, nNAbs do not directly neutralise infection but instead, through their Fc domains, may mediate interaction with other immune effectors to induce clearance of viral particles or virally infected cells. nNAbs may also interrupt viral replication within infected cells. Developing technologies of antibody modification and functionalisation may lead to innovative biologics that harness the activities of nNAbs for antiviral prophylaxis and therapeutics. In this review, we discuss specific examples of nNAb actions in viral infections where they have known importance. We also discuss the potential detrimental effects of such responses. Finally, we explore new technologies for nNAb functionalisation to increase efficacy or introduce favourable characteristics for their therapeutic applications. Full article

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

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21 pages, 354 KiB

Open AccessEditor’s ChoiceReview

Factors Governing B Cell Recognition of Autoantigen and Function in Type 1 Diabetes

by Lindsay E. Bass and Rachel H. Bonami

Antibodies 2024, 13(2), 27; https://doi.org/10.3390/antib13020027 - 1 Apr 2024

Cited by 1 | Viewed by 3087

Abstract

Islet autoantibodies predict type 1 diabetes (T1D) but can be transient in murine and human T1D and are not thought to be directly pathogenic. Rather, these autoantibodies signal B cell activity as antigen-presenting cells (APCs) that present islet autoantigen to diabetogenic T cells [...] Read more.

Islet autoantibodies predict type 1 diabetes (T1D) but can be transient in murine and human T1D and are not thought to be directly pathogenic. Rather, these autoantibodies signal B cell activity as antigen-presenting cells (APCs) that present islet autoantigen to diabetogenic T cells to promote T1D pathogenesis. Disrupting B cell APC function prevents T1D in mouse models and has shown promise in clinical trials. Autoantigen-specific B cells thus hold potential as sophisticated T1D biomarkers and therapeutic targets. B cell receptor (BCR) somatic hypermutation is a mechanism by which B cells increase affinity for islet autoantigen. High-affinity B and T cell responses are selected in protective immune responses, but immune tolerance mechanisms are known to censor highly autoreactive clones in autoimmunity, including T1D. Thus, different selection rules often apply to autoimmune disease settings (as opposed to protective host immunity), where different autoantigen affinity ceilings are tolerated based on variations in host genetics and environment. This review will explore what is currently known regarding B cell signaling, selection, and interaction with T cells to promote T1D pathogenesis. Full article

(This article belongs to the Special Issue Autoimmune and Inflammatory Rheumatic Diseases: Potential Biomarkers, Antibodies Response and New Therapies)

16 pages, 3136 KiB

Open AccessEditor’s ChoiceArticle

Anti-CD99 Antibody Therapy Triggers Macrophage-Dependent Ewing Cell Death In Vitro and Myeloid Cell Recruitment In Vivo

by Allison F. O’Neill, Evelyn M. Nguyen, Evelyn D. Maldonado, Matthew R. Chang, Jiusong Sun, Quan Zhu and Wayne A. Marasco

Antibodies 2024, 13(1), 24; https://doi.org/10.3390/antib13010024 - 18 Mar 2024

Cited by 1 | Viewed by 2665

Abstract

Background: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. Methods: We [...] Read more.

Background: Ewing sarcoma is a rare tumor of the bone or soft tissues characterized by diffuse membranous staining for CD99. As this tumor remains incurable in the metastatic, relapsed, and refractory settings, we explored the downstream immune implications of targeting CD99. Methods: We discovered a human anti-CD99 antibody (NOA2) by phagemid panning and investigated NOA2 immune cell-mediated cytotoxicity in vitro and in vivo focusing on the myeloid cell compartment, given that M2 macrophages are present in human tumors and associated with a poor prognosis. Results: NOA2 is capable of inducing immune effector cell-mediated Ewing death in vitro via engagement of macrophages. Mice with metastatic Ewing tumors, treated with NOA2, experience tumor growth arrest and an associated increase in intratumoral macrophages. Further, incubation of macrophages and Ewing cells with NOA2, in conjunction with anti-PILRα antibody blockade in vitro, results in the reactivation of previously dormant macrophages possibly due to interrupted binding of Ewing CD99 to macrophage PILRα. Conclusions: These studies are the first to demonstrate the role of human immune effector cells in anti-CD99-mediated Ewing tumor death. We propose that the engagement of CD99 by NOA2 results in the recruitment of intratumoral macrophages. In addition, interruption of the CD99:PILRα checkpoint axis may be a relevant therapeutic approach to activate tumor-associated macrophages. Full article

(This article belongs to the Topic Anti-Tumor Immune Responses 2.0)

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16 pages, 1179 KiB

Open AccessEditor’s ChoiceReview

Diagnosis and Management of Catastrophic Antiphospholipid Syndrome and the Potential Impact of the 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria

by Lucas Jacobs, Nader Wauters, Yahya Lablad, Johann Morelle and Maxime Taghavi

Antibodies 2024, 13(1), 21; https://doi.org/10.3390/antib13010021 - 12 Mar 2024

Cited by 5 | Viewed by 6459

Abstract

Catastrophic antiphospholipid syndrome (CAPS) is a rare and life-threatening condition characterized by the persistence of antiphospholipid antibodies and occurrence of multiple vascular occlusive events. CAPS currently remains a diagnostic challenge and requires urgent treatment. The diagnosis of CAPS is made difficult by classification [...] Read more.

Catastrophic antiphospholipid syndrome (CAPS) is a rare and life-threatening condition characterized by the persistence of antiphospholipid antibodies and occurrence of multiple vascular occlusive events. CAPS currently remains a diagnostic challenge and requires urgent treatment. The diagnosis of CAPS is made difficult by classification criteria used as diagnostic criteria in clinical practice, knowledge derived from retrospective data and case reports, confounding clinical and biological features, and its rapid onset and mortality. The absence of prospective studies of CAPS limits the strength of evidence for guideline treatment protocols. This comprehensive review summarizes the current understanding of the disease, and discusses how the 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria impact the definition and therapeutic management of CAPS, which is considered the most severe form of APS. The correct integration of 2023 ACR/EULAR APS classification criteria is poised to facilitate CAPS diagnosis, particularly in critical situations, offering a promising avenue for improved outcomes. Full article

(This article belongs to the Special Issue Antiphospholipid Antibodies)

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19 pages, 1201 KiB

Open AccessEditor’s ChoiceReview

Performance Characteristics and Limitations of the Available Assays for the Detection and Quantitation of Monoclonal Free Light Chains and New Emerging Methodologies

by Hannah V. Giles and Kamaraj Karunanithi

Antibodies 2024, 13(1), 19; https://doi.org/10.3390/antib13010019 - 11 Mar 2024

Cited by 1 | Viewed by 2601

Abstract

Light chain measurements form an essential component of the testing strategy for the detection and monitoring of patients with suspected and/or proven plasma cell disorders. Urine-based electrophoretic assays remain at the centre of the international guidelines for response assessment but the supplementary role [...] Read more.

Light chain measurements form an essential component of the testing strategy for the detection and monitoring of patients with suspected and/or proven plasma cell disorders. Urine-based electrophoretic assays remain at the centre of the international guidelines for response assessment but the supplementary role of serum-free light chain (FLC) assays in response assessment and the detection of disease progression due to their increased sensitivity has been increasingly recognised since their introduction in 2001. Serum FLC assays have also been shown to be prognostic across the spectrum of plasma cell disorders and are now incorporated into risk stratification scores for patients with monoclonal gammopathy of undetermined significance (MGUS), smouldering multiple myeloma, and light chain amyloidosis (AL amyloidosis), as well as being incorporated into the criteria for defining symptomatic multiple myeloma. There are now multiple different commercially available serum FLC assays available with differing performance characteristics, which are discussed in this review, along with the implications of these for patient monitoring. Finally, newer methodologies for the identification and characterisation of monoclonal FLC, including modifications to electrophoretic techniques, mass spectrometry-based assays and Amylite, are also described along with the relevant published data available regarding the performance of each assay. Full article

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

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21 pages, 5864 KiB

Open AccessEditor’s ChoiceArticle

Episomal Vectors for Stable Production of Recombinant Proteins and Engineered Antibodies

by Ian Fallahee and Daniel Hawiger

Antibodies 2024, 13(1), 18; https://doi.org/10.3390/antib13010018 - 11 Mar 2024

Viewed by 3071

Abstract

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody–drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of [...] Read more.

There is tremendous interest in the production of recombinant proteins, particularly bispecific antibodies and antibody–drug conjugates for research and therapeutic use. Here, we demonstrate a highly versatile plasmid system that allows the rapid generation of stable Expi293 cell pools by episomal retention of transfected DNA. By linking protein expression to puromycin resistance through an attenuated internal ribosome entry site, we achieve stable cell pools producing proteins of interest. In addition, split intein–split puromycin-mediated selection of two separate protein expression cassettes allows the stable production of bispecific antibody-like molecules or antibodies with distinct C-terminal heavy chain modifications, such as an antigen on one chain and a sortase tag on the other chain. We also use this novel expression system to generate stable Expi293 cell pools that secrete sortase A Δ59 variant Srt4M. Using these reagents, we prepared a site-specific drug-to-antibody ratio of 1 antibody–siRNA conjugate. We anticipate the simple, robust, and rapid stable protein expression systems described here being useful for a wide variety of applications. Full article

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

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13 pages, 1835 KiB

Open AccessEditor’s ChoiceArticle

Platform-Specific Fc N-Glycan Profiles of an Antisperm Antibody

by Ellena Nador, Chaoshuang Xia, Philip J. Santangelo, Kevin J. Whaley, Catherine E. Costello and Deborah J. Anderson

Antibodies 2024, 13(1), 17; https://doi.org/10.3390/antib13010017 - 6 Mar 2024

Cited by 1 | Viewed by 2295

Abstract

IgG Fc N-glycosylation is necessary for effector functions and is an important component of quality control. The choice of antibody manufacturing platform has the potential to significantly influence the Fc glycans of an antibody and consequently alter their activity and clinical profile. [...] Read more.

