Screening pertactin-specific antibodies and evaluating competitive epitope recognition by native mass spectrometry

Chemical Science · 2026-01-01

vaccine antigen mature pertactin (Prn), offering in-depth characterization of binding affinity, stoichiometry, and competition. We implemented variable temperature electrospray ionization to evaluate thermally induced unfolding and stability of different mAb·Prn complexes, while biolayer interferometry (BLI) and competition experiments were employed to provide complementary information about binding kinetics and mapping of distinct epitopes on Prn. Finally, we used nMS to evaluate the interactions of individual mAbs with Prn variants as a predictor for therapeutic action. Our results demonstrate the utility of nMS in combination with other techniques as a powerful approach for understanding the interactions of protective mAb binding to Prn, providing insight into mechanisms of vaccine-induced protection.

Mouse Fc-FcγRIV structure guides Fc engineering for cross-species FcγR recognition

bioRxiv (Cold Spring Harbor Laboratory) · 2026-05-15

Abstract Antibody-dependent cellular cytotoxicity (ADCC) is a major mechanism of action for many FDA-approved therapeutic antibodies that is driven by interactions between the antibody Fc and Fcγ receptors (FcγRs) on immune effector cells. Murine models used for preclinical antibody evaluation currently have limited predictive value for clinical ADCC performance due to interspecies differences in Fc-FcγR interactions. The molecular determinants governing Fc-FcγR engagement in mice remain poorly defined, complicating the interpretation of murine ADCC data and its clinical relevance. To address this, we present the high-resolution crystal structure of the receptor that regulates Fc-mediated cytotoxicity in mice, mouse FcγRIV, alone and in complex with mouse IgG2a Fc. This complex preserves key features of the human IgG1 Fc-human FcγRIIIa interface which mediates ADCC in humans including salt bridges, hydrogen bonds, and a proline sandwich. However, subtle variations in receptor orientation, Fc-FcγR electrostatics, and glycan positions reduce human IgG1 Fc- mouse FcγRIV binding affinity, resulting in species-restricted Fc-FcγR mediated immune responses. Modeling of human IgG1 Fc interactions with mouse FcγRIV predicted steric clashes, suggesting opportunities to modulate the interaction. One structure-guided substitution variant of human IgG1, Fc humo , maintains comparable human FcγRIIIa engagement with enhanced binding to and activation of mouse FcγRIV, relative to human IgG1 Fc. This study provides proof-of-concept for engineering human Fc domains for cross-species FcγR recognition and provides a strategic framework to improve the predictive power of in vivo preclinical models.

Antibodies blocking PlGF or VEGF interactions with the NRP1 receptor mediate anti-proliferative effects

Journal of Biological Chemistry · 2025-10-26

Abstract Antibodies blocking the function of vascular endothelial growth factor A (VEGFA) remain a promising therapeutic strategy, especially when combined with check-point inhibitors, but their efficacy is limited by tumor resistance. This can occur via multiple mechanisms, including upregulation of placental growth factor 2 (PlGF-2), an alternative ligand for VEGF receptor 1 (VEGFR1) and neuropilin receptor 1 (NRP1). Activity of both growth factors is mediated by interactions with multiple receptors and extra-cellular matrix components, which complicates efforts to understand their contributions to cancer progression. To complement existing antibodies, we discovered those blocking interactions between PlGF-2 or VEGFA and their shared NRP1 receptor in the presence of heparin. Limiting angiogenesis to promote vascular normalization is one mechanism of anti-VEGF protection; here, anti-VEGFA antibodies blocking interactions with VEGFR1 and NRP1 reduced HUVEC tube formation in a physiological angiogenesis model. By contrast, antibodies binding PlGF-2 or VEGFA to block NRP1 significantly reduced proliferation of Caki-I kidney carcinoma cells in vitro , indicating this receptor mediates additional effects. Interestingly, one antibody exhibited dual-reactive binding to VEGFA and PlGF-2, suggesting a novel therapeutic strategy to prevent PlGF-driven VEGF-resistance. Overall, these antibodies define new mechanisms to disrupt PlGF activity and support a role for NRP1 in cell proliferation. Key Results New antibodies binding VEGFA or PlGF selectively block NRP1-receptor interactions. Antibody blockade of VEGFA binding to NRP1 reduced HUVEC angiogenesis. Blocking growth factor interactions with NRP1 reduced Caki-I renal carcinoma proliferation. Identified an antibody with dual-reactive binding to VEGFA and PlGF.

