Antibodies

13 pages, 1414 KiB

Open AccessBrief Report

Characterization of a Trispecific PD-L1 Blocking Antibody That Exhibits EGFR-Conditional 4-1BB Agonist Activity

by Laura Rubio-Pérez, Susana Frago, Marta Compte, Rocío Navarro, Seandean L. Harwood, Rodrigo Lázaro-Gorines, Marina Gómez-Rosel, Oana Hangiu, Noelia Silva-Pilipich, Lucía Vanrell, Cristian Smerdou and Luis Álvarez-Vallina

Antibodies 2024, 13(2), 34; https://doi.org/10.3390/antib13020034 - 24 Apr 2024

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Abstract

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further [...] Read more.

Immune checkpoint blockade has changed the treatment paradigm for advanced solid tumors, but the overall response rates are still limited. The combination of checkpoint blockade with anti-4-1BB antibodies to stimulate tumor-infiltrating T cells has shown anti-tumor activity in human trials. However, the further clinical development of these antibodies has been hampered by significant off-tumor toxicities. Here, we generated an anti-4-1BB/EGFR/PD-L1 trispecific antibody consisting of a triple-targeting tandem trimerbody (TT) fused to an engineered silent Fc region. This antibody (IgTT-4E1-S) was designed to combine the blockade of the PD-L1/PD-1 axis with conditional 4-1BB costimulation specifically confined to the tumor microenvironment (TME). The antibody demonstrated simultaneous binding to purified EGFR, PD-L1, and 4-1BB in solution, effective blockade of the PD-L1/PD1 interaction, and potent 4-1BB-mediated costimulation, but only in the presence of EGFR-expressing cells. These results demonstrate the feasibility of IgTT-4E1-S specifically blocking the PD-L1/PD-1 axis and inducing EGFR-conditional 4-1BB agonist activity. Full article

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

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14 pages, 8324 KiB

Open AccessFeature PaperArticle

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

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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, 676 KiB

Open AccessReview

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

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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 AccessArticle

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

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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 AccessArticle

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

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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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12 pages, 1393 KiB

Open AccessArticle

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

by Tahereh Keshvari, Stanislav Melnik, Lin Sun, Ali Niazi, Farzaneh Aram, Ali Moghadam, Benjamin Kogelmann, Gordana Wozniak-Knopp, Somanath Kallolimath, Amin Ramezani and Herta Steinkellner

Antibodies 2024, 13(2), 29; https://doi.org/10.3390/antib13020029 - 07 Apr 2024

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Abstract

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells [...] Read more.

Aflibercept is a therapeutic recombinant fusion protein comprising extracellular domains of human vascular endothelial growth factor receptors (VEGFRs) and IgG1-Fc. It is a highly glycosylated protein with five N-glycosylation sites that might impact it structurally and/or functionally. Aflibercept is produced in mammalian cells and exhibits large glycan heterogeneity, which hampers glycan-associated investigations. Here, we report the expression of aflibercept in a plant-based system with targeted N-glycosylation profiles. Nicotiana benthamiana-based glycoengineering resulted in the production of aflibercept variants carrying designed carbohydrates, namely, N-glycans with terminal GlcNAc and sialic acid residues, herein referred to as AFLI^GnGn and AFLI^Sia, respectively. Both variants were transiently expressed in unusually high amounts (2 g/kg fresh leaf material) in leaves and properly assembled to dimers. Mass spectrometric site-specific glycosylation analyses of purified aflibercept showed the presence of two to four glycoforms in a consistent manner. We also demonstrate incomplete occupancy of some glycosites. Both AFLI^GnGn and AFLI^Sia displayed similar binding potency to VEGF₁₆₅, with a tendency of lower binding to variants with increased sialylation. Collectively, we show the expression of functionally active aflibercept in significant amounts with controlled glycosylation. The results provide the basis for further studies in order to generate optimized products in the best-case scenario. Full article

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17 pages, 1931 KiB

Open AccessReview

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 - 03 Apr 2024

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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 AccessReview

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 - 01 Apr 2024

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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)

12 pages, 1258 KiB

Open AccessCommunication

Case Series: Efficacy of Polyclonal Intravenous Immunoglobulin for Refractory Clostridioides difficile Infection

by Sophie A. Ragan, Caitlin Doyle, Neha Datta, Heather Abdic, Mark H. Wilcox, Ros Montgomery, Shanika A. Crusz, Yashwant R. Mahida and Tanya M. Monaghan

Antibodies 2024, 13(2), 26; https://doi.org/10.3390/antib13020026 - 01 Apr 2024

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Abstract

Background: Intravenous immunoglobulin (IVIg) for Clostridioides difficile infection (CDI) no longer features in treatment guidelines. However, IVIg is still used by some clinicians for severe or recurrent CDI (rCDI) cases. The main objective of this study was to investigate the efficacy of IVIg [...] Read more.

Background: Intravenous immunoglobulin (IVIg) for Clostridioides difficile infection (CDI) no longer features in treatment guidelines. However, IVIg is still used by some clinicians for severe or recurrent CDI (rCDI) cases. The main objective of this study was to investigate the efficacy of IVIg and to identify possible predictors of disease resolution post IVIg administration for patients with CDI. Methods: This retrospective observational cohort study of patients ≥2 years old hospitalised with severe, relapsing, or rCDI treated with IVIg therapy was performed in a large UK tertiary hospital between April 2018 and March 2023. Scanned electronic notes from patient admissions and clinical reporting systems were used to collect relevant data. Results: In total, 20/978 patients diagnosed with CDI over the 5-year study were treated with IVIg. Twelve (60%) had hospital-onset CDI. Eleven of the twenty patients (55%) responded to treatment, with a mean of 8.6 (SD 10.7) days to disease resolution. Sixteen (80%) patients were treated for severe CDI and four (20%) for rCDI (n = 3) and relapsing CDI (n = 1). There were no statistically significant differences in possible independent predictors of disease resolution post IVIg administration between groups. There was an average of 6.2 (4.9) days to IVIg administration after diagnosis with no difference between responders and non-responders (p = 0.88) and no further significant difference in additional indicators. Four (36%) of the responders were immunosuppressed compared to just one (11%) of the non-responders (p = 0.15). Six of the responders (two with recurrent and four with severe CDI) improved rapidly within 2 days, and three of these were immunosuppressed. Conclusion: We observed disease resolution post IVIg therapy in over 50% of patients with refractory CDI. Our data also support a potential enhanced effect of IVIg in immunosuppressed individuals. Thus, the role of IVIg for CDI treatment, particularly in the immunosuppressed, warrants future case–control studies coupled to mechanistic investigations to improve care for this ongoing significant healthcare-associated infection. Full article

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

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Antibodies, Volume 13, Issue 2 (June 2024) – 9 articles

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