IgG Fc N-glycosylation is necessary for effector functions and is an important component of quality control. The choice of antibody manufacturing platform has the potential to significantly influence the Fc glycans of an antibody and consequently alter their activity and clinical profile. The Human Contraception Antibody (HCA) is an IgG1 antisperm monoclonal antibody (mAb) currently in clinical development as a novel, non-hormonal contraceptive. Part of its development is selecting a suitable expression platform to manufacture HCA for use in the female reproductive tract. Here, we compared the Fc glycosylation of HCA produced in two novel mAb manufacturing platforms, namely transgenic tobacco plants (Nicotiana benthamiana; HCA-N) and mRNA-mediated expression in human vaginal cells (HCA_mRNA). The Fc N-glycan profiles of the two HCA products were determined using mass spectrometry. Major differences in site occupancy, glycan types, and glycoform distributions were revealed. To address how these differences affect Fc function, antibody-dependent cellular phagocytosis (ADCP) assays were performed. The level of sperm phagocytosis was significantly lower in the presence of HCA-N than HCA_mRNA. This study provides evidence that the two HCA manufacturing platforms produce functionally distinct HCAs; this information could be useful for the selection of an optimal platform for HCA clinical development and for mAbs in general. Full article

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15 pages, 2395 KiB

Open AccessEditor’s ChoiceReview

Immunogenicity and Loss of Effectiveness of Biologic Therapy for Inflammatory Bowel Disease Patients Due to Anti-Drug Antibody Development

by Tsvetelina Velikova, Metodija Sekulovski and Monika Peshevska-Sekulovska

Antibodies 2024, 13(1), 16; https://doi.org/10.3390/antib13010016 - 26 Feb 2024

Cited by 1 | Viewed by 3478

Abstract

Many patients with inflammatory bowel disease (IBD) experience a loss of effectiveness to biologic therapy (i.e., anti-TNF therapy, etc.). Therefore, in addition to the adverse effects of the treatment, these patients also face failure to achieve and maintain remission. Immunogenicity, the process of [...] Read more.

Many patients with inflammatory bowel disease (IBD) experience a loss of effectiveness to biologic therapy (i.e., anti-TNF therapy, etc.). Therefore, in addition to the adverse effects of the treatment, these patients also face failure to achieve and maintain remission. Immunogenicity, the process of production of antibodies to biological agents, is fundamental to the evolution of loss of response to treatment in IBD patients. The presence of these antibodies in patients is linked to decreased serum drug levels and inhibited biological activity. However, immunogenicity rates exhibit significant variability across inflammatory disease states, immunoassay formats, and time periods. In this review, we aimed to elucidate the immunogenicity and immune mechanisms of antibody formation to biologics, the loss of therapy response, clinical results of biological treatment for IBD from systematic reviews and meta-analyses, as well as to summarize the most recent strategies for overcoming immunogenicity and approaches for managing treatment failure in IBD. Full article

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19 pages, 3348 KiB

Open AccessEditor’s ChoiceArticle

Development of a Bispecific IgG1 Antibody Targeting BCMA and PDL1

by Irene Cattaneo, Sylvie Choblet, Rut Valgardsdottir, Muriel Roth, Annamaria Massafra, Marten Beeg, Marco Gobbi, Martine Duonor-Cerutti and Josée Golay

Antibodies 2024, 13(1), 15; https://doi.org/10.3390/antib13010015 - 20 Feb 2024

Cited by 1 | Viewed by 2557

Abstract

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. [...] Read more.

We designed, produced, and purified a novel IgG1-like, bispecific antibody (bsAb) directed against B-cell maturation antigen (BCMA), expressed by multiple myeloma (MM) cells, and an immune checkpoint inhibitor (ICI), PDL1, expressed in the MM microenvironment. The BCMA×PDL1 bsAb was fully characterized in vitro. BCMA×PDL1 bound specifically and simultaneously, with nM affinity, to both native membrane-bound antigens and to the recombinant soluble antigen fragments, as shown by immunophenotyping analyses and surface plasmon resonance (SPR), respectively. The binding affinity of bsAb for PDL1 and BCMA was similar to each other, but PDL1 affinity was about 10-fold lower in the bsAb compared to parent mAb, probably due to the steric hindrance associated with the more internal anti-PDL1 Fab. The bsAb was also able to functionally block both antigen targets with IC₅₀ in the nM range. The bsAb Fc was functional, inducing human-complement-dependent cytotoxicity as well as ADCC by NK cells in 24 h killing assays. Finally, BCMA×PDL1 was effective in 7-day killing assays with peripheral blood mononuclear cells as effectors, inducing up to 75% of target MM cell line killing at a physiologically attainable, 6 nM, concentration. These data provide the necessary basis for future optimization and in vivo testing of this novel bsAb. Full article

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

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17 pages, 1342 KiB

Open AccessEditor’s ChoiceReview

SARS-CoV-2: A Glance at the Innate Immune Response Elicited by Infection and Vaccination

by Nicola Manfrini, Samuele Notarbartolo, Renata Grifantini and Elisa Pesce

Antibodies 2024, 13(1), 13; https://doi.org/10.3390/antib13010013 - 8 Feb 2024

Cited by 4 | Viewed by 3559

Abstract

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, [...] Read more.

The COVID-19 pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to almost seven million deaths worldwide. SARS-CoV-2 causes infection through respiratory transmission and can occur either without any symptoms or with clinical manifestations which can be mild, severe or, in some cases, even fatal. Innate immunity provides the initial defense against the virus by sensing pathogen-associated molecular patterns and triggering signaling pathways that activate the antiviral and inflammatory responses, which limit viral replication and help the identification and removal of infected cells. However, temporally dysregulated and excessive activation of the innate immune response is deleterious for the host and associates with severe COVID-19. In addition to its defensive role, innate immunity is pivotal in priming the adaptive immune response and polarizing its effector function. This capacity is relevant in the context of both SARS-CoV-2 natural infection and COVID-19 vaccination. Here, we provide an overview of the current knowledge of the innate immune responses to SARS-CoV-2 infection and vaccination. Full article

(This article belongs to the Special Issue SARS-CoV-2: Immune Response Elicited by Infection or Vaccination)

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20 pages, 3632 KiB

Open AccessEditor’s ChoiceArticle

An Engineered Mouse Model That Generates a Diverse Repertoire of Endogenous, High-Affinity Common Light Chain Antibodies

by Yinghui Rong, I-Ling Chen, Lance Larrabee, Manali S. Sawant, Germaine Fuh and Patrick Koenig

Antibodies 2024, 13(1), 14; https://doi.org/10.3390/antib13010014 - 8 Feb 2024

Viewed by 3770

Abstract

Bispecific antibodies have gained increasing popularity as therapeutics as they enable novel activities that cannot be achieved with monospecific antibodies. Some of the most popular bispecific formats are molecules in which two Fab arms with different antigen specificities are combined into one IgG-like [...] Read more.

Bispecific antibodies have gained increasing popularity as therapeutics as they enable novel activities that cannot be achieved with monospecific antibodies. Some of the most popular bispecific formats are molecules in which two Fab arms with different antigen specificities are combined into one IgG-like molecule. One way to produce these bispecific molecules requires the discovery of antibodies against the two antigens of interest that share a common light chain. Here, we present the generation and characterization of a common light chain mouse model, in which the endogenous IGKJ cluster is replaced with a prearranged, modified murine IGKV10-96/IGKJ1 segment. We demonstrate that genetic modification does not impact B-cell development. Upon immunization with ovalbumin, the animals generate an antibody repertoire with VH gene segment usage of a similar diversity to wildtype mice, while the light chain diversity is restricted to antibodies derived from the prearranged IGKV10-96/IGKJ1 germline. We further show that the clonotype diversity of the common light chain immune repertoire matches the diversity of immune repertoire isolated from wildtype mice. Finally, the common light chain anti-ovalbumin antibodies have only slightly lower affinities than antibodies isolated from wildtype mice, demonstrating the suitability of these animals for antibody discovery for bispecific antibody generation. Full article

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

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15 pages, 311 KiB

Open AccessEditor’s ChoiceReview

Immune-Mediated Necrotizing Myopathy (IMNM): A Story of Antibodies

by Sarah Julien, Inès Challier, Marine Malleter, Fabienne Jouen, Laurent Drouot and Olivier Boyer

Antibodies 2024, 13(1), 12; https://doi.org/10.3390/antib13010012 - 7 Feb 2024

Viewed by 4606

Abstract

Immune-mediated necrotizing myopathy (IMNM) is a rare and severe disease that corresponds to a specific entity of idiopathic inflammatory myopathy. Patients with IMNM suffer from proximal muscle weakness, and present high levels of creatine kinase and necrotic myofibers. Anti-Signal Recognition Particle (SRP) and [...] Read more.