Structural basis for neutralizing antibody binding to pertussis toxin

Proceedings of the National Academy of Sciences · 2025-04-02 · 5 citations

Pertussis toxin (PT) is a key protective antigen in vaccine- and natural immunity-mediated protection from Bordetella pertussis infection. Despite its importance, no PT-neutralizing epitopes have been characterized structurally. To define neutralizing epitopes and identify key structural elements to preserve during PT antigen design, we determined a 3.6 Å cryoelectron microscopy structure of genetically detoxified PT (PTg) bound to hu11E6 and hu1B7, two potently neutralizing anti-PT antibodies with complementary mechanisms: disruption of toxin adhesion to cells and intracellular activities, respectively. Hu11E6 binds the paralogous S2 and S3 subunits of PTg via a conserved epitope but surprisingly did not span the previously identified sialic acid–binding site implicated in toxin adhesion. Hu11E6 specifically prevented PTg binding to sialylated N-glycans and a sialylated model receptor, as demonstrated by high-throughput glycan array analysis and ELISA, while a T cell activation assay showed that it blocks PTg mitogenic activities to define its neutralizing mechanism. Hu1B7 bound a quaternary epitope spanning the S1 and S5 subunits, although functional studies of hu1B7 variants suggested that S5 binding is not involved in its PT neutralization mechanism. These results structurally define neutralizing epitopes on PT, improving our molecular understanding of immune protection from B. pertussis and providing key information for the future development of PT immunogens.

Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

bioRxiv (Cold Spring Harbor Laboratory) · 2025-04-18 · 1 citations

A key mechanism of antiviral antibodies is to bind cell-surface viral antigens and activate cellular immunity to clear infected cells, yet antibodies targeting human cytomegalovirus (HCMV) have exhibited limited efficacy. This appears due to HCMV's multiple immune evasion mechanisms, including viral receptors (vFcγRs) which bind human IgG Fc domains to co-operatively inhibit Fc activation of host Fcγ receptors and impair Fc-mediated effector functions. We biochemically characterized and evaluated the functions of two highly conserved vFcγRs, gp34 and gp68, and mapped their binding epitopes on the Fc domain. Based on this information, we then engineered Fc variants that retain binding to CD16A, which is essential for NK activation, and to FcRn but have markedly attenuated binding to gp34 and gp68. IgG1 antibodies targeting the gB fusogen with engineered Fc domains were not internalized by infected cells, mediated enhanced CD16A activation and limited viral spread in HCMV-infected fibroblasts more effectively than wild-type Fc. Together, this work demonstrates a strategy to enhance the efficacy of antibody therapies to clear HCMV infections. Highlights: Host and HCMV FcR compete for IgG1 binding but engage different residues.Fc-engineering abrogates viral FcR antagonism while retaining CD16A activation.Antibodies that resist vFcR capture promote superior ADCC against infected cells.Designer Fc domains complement Fabs to create enhanced disease-specific therapies.

SARS-CoV-2 humoral immune responses in convalescent individuals over 12 months reveal severity-dependent antibody dynamics