Immune-mediated necrotizing myopathy (IMNM) is a rare and severe disease that corresponds to a specific entity of idiopathic inflammatory myopathy. Patients with IMNM suffer from proximal muscle weakness, and present high levels of creatine kinase and necrotic myofibers. Anti-Signal Recognition Particle (SRP) and anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase autoantibodies (HMGCR) have recently been identified in two thirds of patients with IMNM and are used as a hallmark of the disease. In this review, we provide a detailed description of these antibodies and the tests used to detect them in the serum of patients. Based on in vitro studies and mouse models of IMNM, we discuss the role of autoantibodies in the pathogenesis of the disease. Finally, in the light of the latest knowledge, we conclude with a review of recent therapeutic approaches in IMNM. Full article

18 pages, 1032 KiB

Open AccessEditor’s ChoiceReview

Immunotherapy Strategy for Systemic Autoimmune Diseases: Betting on CAR-T Cells and Antibodies

by Vitaly Chasov, Ekaterina Zmievskaya, Irina Ganeeva, Elvina Gilyazova, Damir Davletshin, Marat Khaliulin, Emmanuel Kabwe, Yuriy N. Davidyuk, Aygul Valiullina, Albert Rizvanov and Emil Bulatov

Antibodies 2024, 13(1), 10; https://doi.org/10.3390/antib13010010 - 1 Feb 2024

Cited by 9 | Viewed by 6749

Abstract

Systemic autoimmune diseases (SAIDs), such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and rheumatoid arthritis (RA), are fully related to the unregulated innate and adaptive immune systems involved in their pathogenesis. They have similar pathogenic characteristics, including the interferon signature, loss of [...] Read more.

Systemic autoimmune diseases (SAIDs), such as systemic lupus erythematosus (SLE), systemic sclerosis (SSc) and rheumatoid arthritis (RA), are fully related to the unregulated innate and adaptive immune systems involved in their pathogenesis. They have similar pathogenic characteristics, including the interferon signature, loss of tolerance to self-nuclear antigens, and enhanced tissue damage like necrosis and fibrosis. Glucocorticoids and immunosuppressants, which have limited specificity and are prone to tolerance, are used as the first-line therapy. A plethora of novel immunotherapies have been developed, including monoclonal and bispecific antibodies, and other biological agents to target cellular and soluble factors involved in disease pathogenesis, such as B cells, co-stimulatory molecules, cytokines or their receptors, and signaling molecules. Many of these have shown encouraging results in clinical trials. CAR-T cell therapy is considered the most promising technique for curing autoimmune diseases, with recent successes in the treatment of SLE and SSc. Here, we overview novel therapeutic approaches based on CAR-T cells and antibodies for targeting systemic autoimmune diseases. Full article

(This article belongs to the Special Issue Autoimmune and Inflammatory Rheumatic Diseases: Potential Biomarkers, Antibodies Response and New Therapies)

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18 pages, 2800 KiB

Open AccessEditor’s ChoiceArticle

TLR2 and TLR9 Blockade Using Specific Intrabodies Inhibits Inflammation-Mediated Pancreatic Cancer Cell Growth

by Amrendra K. Ajay, Martin Gasser, Li-Li Hsiao, Thomas Böldicke and Ana Maria Waaga-Gasser

Antibodies 2024, 13(1), 11; https://doi.org/10.3390/antib13010011 - 1 Feb 2024

Viewed by 2602

Abstract

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor [...] Read more.

Pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) remains a deadly cancer worldwide with a need for new therapeutic approaches. A dysregulation in the equilibrium between pro- and anti-inflammatory responses with a predominant immunosuppressive inflammatory reaction in advanced stage tumors seem to contribute to tumor growth and metastasis. The current therapies do not include strategies against pro-tumorigenic inflammation in cancer patients. We have shown that the upregulated cell surface expression of Toll-like Receptor (TLR) 2 and of TLR9 inside PDAC cells maintain chronic inflammatory responses, support chemotherapeutic resistance, and mediate tumor progression in human pancreatic cancer. We further demonstrated intracellular TLR2 and TLR9 targeting using specific intrabodies, which resulted in downregulated inflammatory signaling. In this study, we tested, for the first time, an intrabody-mediated TLR blockade in human TLR2- and TLR9-expressing pancreatic cancer cells for its effects on inflammatory signaling-mediated tumor growth. Newly designed anti-TLR2- and anti-TLR9-specific intrabodies inhibited PDAC growth. Co-expression analysis of the intrabodies and corresponding human TLRs showed efficient retention and accumulation of both intrabodies within the endoplasmic reticulum (ER), while co-immunoprecipitation studies indicated both intrabodies interacting with their cognate TLR antigen within the pancreatic cancer cells. Cancer cells with attenuated proliferation expressing accumulated TLR2 and TRL9 intrabodies demonstrated reduced STAT3 phosphorylation signaling, while apoptotic markers Caspases 3 and 8 were upregulated. To conclude, our results demonstrate the TLR2 and TLR9-specific intrabody-mediated signaling pathway inhibition of autoregulatory inflammation inside cancer cells and their proliferation, resulting in the suppression of pancreatic tumor cell growth. These findings underscore the potential of specific intrabody-mediated TLR inhibition in the ER relevant for tumor growth inhibition and open up a new therapeutic intervention strategy for the treatment of pancreatic cancer. Full article

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

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22 pages, 2082 KiB

Open AccessEditor’s ChoiceArticle

Diversity in the HLA-I Recognition of HLA-F Monoclonal Antibodies: HLA-F or HLA-Ib Monospecific, HLA-E or HLA-G Bispecific Antibodies with or without HLA-Ia Reactivity

by Mepur H. Ravindranath, Narendranath M. Ravindranath, Carly J. Amato-Menker, Fatiha El Hilali and Edward J. Filippone

Antibodies 2024, 13(1), 8; https://doi.org/10.3390/antib13010008 - 31 Jan 2024

Viewed by 2226

Abstract

Previous investigators have used various anti-HLA-F monoclonal antibodies (mAbs) to demonstrate that the tissue distribution of HLA-F is highly restricted. Notably, these mAbs differed in their immunodiagnostic capabilities. Specifically, mAbs Fpep1.1 and FG1 detected HLA-F intracellularly in B cells but not on the [...] Read more.

Previous investigators have used various anti-HLA-F monoclonal antibodies (mAbs) to demonstrate that the tissue distribution of HLA-F is highly restricted. Notably, these mAbs differed in their immunodiagnostic capabilities. Specifically, mAbs Fpep1.1 and FG1 detected HLA-F intracellularly in B cells but not on the cell surface, whereas mAb 3D11 detected HLA-F on the cell surface. The presence of HLA-F on T cells was recognized by mAb FG1 but not by mAb Fpep1.1. mAb 3D11 detected HLA-F on the cell surface of activated B cells and on peripheral blood lymphocytes, but not on the normal cells. Importantly, mAb 3D11 revealed that HLA-F exists as a heavy chain (HC) monomer, rather than as an HC associated with B2m. Although these mAbs are believed to be specific to HLA-F, their monospecificity has not been formally established, which is critical for immunodiagnostic and therapeutic purposes. Previously, we investigated the diversity of HLA class I reactivities of anti-HLA-E mAbs using HLA-I coated multiplex bead assays on a Luminex platform. We reported that more than 80% of the HLA-E mAbs were cross-reactive with other HLA-I molecules, with exceptionally few truly HLA-E-monospecific mAbs. In the present investigation, we generated IgG mAbs against HCs of HLA-F in Balb/C mice and examined the cross-reactivity of anti-HLA-F mAbs with other HLA-I alleles using a multiplex bead assay on the Luminex platform. Beads coated with an array of HLA homo- and heterodimers of different HLA-Ia (HLA-A, HLA-B, and HLA-C) and Ib (HLA-E, HLA-F, and HLA-G) alleles were used to examine the binding of the anti-HLA-F mAbs. Only two mAbs were HLA-F monospecific, and five were HLA-Ib restricted. Several anti-HLA-F mAbs cross-reacted with HLA-E (n = 4), HLA-G (n = 3), HLA-Ia alleles (n = 9), HLA-G and HLA-Ia (n = 2), and HLA-Ib and HLA-Ia (n = 6). This monospecificity and polyreactivity were corroborated by the presence of HLA-F monospecific and HLA-I-shared sequences. This study emphasizes the need to monitor the mono-specificity of HLA-F for reliable immunodiagnostics and passive immunotherapy. Full article

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13 pages, 1134 KiB

Open AccessEditor’s ChoiceArticle

Immune Responses to Anti-Hepatitis C Virus Antibodies during Pre-Liver Transplantation Direct-Acting Antiviral Therapy in Hepatitis C Virus-Infected Recipients Associated with Post-Liver Transplantation Allograft Injury

by Shu-Hsien Lin, Kun-Ta Wu, Chih-Chi Wang, Kuang-Tzu Huang, Li-Wen Hsu, Hock-Liew Eng and King-Wah Chiu

Antibodies 2024, 13(1), 7; https://doi.org/10.3390/antib13010007 - 16 Jan 2024

Cited by 1 | Viewed by 2474

Abstract

Background and Aims: The impact of antibody responses following direct-acting antiviral (DAA) therapy in hepatitis C virus (HCV)-infected recipients before and after liver transplantation (LT) is still undetermined. Methods: In this observational cohort study, we aimed to explore the association between changes in [...] Read more.

Background and Aims: The impact of antibody responses following direct-acting antiviral (DAA) therapy in hepatitis C virus (HCV)-infected recipients before and after liver transplantation (LT) is still undetermined. Methods: In this observational cohort study, we aimed to explore the association between changes in anti-HCV antibody titers following pre-LT DAA therapy and allograft injury, including biliary complications (BCs) and acute cellular rejection (ACR). Results: A total of 153 cases were enrolled from January 2015 to February 2021. Serum anti-HCV antibody titers were assessed before and after (day 30) LT. Among all recipients, 31/153 (20.3%) had pre-LT DAA therapy (the DAA group) and 122/153 (79.7%) did not undergo pre-LT DAA therapy (the DAA-naïve group). A higher incidence of post-LT BCs was observed in the DAA group (p = 0.028). Compared with the DAA-naïve group, the DAA group had a significantly higher mean level of anti-HCV titer upregulation (p = 0.0024); furthermore, among the recipients with BCs (n = 28) and ACR (n = 41), those in the DAA group exhibited significantly higher mean levels of anti-HCV antibody titer upregulation (p < 0.005). Conclusions: In conclusion, we speculate that the upregulation of anti-HCV antibody titers, which might have been induced via the restoration of HCV-specific immune responses through pre-LT DAA therapy, was associated with post-LT allograft injury. Full article

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4 pages, 165 KiB

Open AccessEditor’s ChoiceCommentary

Is It Time to Reconsider Rituximab Dosing Regimens for Pemphigus Vulgaris?

by Christian Ciolfi, Jacopo Tartaglia and Mauro Alaibac

Antibodies 2024, 13(1), 4; https://doi.org/10.3390/antib13010004 - 5 Jan 2024

Viewed by 2842

Abstract

Rituximab is currently approved for patients affected by moderate-to-severe pemphigus vulgaris, a severe autoimmune blistering skin disease that can be life-threatening. The standard rituximab dosing regimens, originally established for B-cell non-Hodgkin’s lymphomas, have been recognized to exceed the effective dose required for inducing [...] Read more.

Rituximab is currently approved for patients affected by moderate-to-severe pemphigus vulgaris, a severe autoimmune blistering skin disease that can be life-threatening. The standard rituximab dosing regimens, originally established for B-cell non-Hodgkin’s lymphomas, have been recognized to exceed the effective dose required for inducing B-cell depletion, considering that the B-cell burden in pemphigus vulgaris is considerably lower than in lymphoproliferative disorders. We herein report our experience with very ultra-low-dose rituximab in two patients affected by pemphigus vulgaris. Full article

24 pages, 5307 KiB

Open AccessEditor’s ChoiceArticle

Isolation and Characterization of Neutralizing Monoclonal Antibodies from a Large Panel of Murine Antibodies against RBD of the SARS-CoV-2 Spike Protein

by Emanuela D’Acunto, Alessia Muzi, Silvia Marchese, Lorena Donnici, Valerio Chiarini, Federica Bucci, Emiliano Pavoni, Fabiana Fosca Ferrara, Manuela Cappelletti, Roberto Arriga, Silvia Maria Serrao, Valentina Peluzzi, Eugenia Principato, Mirco Compagnone, Eleonora Pinto, Laura Luberto, Daniela Stoppoloni, Armin Lahm, Rüdiger Groß, Alina Seidel, Lukas Wettstein, Jan Münch, Andrew Goodhead, Judicael Parisot, Raffaele De Francesco, Gennaro Ciliberto, Emanuele Marra, Luigi Aurisicchio and Giuseppe Roscilli Show full author list Hide full author list

Antibodies 2024, 13(1), 5; https://doi.org/10.3390/antib13010005 - 5 Jan 2024

Cited by 1 | Viewed by 6306

Abstract

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance [...] Read more.

The COVID-19 pandemic, once a global crisis, is now largely under control, a testament to the extraordinary global efforts involving vaccination and public health measures. However, the relentless evolution of SARS-CoV-2, leading to the emergence of new variants, continues to underscore the importance of remaining vigilant and adaptable. Monoclonal antibodies (mAbs) have stood out as a powerful and immediate therapeutic response to COVID-19. Despite the success of mAbs, the evolution of SARS-CoV-2 continues to pose challenges and the available antibodies are no longer effective. New variants require the ongoing development of effective antibodies. In the present study, we describe the generation and characterization of neutralizing mAbs against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein by combining plasmid DNA and recombinant protein vaccination. By integrating genetic immunization for rapid antibody production and the potent immune stimulation enabled by protein vaccination, we produced a rich pool of antibodies, each with unique binding and neutralizing specificities, tested with the ELISA, BLI and FACS assays and the pseudovirus assay, respectively. Here, we present a panel of mAbs effective against the SARS-CoV-2 variants up to Omicron BA.1 and BA.5, with the flexibility to target emerging variants. This approach ensures the preparedness principle is in place to address SARS-CoV-2 actual and future infections. Full article

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

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12 pages, 988 KiB

Open AccessEditor’s ChoiceArticle

Clinical and Analytical Performance of ELISA Salivary Serologic Assay to Detect SARS-CoV-2 IgG in Children and Adults

by Andrea Padoan, Chiara Cosma, Costanza Di Chiara, Giulia Furlan, Stefano Gastaldo, Ilaria Talli, Daniele Donà, Daniela Basso, Carlo Giaquinto and Mario Plebani

Antibodies 2024, 13(1), 6; https://doi.org/10.3390/antib13010006 - 5 Jan 2024

Cited by 1 | Viewed by 2221

Abstract

Saliva is a promising matrix with several purposes. Our aim is to verify if salivary anti-SARS-CoV-2 antibody determination is suitable for monitoring immune responses. One hundred eighty-seven subjects were enrolled at University-Hospital Padova: 105 females (56.1%) and 82 males (43.9%), 95 (50.8%) children [...] Read more.

Saliva is a promising matrix with several purposes. Our aim is to verify if salivary anti-SARS-CoV-2 antibody determination is suitable for monitoring immune responses. One hundred eighty-seven subjects were enrolled at University-Hospital Padova: 105 females (56.1%) and 82 males (43.9%), 95 (50.8%) children and 92 (49.2%) adults. Subjects self-collected saliva using Salivette; nineteen subjects collected three different samples within the day. A serum sample was obtained for all individuals. The N/S anti-SARS-CoV-2 salivary IgG (sal-IgG) and serum anti-SARS-CoV-2 S-RBD IgG (ser-IgG) were used for determining anti-SARS-CoV-2 antibodies. The mean (min–max) age was 9.0 (1–18) for children and 42.5 (20–61) for adults. Of 187 samples, 63 were negative for sal-IgG (33.7%), while 7 were negative for ser-IgG (3.7%). Spearman’s correlation was 0.56 (p < 0.001). Sal-IgG and ser-IgG levels were correlated with age but not with gender, comorbidities, prolonged therapy, previous SARS-CoV-2 infection, or time from last COVID-19 infection/vaccination. The repeatability ranged from 23.8% (7.4 kAU/L) to 4.0% (3.77 kAU/L). The linearity of the assay was missed in 4/6 samples. No significant intrasubject differences were observed in sal-IgG across samples collected at different time points. Sal-IgG has good agreement with ser-IgG. Noninvasive saliva collection represents an alternative method for antibody measurement, especially in children. Full article

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14 pages, 658 KiB

Open AccessEditor’s ChoiceReview

Antiphospholipid Antibodies Associated with Native Arteriovenous Fistula Complications in Hemodialysis Patients: A Comprehensive Review of the Literature

by Maxime Taghavi, Abla Jabrane, Lucas Jacobs, Maria Do Carmo Filomena Mesquita, Anne Demulder and Joëlle Nortier

Antibodies 2024, 13(1), 1; https://doi.org/10.3390/antib13010001 - 2 Jan 2024

Cited by 3 | Viewed by 2921

Abstract

Antiphospholipid antibody (aPL)-persistent positivity is frequent in hemodialysis (HD) patients. Native arteriovenous fistula (AVF) complications such as stenosis and thrombosis are among the most important causes of morbidity and mortality in hemodialysis patients. The association between aPL positivity and AVF thrombosis seems to [...] Read more.

Antiphospholipid antibody (aPL)-persistent positivity is frequent in hemodialysis (HD) patients. Native arteriovenous fistula (AVF) complications such as stenosis and thrombosis are among the most important causes of morbidity and mortality in hemodialysis patients. The association between aPL positivity and AVF thrombosis seems to now be well established. However, whether aPL positivity is associated with other AVF complications, such as maturation failure or stenosis, is not well known. Given the significant impact of AVF failure on patient’s prognosis, it is of interest to further investigate this particular point in order to improve prevention, surveillance and treatment, and, ultimately, the patient’s outcome. This literature review aims to report the recent literature on aPL-associated native AVF complications. Full article

(This article belongs to the Special Issue Autoimmune and Inflammatory Rheumatic Diseases: Potential Biomarkers, Antibodies Response and New Therapies)

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15 pages, 3888 KiB

Open AccessEditor’s ChoiceArticle

A Novel Anti-CD47 Nanobody Tetramer for Cancer Therapy

by Nataliya M. Ratnikova, Yulia Kravchenko, Anna Ivanova, Vladislav Zhuchkov, Elena Frolova and Stepan Chumakov

Antibodies 2024, 13(1), 2; https://doi.org/10.3390/antib13010002 - 2 Jan 2024

Cited by 2 | Viewed by 3617

Abstract

CD47 acts as a defense mechanism for tumor cells by sending a “don’t eat me” signal via its bond with SIRPα. With CD47’s overexpression linked to poor cancer outcomes, its pathway has become a target in cancer immunotherapy. Though monoclonal antibodies offer specificity, [...] Read more.