Communications Medicine · 2025-05-02 · 9 citations

Defining the kinetics of SARS-CoV-2 antibody responses is critical for informing the management of reinfections, vaccinations, and therapeutics of Coronavirus disease 2019 (COVID-19). Using four antibody assays, we evaluated antibody titers against SARS-CoV-2 nucleocapsid (N), spike (S), and receptor binding domain (RBD) in 98 convalescent participants with varying COVID-19 disease severities (asymptomatic, mild, moderate or severe) at 1, 3, 6, and 12-months post-SARS-CoV-2-positive PCR and in 17 non-vaccinated, non-infected controls. Increasing acute COVID-19 disease severity correlates with higher anti-N and anti-RBD titers throughout 12 months post-infection. Anti-N and anti-RBD titers decline over time in all participants, except for increased anti-RBD titers post-vaccination, with hospitalized participants exhibiting faster decay rates. Less than 50% of participants retain anti-N titers above controls at 12 months, with non-hospitalized participants falling below controls sooner. Nearly all participants maintain anti-RBD titers above controls for 12 months, suggesting long-term protection against severe reinfections. Nonetheless, by 6 months, few participants retain >50% of their initial 1-month anti-N or anti-RBD titers. Notably, vaccine-induced anti-RBD titers are higher in non-hospitalized participants. Lastly, early convalescent titers correlate with age but not with Post-Acute Sequelae of SARS-CoV-2 infection (PASC) status or steroid use. Hospitalized participants initially develop higher anti-SARS-CoV-2 antibody titers that decline faster relative to non-hospitalized participants. While anti-N titers fall below control levels in some participants, anti-RBD titers remain above controls over 12 months, demonstrating long-lived antibody responses known to protect against severe disease. These findings advance our understanding of COVID-19 antibody dynamics. This study explores how the immune system responds to COVID-19 over time by measuring antibodies, small proteins that help fight infection. We studied 98 participants who recovered from COVID-19 with varying illness severities and 17 who were never infected. Blood samples were collected over a year to track changes in antibody levels. Our results show that severe illness leads to higher antibody levels, though with a more precipitous antibody decline than in milder cases. Notably, antibodies that offer long-term protection against severe COVID-19 remain high for 12 months after the infection in most individuals. Lastly, vaccination boosts antibody levels, particularly in individuals with milder illness. Our research enhances understanding of immunity post COVID-19 and informs vaccination and reinfection prevention strategies. Siles Alvarado and Schuler et al. examine the long-term dynamics of SARS-CoV-2 antibodies in individuals with varying COVID-19 severities over 12 months. While anti-N antibodies decline, anti-RBD antibodies persist, offering insights into the evolving immunity post-COVID.

Selective decoupling of IgG1 binding to viral Fc receptors restores antibody-mediated NK cell activation against HCMV

Cell Reports · 2025-12-01 · 3 citations

Antibodies binding cell-surface antigens to activate cellular immunity are an important mechanism of anti-viral protection, yet antibodies targeting cells infected by human cytomegalovirus (HCMV) exhibit limited efficacy. This is due to HCMV immune evasion mechanisms, including viral receptors (vFcγRs) that bind human immunoglobulin G Fc domains to inhibit host Fcγ receptor activation and impair Fc-mediated immune functions. Here, we biochemically characterize two conserved vFcγRs, gp34 and gp68, and map their Fc binding sites. We then engineer Fc variants to retain binding to host Fc receptors CD16A and FcRn but exhibit markedly reduced gp34/gp68 interactions. Antibodies targeting the gB fusogen with engineered Fc domains are not internalized by infected cells, mediate enhanced immune cell activation, and limit viral spread in HCMV-infected fibroblasts more effectively than antibodies with wild-type Fc. This work demonstrates a strategy to enhance therapeutic antibody control of HCMV infections and other herpesvirus infections with similar immune-evasion mechanisms.

Screening pertactin-specific antibodies and evaluating competitive epitope recognition by native mass spectrometry

ChemRxiv · 2025-06-23

Structural characterization of antigen-antibody interactions is critical for understanding protective vaccine responses and development of therapeutic monoclonal antibodies (mAb). Traditional biophysical and biochemical techniques often require the immobilization of one binding partner or provide ensemble-averaged measurements, constraints which may limit the ability to probe multiple facets of antigen-antibody interactions. Native mass spectrometry (MS) offers a versatile alternative, providing a comprehensive view of antigen-antibody complexes. Here, we utilized native MS to screen the interactions between a small panel of monoclonal antibodies (mAbs) and the Bordetella pertussis vaccine antigen mature pertactin (Prn), offering an in-depth characterization of binding stoichiometry, cooperativity, and competition. We implemented variable temperature electrospray ionization to evaluate thermal induced unfolding and stability of different mAb•Prn complexes, while biolayer interferometry (BLI) and competition experiments were employed to provide complementary information about binding kinetics and mapping of distinct epitopes on Prn. Finally, we used native MS to evaluate the interactions of individual mAbs with Prn variants as a predictor for therapeutic action. Our results demonstrate the utility of native MS, in combination with complementary techniques, as a powerful approach for understanding the interactions of protective mAbs binding Prn, which can ultimately contribute to the development of more effective vaccines to prevent pertussis infection.