CD47 acts as a defense mechanism for tumor cells by sending a “don’t eat me” signal via its bond with SIRPα. With CD47’s overexpression linked to poor cancer outcomes, its pathway has become a target in cancer immunotherapy. Though monoclonal antibodies offer specificity, they have limitations like the large size and production costs. Nanobodies, due to their small size and unique properties, present a promising therapeutic alternative. In our study, a high-affinity anti-CD47 nanobody was engineered from an immunized alpaca. We isolated a specific VHH from the phage library, which has nanomolar affinity to SIRPα, and constructed a streptavidin-based tetramer. The efficacy of the nanobody and its derivative was evaluated using various assays. The new nanobody demonstrated higher affinity than the monoclonal anti-CD47 antibody, B6H12.2. The nanobody and its derivatives also stimulated substantial phagocytosis of tumor cell lines and induced apoptosis in U937 cells, a response confirmed in both in vitro and in vivo settings. Our results underscore the potential of the engineered anti-CD47 nanobody as a promising candidate for cancer immunotherapy. The derived nanobody could offer a more effective, cost-efficient alternative to conventional antibodies in disrupting the CD47–SIRPα axis, opening doors for its standalone or combinatorial therapeutic applications in oncology. Full article

(This article belongs to the Special Issue Novel Strategies and Technologies for the Development of Tumor-Specific Antibodies)

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17 pages, 5284 KiB

Open AccessEditor’s ChoiceArticle

Rapid Generation of Murine Bispecific Antibodies Using FAST-Ig^TM for Preclinical Screening of HER2/CD3 T-Cell Engagers

by Hikaru Koga, Haruka Kuroi, Rena Hirano, Hiroyuki Hirayama, Yoshiaki Nabuchi and Taichi Kuramochi

Antibodies 2024, 13(1), 3; https://doi.org/10.3390/antib13010003 - 2 Jan 2024

Viewed by 3520

Abstract

Bispecific antibodies (BsAbs) can bind to two different antigens, enabling therapeutic concepts that cannot be achieved with monoclonal antibodies. Immuno-competent mice are essential for validating drug discovery concepts, necessitating the development of surrogate mouse BsAbs. In this study, we explored the potential of [...] Read more.

Bispecific antibodies (BsAbs) can bind to two different antigens, enabling therapeutic concepts that cannot be achieved with monoclonal antibodies. Immuno-competent mice are essential for validating drug discovery concepts, necessitating the development of surrogate mouse BsAbs. In this study, we explored the potential of FAST-Ig^TM, a previously reported BsAb technology, for mouse BsAb production. We investigated charge-based orthogonal Fab mutations to facilitate the correct assembly of heavy and light chains of mouse antibodies and employed knobs-into-holes mutations to facilitate the heterodimerization of heavy chains. We combined five anti-CD3 and two anti-HER2 antibodies in mouse IgG1 and IgG2a subclasses. These 20 BsAbs were analyzed using mass spectrometry or ion exchange chromatography to calculate the percentages of BsAbs with correct chain pairing (BsAb yields). Using FAST-Ig, 19 out of the 20 BsAbs demonstrated BsAb yields of 90% or higher after simple protein A purification from transiently expressed antibodies in Expi293F cells. Importantly, the mouse BsAbs maintained their fundamental physicochemical properties and affinity against each antigen. A Jurkat NFAT-luciferase reporter cell assay demonstrated the combined effects of epitope, affinity, and subclasses. Our findings highlight the potential of FAST-Ig technology for efficiently generating mouse BsAbs for preclinical studies. Full article

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

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22 pages, 2515 KiB

Open AccessEditor’s ChoiceReview

Trends in the Development of Antibody-Drug Conjugates for Cancer Therapy

by Chi Hun Song, Minchan Jeong, Hyukmin In, Ji Hoe Kim, Chih-Wei Lin and Kyung Ho Han

Antibodies 2023, 12(4), 72; https://doi.org/10.3390/antib12040072 - 3 Nov 2023

Cited by 8 | Viewed by 5266

Abstract

In cancer treatment, the first-generation, cytotoxic drugs, though effective against cancer cells, also harmed healthy ones. The second-generation targeted cancer cells precisely to inhibit their growth. Enter the third-generation, consisting of immuno-oncology drugs, designed to combat drug resistance and bolster the immune system’s [...] Read more.

In cancer treatment, the first-generation, cytotoxic drugs, though effective against cancer cells, also harmed healthy ones. The second-generation targeted cancer cells precisely to inhibit their growth. Enter the third-generation, consisting of immuno-oncology drugs, designed to combat drug resistance and bolster the immune system’s defenses. These advanced therapies operate by obstructing the uncontrolled growth and spread of cancer cells through the body, ultimately eliminating them effectively. Within the arsenal of cancer treatment, monoclonal antibodies offer several advantages, including inducing cancer cell apoptosis, precise targeting, prolonged presence in the body, and minimal side effects. A recent development in cancer therapy is Antibody-Drug Conjugates (ADCs), initially developed in the mid-20th century. The second generation of ADCs addressed this issue through innovative antibody modification techniques, such as DAR regulation, amino acid substitutions, incorporation of non-natural amino acids, and enzymatic drug attachment. Currently, a third generation of ADCs is in development. This study presents an overview of 12 available ADCs, reviews 71 recent research papers, and analyzes 128 clinical trial reports. The overarching objective is to gain insights into the prevailing trends in ADC research and development, with a particular focus on emerging frontiers like potential targets, linkers, and drug payloads within the realm of cancer treatment. Full article

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

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14 pages, 2437 KiB

Open AccessEditor’s ChoiceReview

Recent Progress in Antibody Epitope Prediction

by Xincheng Zeng, Ganggang Bai, Chuance Sun and Buyong Ma

Antibodies 2023, 12(3), 52; https://doi.org/10.3390/antib12030052 - 8 Aug 2023

Cited by 11 | Viewed by 7370

Abstract

Recent progress in epitope prediction has shown promising results in the development of vaccines and therapeutics against various diseases. However, the overall accuracy and success rate need to be improved greatly to gain practical application significance, especially conformational epitope prediction. In this review, [...] Read more.

Recent progress in epitope prediction has shown promising results in the development of vaccines and therapeutics against various diseases. However, the overall accuracy and success rate need to be improved greatly to gain practical application significance, especially conformational epitope prediction. In this review, we examined the general features of antibody–antigen recognition, highlighting the conformation selection mechanism in flexible antibody–antigen binding. We recently highlighted the success and warning signs of antibody epitope predictions, including linear and conformation epitope predictions. While deep learning-based models gradually outperform traditional feature-based machine learning, sequence and structure features still provide insight into antibody–antigen recognition problems. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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14 pages, 2257 KiB

Open AccessEditor’s ChoiceArticle

Suppression of MUC1-Overexpressing Tumors by a Novel MUC1/CD3 Bispecific Antibody

by Jun Fang, Shifa Lai, Haoyang Yu and Lan Ma

Antibodies 2023, 12(3), 47; https://doi.org/10.3390/antib12030047 - 13 Jul 2023

Cited by 4 | Viewed by 3027

Abstract

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies [...] Read more.

Mucin1 (MUC1) is abnormally glycosylated and overexpressed in a variety of epithelial cancers and plays a critical role in tumor progression. MUC1 has received remark attention as an oncogenic molecule and is considered a valuable tumor target for immunotherapy, while many monoclonal antibodies (mAbs) targeting MUC1-positive cancers in clinical studies lack satisfactory results. It would be highly desirable to develop an effective therapy against MUC1-expressing cancers. In this study, we constructed a novel T cell-engaging bispecific antibody (BsAb) targeting MUC1 and CD3 with the Fab-ScFv-IgG format. A high quality of MUC1-CD3 BsAb can be acquired through a standard method. Our study suggested that this BsAb could specifically bind to MUC1- and CD3-positive cells and efficiently enhance T cell activation, cytokine release, and cytotoxicity. Furthermore, our study demonstrated that this BsAb could potently redirect T cells to eliminate MUC1-expressing tumor cells in vitro and significantly suppress MUC1-positive tumor growth in a xenograft mouse model. Thus, T cell-engaging MUC1/CD3 BsAb could be an effective therapeutic approach to combat MUC1-positive tumors and our MUC1/CD3 BsAb could be a promising candidate in clinical applications for the treatment of MUC1-positive cancer patients. Full article

(This article belongs to the Special Issue Novel Strategies and Technologies for the Development of Tumor-Specific Antibodies)

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21 pages, 923 KiB

Open AccessEditor’s ChoiceReview

Expanding the Reach of Monoclonal Antibodies: A Review of Synthetic Nucleic Acid Delivery in Immunotherapy

by Christopher Chung, Sagar B. Kudchodkar, Curtis N. Chung, Young K. Park, Ziyang Xu, Norbert Pardi, Mohamed Abdel-Mohsen and Kar Muthumani

Antibodies 2023, 12(3), 46; https://doi.org/10.3390/antib12030046 - 6 Jul 2023

Cited by 11 | Viewed by 8568

Abstract

Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These [...] Read more.

Harnessing the immune system to combat disease has revolutionized medical treatment. Monoclonal antibodies (mAbs), in particular, have emerged as important immunotherapeutic agents with clinical relevance in treating a wide range of diseases, including allergies, autoimmune diseases, neurodegenerative disorders, cancer, and infectious diseases. These mAbs are developed from naturally occurring antibodies and target specific epitopes of single molecules, minimizing off-target effects. Antibodies can also be designed to target particular pathogens or modulate immune function by activating or suppressing certain pathways. Despite their benefit for patients, the production and administration of monoclonal antibody therapeutics are laborious, costly, and time-consuming. Administration often requires inpatient stays and repeated dosing to maintain therapeutic levels, limiting their use in underserved populations and developing countries. Researchers are developing alternate methods to deliver monoclonal antibodies, including synthetic nucleic acid-based delivery, to overcome these limitations. These methods allow for in vivo production of monoclonal antibodies, which would significantly reduce costs and simplify administration logistics. This review explores new methods for monoclonal antibody delivery, including synthetic nucleic acids, and their potential to increase the accessibility and utility of life-saving treatments for several diseases. Full article

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

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21 pages, 2940 KiB

Open AccessEditor’s ChoiceReview

Proteolysis-Targeting Chimera (PROTAC) Delivery into the Brain across the Blood-Brain Barrier

by Toshihiko Tashima

Antibodies 2023, 12(3), 43; https://doi.org/10.3390/antib12030043 - 26 Jun 2023

Cited by 9 | Viewed by 7050

Abstract

Drug development for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease has challenging difficulties due to the pharmacokinetic impermeability based on the blood-brain barrier (BBB) as well as the blurriness of pharmacodynamic targets based on their unclarified pathogenesis and complicated [...] Read more.

Drug development for neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease has challenging difficulties due to the pharmacokinetic impermeability based on the blood-brain barrier (BBB) as well as the blurriness of pharmacodynamic targets based on their unclarified pathogenesis and complicated progression mechanisms. Thus, in order to produce innovative central nervous system (CNS) agents for patients suffering from CNS diseases, effective, selective delivery of CNS agents into the brain across the BBB should be developed. Currently, proteolysis-targeting chimeras (PROTACs) attract rising attention as a new modality to degrade arbitrary intracellular proteins by the ubiquitin-proteasome system. The internalizations of peptide-based PROTACs by cell-penetrating peptides and that of small molecule-based PROTACs through passive diffusion lack cell selectivity. Therefore, these approaches may bring off-target side effects due to wrong distribution. Furthermore, efflux transporters such as multiple drug resistance 1 (MDR1) expressed at the BBB might interrupt the entry of small molecule-based PROTACs into the brain. Nonetheless, intelligent delivery using machinery systems to absorb the nutrition into the brain for homeostasis, such as carrier-mediated transport (CMT) or receptor-mediated transcytosis (RMT), can be established. PROTACs with N-containing groups that are recognized by the proton-coupled organic cation antiporter might cross the BBB through CMT. PROTAC-antibody conjugates (PACs) might cross the BBB through RMT. Subsequently, such small molecule-based PROTACs released in the brain interstitial fluid would be transported into cells such as neurons through passive diffusion and then demonstrate arbitrary protein degradation. In this review, I introduce the potential and advantages of PROTAC delivery into the brain across the BBB through CMT or RMT using PACs in a non-invasive way. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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13 pages, 623 KiB

Open AccessEditor’s ChoiceReview

The Role of Bispecific Antibodies in Relapsed Refractory Multiple Myeloma: A Systematic Review

by Razwana Khanam, Omer S. Ashruf, Syed Hamza Bin Waqar, Zunairah Shah, Saba Batool, Rameesha Mehreen, Pranali Pachika, Zinath Roksana, Mohammad Ebad Ur Rehman and Faiz Anwer

Antibodies 2023, 12(2), 38; https://doi.org/10.3390/antib12020038 - 29 May 2023

Cited by 8 | Viewed by 4747

Abstract

Multiple myeloma is a heterogeneous clonal malignant plasma cell disorder, which remains incurable despite the therapeutic armamentarium’s evolution. Bispecific antibodies (BsAbs) can bind simultaneously to the CD3 T-cell receptor and tumor antigen of myeloma cells, causing cell lysis. This systematic review of phase [...] Read more.

Multiple myeloma is a heterogeneous clonal malignant plasma cell disorder, which remains incurable despite the therapeutic armamentarium’s evolution. Bispecific antibodies (BsAbs) can bind simultaneously to the CD3 T-cell receptor and tumor antigen of myeloma cells, causing cell lysis. This systematic review of phase I/II/III clinical trials aimed to analyze the efficacy and safety of BsAbs in relapsed refractory multiple myeloma (RRMM). A thorough literature search was performed using PubMed, Cochrane Library, EMBASE, and major conference abstracts. A total of 18 phase I/II/III studies, including 1283 patients, met the inclusion criteria. Among the B-cell maturation antigen (BCMA)-targeting agents across 13 studies, the overall response rate (ORR) ranged between 25% and 100%, with complete response/stringent complete response (CR/sCR) between 7 and 38%, very good partial response (VGPR) between 5 and 92%, and partial response (PR) between 5 and 14%. Among the non-BCMA-targeting agents across five studies, the ORR ranged between 60 and 100%, with CR/sCR seen in 19–63%, and VGPR in 21–65%. The common adverse events were cytokine release syndrome (17–82%), anemia (5–52%), neutropenia (12–75%), and thrombocytopenia (14–42%). BsAbs have shown promising efficacy against RRMM cohorts with a good safety profile. Upcoming phase II/III trials are much awaited, along with the study of other agents in concert with BsAbs to gauge response. Full article

(This article belongs to the Special Issue Novel Strategies and Technologies for the Development of Tumor-Specific Antibodies)

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9 pages, 985 KiB

Open AccessEditor’s ChoiceCommunication

Detection of SARS-CoV-2 Antibodies: Comparison of Enzyme Immunoassay, Surrogate Neutralization and Virus Neutralization Test

by Tatjana Vilibic-Cavlek, Maja Bogdanic, Ema Borko, Zeljka Hruskar, Denis Zilic, Thomas Ferenc, Irena Tabain, Ljubo Barbic, Mateja Vujica Ferenc, Ivana Ferencak and Vladimir Stevanovic

Antibodies 2023, 12(2), 35; https://doi.org/10.3390/antib12020035 - 10 May 2023

Cited by 4 | Viewed by 2338

Abstract

Background: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. Methods: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and [...] Read more.

Background: Since sensitivity and specificity vary widely between tests, SARS-CoV-2 serology results should be interpreted with caution. Methods: The study included serum samples from patients who had recovered from COVID-19 (n = 71), individuals vaccinated against SARS-CoV-2 (n = 84), and asymptomatic individuals (n = 33). All samples were tested for the presence of binding antibodies (enzyme immunoassay; EIA), neutralizing (NT) antibodies (virus neutralization test; VNT), and surrogate NT (sNT) antibodies (surrogate virus neutralization test; sVNT) of SARS-CoV-2. Results: SARS-CoV-2-binding antibodies were detected in 71 (100%) COVID-19 patients, 77 (91.6%) vaccinated individuals, and 4 (12.1%) control subjects. Among EIA-positive samples, VNT was positive (titer ≥ 8) in 100% of COVID-19 patients and 63 (75.0%) of the vaccinated individuals, while sVNT was positive (>30% inhibition) in 62 (87.3%) patients and 59 (70.2%) vaccinated individuals. The analysis of antibody levels showed a significant moderate positive correlation between EIA and VNT, a moderate positive correlation between EIA and sVNT, and a strong positive correlation between VNT and sVNT. The proportion of positive sVNT detection rate was associated with VNT titer. The lowest positivity (72.4%/70.8%) was detected in samples with low NT titers (8/16) and increased progressively from 88.2% in samples with titer 32 to 100% in samples with titer 256. Conclusions: sVNT appeared to be a reliable method for the assessment COVID-19 serology in patients with high antibody levels, while false-negative results were frequently observed in patients with low NT titers. Full article

(This article belongs to the Special Issue SARS-CoV-2: Immune Response Elicited by Infection or Vaccination)

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16 pages, 1239 KiB

Open AccessEditor’s ChoiceReview

CCR6 as a Potential Target for Therapeutic Antibodies for the Treatment of Inflammatory Diseases

by Sara Gómez-Melero and Javier Caballero-Villarraso

Antibodies 2023, 12(2), 30; https://doi.org/10.3390/antib12020030 - 20 Apr 2023

Cited by 11 | Viewed by 6670

Abstract

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor (GPCR) involved in a wide range of biological processes. When CCR6 binds to its sole ligand CCL20, a signaling network is produced. This pathway is implicated in mechanisms related to many diseases, [...] Read more.