Structural Basis for Antibody Neutralization of Pertussis Toxin

bioRxiv (Cold Spring Harbor Laboratory) · 2024-09-23 · 1 citations

SUMMARY/ABSTRACT Pertussis toxin (PT) is a key protective antigen in vaccine- and natural immunity-mediated protection from Bordetella pertussis infection. Despite its importance, no PT-neutralizing epitopes have been characterized structurally. To define neutralizing epitopes and identify key structural elements to preserve during PT antigen design, we determined a 3.6 Å cryo-electron microscopy structure of genetically detoxified PT (PTg) bound to hu11E6 and hu1B7, two potently neutralizing anti-PT antibodies with complementary mechanisms: disruption of toxin adhesion to cells and intracellular activities, respectively. Hu11E6 bound the paralogous S2 and S3 subunits of PTg via a conserved epitope, but surprisingly did not span the sialic acid binding site implicated in toxin adhesion. High-throughput glycan array analysis showed that hu11E6 specifically prevents PTg binding to sialylated N-glycans, while a T cell activation assay showed that hu11E6 blocks PTg mitogenic activities to define the neutralizing mechanism. Hu1B7 bound a quaternary epitope spanning the S1 and S5 subunits, although functional studies of hu1B7 variants suggested that S5 binding is not involved in its PT neutralization mechanism. These results are the first to structurally define neutralizing epitopes on PT, improving our molecular understanding of immune protection from B. pertussis and providing key information for the future development of PT immunogens. SIGNIFICANCE Antibodies neutralizing pertussis toxin (PT) prevent the severe clinical symptoms associated with infection by Bordetella pertussis . However, the molecular basis of effective PT-targeted immunity is poorly understood. To gain insight into PT-inhibitory mechanisms, we determined the cryo-electron microscopy structure of genetically detoxified PT (PTg) with two potently neutralizing antibodies to precisely define their epitopes. Carbohydrate-binding studies show that the hu11E6-binding surface on PT interacts with N-linked glycans and that blocking these interactions prevents PT’s T cell mitogenic activities. Hu1B7 binds an epitope near the S1 active site that includes S5 contacts but these do not appear important for neutralization. This work identifies PT-neutralizing epitopes and supports inclusion of the hu1B7 and hu11E6 epitopes in next-generation vaccines and PT-based immunogens.

Display of Native SARS-CoV-2 Spike on Mammalian Cells to Measure Antibody Affinity and ADCC

BIO-PROTOCOL · 2024-01-01

COVID-19 pandemic led to the rapid development of antibody-based therapeutics and vaccines targeting the SARS-CoV-2 spike protein. Several antibodies have been instrumental in protecting vulnerable populations, but their utility was limited by the emergence of spike variants with diminished susceptibility to antibody binding and neutralization. Moreover, these spike variants exhibited reduced neutralization by polyclonal antibodies in vaccinated individuals. Accordingly, the characterization of antibody binding to spike variants is critical to define antibody potency and understand the impact of amino acid changes. A key challenge in this effort is poor spike stability, with most current methods assessing antibody binding using individual domains instead of the intact spike or variants with stabilizing amino acid changes in the ectodomain (e.g., 2P or HexaPro). The use of non-native spike may not accurately predict antibody binding if changes lie within the epitope or alter epitope accessibility by altering spike dynamics. Here, we present methods to characterize antibody affinity for and activity against unmodified SARS-CoV-2 spike protein variants displayed on a mammalian cell membrane that recapitulates the native spike environment on infected cells. These include a flow cytometry-based method to determine the effective antibody binding affinity (KD) and an antibody-dependent cellular cytotoxicity (ADCC) assay to assess Fc-mediated activities. These methods can readily evaluate antibody activity across a panel of spike variants and contribute to our understanding of spike/antibody co-evolution. Key features • Allows rapid characterization of antibody binding to native SARS-CoV-2 spike on the mammalian cell surface. • Describes analysis of antibody binding to multiple native spike variants without stabilizing mutations • Describes analysis of Fc-mediated antibody-dependent cellular cytotoxicity • Requires transient transfection of Expi293F and 293T cells to assess antibody binding and ADCC, a flow cytometer for antibody binding, and a plate reader for ADCC • Protocol is readily adaptable to other viral fusogens and membrane proteins.