The CC chemokine receptor 6 (CCR6) is a G protein-coupled receptor (GPCR) involved in a wide range of biological processes. When CCR6 binds to its sole ligand CCL20, a signaling network is produced. This pathway is implicated in mechanisms related to many diseases, such as cancer, psoriasis, multiple sclerosis, HIV infection or rheumatoid arthritis. The CCR6/CCL20 axis plays a fundamental role in immune homeostasis and activation. Th17 cells express the CCR6 receptor and inflammatory cytokines, including IL-17, IL-21 and IL-22, which are involved in the spread of inflammatory response. The CCL20/CCR6 mechanism plays a crucial role in the recruitment of these pro-inflammatory cells to local tissues. To date, there are no drugs against CCR6 approved, and the development of small molecules against CCR6 is complicated due to the difficulty in screenings. This review highlights the potential as a therapeutic target of the CCR6 receptor in numerous diseases and the importance of the development of antibodies against CCR6 that could be a promising alternative to small molecules in the treatment of CCR6/CCL20 axis-related pathologies. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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19 pages, 1303 KiB

Open AccessEditor’s ChoiceReview

New Insights into Pathogenesis and Treatment of ANCA-Associated Vasculitis: Autoantibodies and Beyond

by Marino Paroli, Chiara Gioia and Daniele Accapezzato

Antibodies 2023, 12(1), 25; https://doi.org/10.3390/antib12010025 - 21 Mar 2023

Cited by 9 | Viewed by 8393

Abstract

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a group of rare systemic diseases affecting small-caliber vessels. The damage caused by AAV mainly involves the lung and kidneys. AAV includes three different types: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis [...] Read more.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is a group of rare systemic diseases affecting small-caliber vessels. The damage caused by AAV mainly involves the lung and kidneys. AAV includes three different types: granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA). Although the different phenotypic forms of AAV share common features, recent studies have shown that there are significant differences in terms of pathogenetic mechanisms involving both the adaptive and innate immune systems. Advances in our understanding of pathogenesis have enabled the development of immuno-targeted therapies. This review illustrates the characteristics of the various forms of AAV and the new therapies available for this disease that can have lethal consequences if left untreated. Full article

(This article belongs to the Special Issue Autoimmune and Inflammatory Rheumatic Diseases: Potential Biomarkers, Antibodies Response and New Therapies)

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13 pages, 2577 KiB

Open AccessEditor’s ChoiceReview

Intracellular Antibodies for Drug Discovery and as Drugs of the Future

by T. H. Rabbitts

Antibodies 2023, 12(1), 24; https://doi.org/10.3390/antib12010024 - 16 Mar 2023

Cited by 7 | Viewed by 4696

Abstract

The application of antibodies in cells was first shown in the early 1990s, and subsequently, the field of intracellular antibodies has expanded to encompass antibody fragments and their use in target validation and as engineered molecules that can be fused to moieties (referred [...] Read more.

The application of antibodies in cells was first shown in the early 1990s, and subsequently, the field of intracellular antibodies has expanded to encompass antibody fragments and their use in target validation and as engineered molecules that can be fused to moieties (referred to as warheads) to replace the Fc effector region of a whole immunoglobulin to elicit intracellular responses, such as cell death pathways or protein degradation. These various forms of intracellular antibodies have largely been used as research tools to investigate function within cells by perturbing protein activity. New applications of such molecules are on the horizon, namely their use as drugs per se and as templates for small-molecule drug discovery. The former is a potential new pharmacology that could harness the power and flexibility of molecular biology to generate new classes of drugs (herein referred to as macrodrugs when used in the context of disease control). Delivery of engineered intracellular antibodies, and other antigen-binding macromolecules formats, into cells to produce a therapeutic effect could be applied to any therapeutic area where regulation, degradation or other kinds of manipulation of target proteins can produce a therapeutic effect. Further, employing single-domain antibody fragments as competitors in small-molecule screening has been shown to enable identification of drug hits from diverse chemical libraries. Compounds selected in this way can mimic the effects of the intracellular antibodies that have been used for target validation. The capability of intracellular antibodies to discriminate between closely related proteins lends a new dimension to drug screening and drug development. Full article

(This article belongs to the Special Issue Antibody Drug and Target Discovery for Cancer Therapies)

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24 pages, 1399 KiB

Open AccessEditor’s ChoiceReview

Intravenous Immunoglobulins as Immunomodulators in Autoimmune Diseases and Reproductive Medicine

by Tsvetelina Velikova, Metodija Sekulovski, Simona Bogdanova, Georgi Vasilev, Monika Peshevska-Sekulovska, Dimitrina Miteva and Tsvetoslav Georgiev

Antibodies 2023, 12(1), 20; https://doi.org/10.3390/antib12010020 - 2 Mar 2023

Cited by 17 | Viewed by 6168

Abstract

Intravenous administration of immunoglobulins has been routinely used for more than 60 years in clinical practice, developed initially as replacement therapy in immunodeficiency disorders. Today, the use of intravenous immunoglobulins (IVIGs) is embedded in the modern algorithms for the management of a few [...] Read more.

Intravenous administration of immunoglobulins has been routinely used for more than 60 years in clinical practice, developed initially as replacement therapy in immunodeficiency disorders. Today, the use of intravenous immunoglobulins (IVIGs) is embedded in the modern algorithms for the management of a few diseases, while in most cases, their application is off-label and thus different from their registered therapeutic indications according to the summary of product characteristics. In this review, we present the state-of-the-art use of IVIGs in various autoimmune conditions and immune-mediated disorders associated with reproductive failure, as approved therapy, based on indications or off-label. IVIGs are often an alternative to other treatments, and the administration of IVIGs continues to expand as data accumulate. Additionally, new insights into the pathophysiology of immune-mediated disorders have been gained. Therefore, the need for immunomodulation has increased, where IVIG therapy represents an option for stimulating, inhibiting and regulating various immune processes. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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14 pages, 1769 KiB

Open AccessEditor’s ChoiceReview

Serological Cross-Reactivity in Zoonotic Flaviviral Infections of Medical Importance

by Priscilla Gomes da Silva, José Augusto Seixas dos Reis, Marcio Nogueira Rodrigues, Quézia da Silva Ardaya and João Rodrigo Mesquita

Antibodies 2023, 12(1), 18; https://doi.org/10.3390/antib12010018 - 24 Feb 2023

Cited by 9 | Viewed by 3842

Abstract

Flaviviruses are enveloped RNA viruses from the family Flaviviridae that comprise many important human pathogenic arboviruses such as Yellow Fever, Dengue, and Zika viruses. Because they belong to the same genus, these viruses show sequence and structural homology among them, which results in [...] Read more.

Flaviviruses are enveloped RNA viruses from the family Flaviviridae that comprise many important human pathogenic arboviruses such as Yellow Fever, Dengue, and Zika viruses. Because they belong to the same genus, these viruses show sequence and structural homology among them, which results in serological cross-reactivity. Upon infection, the immune system produces both species-specific and cross-reactive antibodies, and depending on the virus, in a successive flavivirus infection, cross-reactive antibodies either enhance protection or exacerbate the disease—the latter usually due to antibody-dependent enhancement. These antigenic relationships between different flaviviruses that lead to serological cross-reactivity make them difficult to be identified through serological methods, especially when it comes to successive flavivirus infections. We present here an overview of the main structural, epidemiological, and immunological aspects of flaviviruses, highlighting the role of neutralizing antibodies in fighting viral infections and in the “original antigenic sin” problem. Finally, we draw attention to the importance of developing a rapid serological diagnostic test for flaviviruses with high sensitivity and specificity, especially when considering that cross-reactive immunity can influence the outcome of these infections. Full article

(This article belongs to the Section Humoral Immunity)

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19 pages, 6552 KiB

Open AccessEditor’s ChoiceReview

Non-Affinity Purification of Antibodies

by Tsutomu Arakawa, Yui Tomioka, Masataka Nakagawa, Chiaki Sakuma, Yasunori Kurosawa, Daisuke Ejima, Kouhei Tsumoto and Teruo Akuta

Antibodies 2023, 12(1), 15; https://doi.org/10.3390/antib12010015 - 13 Feb 2023

Cited by 6 | Viewed by 5937

Abstract

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an [...] Read more.

Currently, purification of antibodies is mainly carried out using a platform technology composed primarily of Protein A chromatography as a capture step, regardless of the scale. However, Protein A chromatography has a number of drawbacks, which are summarized in this review. As an alternative, we propose a simple small-scale purification protocol without Protein A that uses novel agarose native gel electrophoresis and protein extraction. For large-scale antibody purification, we suggest mixed-mode chromatography that can in part mimic the properties of Protein A resin, focusing on 4-Mercapto-ethyl-pyridine (MEP) column chromatography. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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13 pages, 6949 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Characterization of Active MMP9 in Chronic Inflammatory Diseases Using a Novel Anti-MMP9 Antibody

by Maile Velasquez, Chris O’Sullivan, Robert Brockett, Amanda Mikels-Vigdal, Igor Mikaelian, Victoria Smith and Andrew E. Greenstein

Antibodies 2023, 12(1), 9; https://doi.org/10.3390/antib12010009 - 18 Jan 2023

Cited by 6 | Viewed by 3732

Abstract

Matrix metalloproteinase 9 (MMP9), a protease implicated in multiple diseases, is secreted as an inactive zymogen and requires proteolytic removal of the pro-domain for activation. The relative levels and functionality of the pro- and active-MMP9 isoforms in tissues are not characterized. We generated [...] Read more.

Matrix metalloproteinase 9 (MMP9), a protease implicated in multiple diseases, is secreted as an inactive zymogen and requires proteolytic removal of the pro-domain for activation. The relative levels and functionality of the pro- and active-MMP9 isoforms in tissues are not characterized. We generated a specific antibody that distinguishes an active form of MMP9, F107-MMP9, from the inactive pro-MMP9 isoform. Using multiple in vitro assays and specimen types, we show that F107-MMP9 expression is localized and disease-specific compared with its more abundant parental pro-form. It is detected around sites of active tissue remodeling, including fistulae of inflammatory bowel and dermal fissures in hidradenitis suppurativa, and is expressed by myeloid cells, including macrophages and neutrophils. Together, our findings provide insights into the distribution and potential role of MMP9 in inflammatory diseases. Full article

(This article belongs to the Special Issue Antibodies: 10th Anniversary)

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16 pages, 1299 KiB

Open AccessEditor’s ChoiceReview

A Review of the Currently Available Antibody Therapy for the Treatment of Coronavirus Disease 2019 (COVID-19)

by Kristin Widyasari and Jinnam Kim

Antibodies 2023, 12(1), 5; https://doi.org/10.3390/antib12010005 - 11 Jan 2023

Cited by 20 | Viewed by 4729

Abstract

Monoclonal antibodies are a promising treatment for COVID-19. However, the emergence of SARS-CoV-2 variants raised concerns about these therapies’ efficacy and long-term viability. Studies reported several antibodies, that received authorization for COVID-19 treatment, are not effective against new variants or subvariants of SARS-CoV-2, [...] Read more.

Monoclonal antibodies are a promising treatment for COVID-19. However, the emergence of SARS-CoV-2 variants raised concerns about these therapies’ efficacy and long-term viability. Studies reported several antibodies, that received authorization for COVID-19 treatment, are not effective against new variants or subvariants of SARS-CoV-2, hence their distribution has to be paused. Here, the authors reviewed the status of the currently available monoclonal antibodies for COVID-19 treatment, their potential as a therapeutic agent, and the challenges ahead. To address these issues, the authors presented general information on SARS-CoV-2 and how monoclonal antibodies work against SARS-CoV-2. The authors then focus on the antibodies that have been deployed for COVID-19 treatment and their current status, as well as the evidence supporting their potential as an early intervention against COVID-19. Lastly, the authors discussed some leading obstacles that hinder the development and administration of monoclonal antibodies for the treatment of COVID-19. Full article

(This article belongs to the Special Issue The Role of Antibodies in SARS-CoV-2 Infection)

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11 pages, 893 KiB

Open AccessEditor’s ChoiceReview

TLR7 and IgM: Dangerous Partners in Autoimmunity

by Timm Amendt and Philipp Yu

Antibodies 2023, 12(1), 4; https://doi.org/10.3390/antib12010004 - 6 Jan 2023

Cited by 3 | Viewed by 4776

Abstract

The B cell antigen receptor (BCR)-repertoire is capable of recognizing a nearly unlimited number of antigens. Inevitably, the random nature of antibody gene segment rearrangement, needed in order to provide mature B cells, will generate autoreactive specificities. Once tolerance mechanisms fail to block [...] Read more.

The B cell antigen receptor (BCR)-repertoire is capable of recognizing a nearly unlimited number of antigens. Inevitably, the random nature of antibody gene segment rearrangement, needed in order to provide mature B cells, will generate autoreactive specificities. Once tolerance mechanisms fail to block the activation and differentiation of autoreactive B cells, harmful autoantibodies may get secreted establishing autoimmune diseases. Besides the hallmark of autoimmunity, namely IgG autoantibodies, IgM autoantibodies are also found in many autoimmune diseases. In addition to pathogenic functions of secreted IgM the IgM-BCR expressing B cell might be the initial check-point where, in conjunction with innate receptor signals, B cell mediated autoimmunity starts it fateful course. Recently, pentameric IgM autoantibodies have been shown to contribute significantly to the pathogenesis of various autoimmune diseases, such as rheumatoid arthritis (RA), autoimmune hemolytic anemia (AIHA), pemphigus or autoimmune neuropathy. Further, recent studies suggest differences in the recognition of autoantigen by IgG and IgM autoantibodies, or propose a central role of anti-ACE2-IgM autoantibodies in severe COVID-19. However, exact mechanisms still remain to be uncovered in detail. This article focuses on summarizing recent findings regarding the importance of autoreactive IgM in establishing autoimmune diseases. Full article

(This article belongs to the Section Humoral Immunity)

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13 pages, 1115 KiB

Open AccessEditor’s ChoiceReview

Specific Autoantibodies and Microvascular Damage Progression Assessed by Nailfold Videocapillaroscopy in Systemic Sclerosis: Are There Peculiar Associations? An Update

by Elvis Hysa, Rosanna Campitiello, Silvia Sammorì, Emanuele Gotelli, Andrea Cere, Giampaola Pesce, Carmen Pizzorni, Sabrina Paolino, Alberto Sulli, Vanessa Smith and Maurizio Cutolo

Antibodies 2023, 12(1), 3; https://doi.org/10.3390/antib12010003 - 4 Jan 2023

Cited by 4 | Viewed by 3092

Abstract

Background: Specific autoantibodies and nailfold videocapillaroscopy (NVC) findings are serum and morphological diagnostic hallmarks of systemic sclerosis (SSc) as well as useful biomarkers which stratify the microvascular progression and prognosis of patients. Methods: The aim of our narrative review is to provide an [...] Read more.

Background: Specific autoantibodies and nailfold videocapillaroscopy (NVC) findings are serum and morphological diagnostic hallmarks of systemic sclerosis (SSc) as well as useful biomarkers which stratify the microvascular progression and prognosis of patients. Methods: The aim of our narrative review is to provide an update and overview of the link between SSc-related autoantibodies, used in clinical practice, and microvascular damage, evaluated by NVC, by exploring the interaction between these players in published studies. A narrative review was conducted by searching relevant keywords related to this field in Pubmed, Medline and EULAR/ACR conference abstracts with a focus on the findings published in the last 5 years. Results: Our search yielded 13 clinical studies and 10 pre-clinical studies. Most of the clinical studies (8/13, 61.5%) reported a significant association between SSc-related autoantibodies and NVC patterns: more specifically anti-centromere autoantibodies (ACA) were associated more often with an “Early” NVC pattern, whereas anti-topoisomerase autoantibodies (ATA) more frequently showed an “Active” or “Late” NVC pattern. Five studies, instead, did not find a significant association between specific autoantibodies and NVC findings. Among the pre-clinical studies, SSc-related autoantibodies showed different mechanisms of damage towards both endothelial cells, fibroblasts and smooth muscle vascular cells. Conclusions: The clinical and laboratory evidence on SSc-related autoantibodies and microvascular damage shows that these players are interconnected. Further clinical and demographic factors (e.g., age, sex, disease duration, treatment and comorbidities) might play an additional role in the SSc-related microvascular injury whose progression appears to be complex and multifactorial. Full article

(This article belongs to the Special Issue New, Old, and Shared Antibody Specificities in Autoimmune Diseases)

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24 pages, 3262 KiB

Open AccessEditor’s ChoiceReview

Delivery of Drugs into Cancer Cells Using Antibody–Drug Conjugates Based on Receptor-Mediated Endocytosis and the Enhanced Permeability and Retention Effect

by Toshihiko Tashima

Antibodies 2022, 11(4), 78; https://doi.org/10.3390/antib11040078 - 19 Dec 2022

Cited by 12 | Viewed by 5092

Abstract

Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by [...] Read more.

Innumerable people worldwide die of cancer every year, although pharmaceutical therapy has actualized many benefits in human health. For background, anti-cancer drug development is difficult due to the multifactorial pathogenesis and complicated pathology of cancers. Cancer cells excrete hydrophobic low-molecular anti-cancer drugs by overexpressed efflux transporters such as multiple drug resistance 1 (MDR1) at the apical membrane. Mutation-driven drug resistance is also developed in cancer. Moreover, the poor distribution of drug to cancer cells is a serious problem, because patients suffer from off-target side effects. Thus, highly selective and effective drug delivery into solid cancer cells across the membrane should be established. It is known that substances (10–100 nm in diameter) such as monoclonal antibodies (mAbs) (approximately 14.2 nm in diameter) or nanoparticles spontaneously gather in solid tumor stroma or parenchyma through the capillary endothelial fenestration, ranging from 200–2000 nm, in neovasculatures due to the enhanced permeability and retention (EPR) effect. Furthermore, cancer antigens, such as HER2, Nectin-4, or TROP2, highly selectively expressed on the surface of cancer cells act as a receptor for receptor-mediated endocytosis (RME) using mAbs against such antigens. Thus, antibody–drug conjugates (ADCs) are promising anti-cancer pharmaceutical agents that fulfill accurate distribution due to the EPR effect and due to antibody–antigen binding and membrane permeability owing to RME. In this review, I introduce the implementation and possibility of highly selective anti-cancer drug delivery into solid cancer cells based on the EPR effect and RME using anti-cancer antigens ADCs with payloads through suitable linkers. Full article

(This article belongs to the Special Issue Targeting Tumor Cells with Antibodies Enhances Anti-Tumor Immunity)

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