Targeting immunodominant Bet v 1 epitopes with monoclonal antibodies prevents the birch allergic response.

BACKGROUND: Blocking the major cat allergen, Fel d 1, with mAbs was effective in preventing an acute cat allergic response. OBJECTIVES: This study sought to extend the allergen-specific antibody approach and demonstrate that a combination of mAbs targeting Bet v 1, the immunodominant and most abundant allergenic protein in birch pollen, can prevent the birch allergic response. METHODS: Bet v 1-specific mAbs, REGN5713, REGN5714, and REGN5715, were isolated using the VelocImmune platform. Surface plasmon resonance, x-ray crystallography, and cryo-electron microscopy determined binding kinetics and structural data. Inhibition of IgE-binding, basophil activation, and mast cell degranulation were assessed via blocking ELISA, flow cytometry, and the passive cutaneous anaphylaxis mouse model. RESULTS: REGN5713, REGN5714, and REGN5715 bind with high affinity and noncompetitively to Bet v 1. A cocktail of all 3 antibodies, REGN5713/14/15, blocks IgE binding to Bet v 1 and inhibits Bet v 1- and birch pollen extract-induced basophil activation ex vivo and mast cell degranulation in vivo. Crystal structures of the complex of Bet v 1 with immunoglobulin antigen-binding fragments of REGN5713 or REGN5715 show distinct interaction sites on Bet v 1. Cryo-electron microscopy reveals a planar and roughly symmetrical complex formed by REGN5713/14/15 bound to Bet v 1. CONCLUSIONS: These data confirm the immunodominance of Bet v 1 in birch allergy and demonstrate blockade of the birch allergic response with REGN5713/14/15. Structural analyses show simultaneous binding of REGN5713, REGN5714, and REGN5715 with substantial areas of Bet v 1 exposed, suggesting that targeting specific epitopes is sufficient to block the allergic response.

Kappa-on-Heavy (KoH) bodies are a distinct class of fully-human antibody-like therapeutic agents with antigen-binding properties.

We describe a Kappa-on-Heavy (KoH) mouse that produces a class of highly diverse, fully human, antibody-like agents. This mouse was made by replacing the germline variable sequences of both the Ig heavy-chain (IgH) and Ig kappa (IgK) loci with the human IgK germline variable sequences, producing antibody-like molecules with an antigen binding site made up of 2 kappa variable domains. These molecules, named KoH bodies, structurally mimic naturally existing Bence-Jones light-chain dimers in their variable domains and remain wild-type in their antibody constant domains. Unlike artificially diversified, nonimmunoglobulin alternative scaffolds (e.g., DARPins), KoH bodies consist of a configuration of normal Ig scaffolds that undergo natural diversification in B cells. Monoclonal KoH bodies have properties similar to those of conventional antibodies but exhibit an enhanced ability to bind small molecules such as the endogenous cardiotonic steroid marinobufagenin (MBG) and nicotine. A comparison of crystal structures of MBG bound to a KoH Fab versus a conventional Fab showed that the KoH body has a much deeper binding pocket, allowing MBG to be held 4 Å further down into the combining site between the 2 variable domains.

Epitope Mapping by HDX-MS Elucidates the Surface Coverage of Antigens Associated with High Blocking Efficiency of Antibodies to Birch Pollen Allergen.

Epitopes of a native pollen allergen protein, birch Bet v1, against four of the noncompeting anti-Bet v1 antibodies individually or in combination, were identified by solution-phase amide backbone H/D exchange (HDX) coupled with high-resolution Q-TOF or Orbitrap mass spectrometry. The HDX results indicates that the four anti-Bet v1 antibodies protected specific regions of Bet v1, explaining the difference in their blocking efficiency of each antibody against Bet v1 binding to polyclonal IgEs in Bet v1 allergic patients. An in-house HDX-MS system was further developed to explore the surface protection of Bet v1 in the presence of all four antibodies with 100% sequence coverage and high redundancy. The data demonstrated that four anti-Bet v1 antibodies were able to simultaneously bind to Bet v1 in solution to provide the most effective blocking for 9 of 10 tested IgE donors in an in vitro antibody-blocking assay. For the first time, we have applied HDX to elucidate the therapeutic advantage of combination antibodies compared with individual antibodies in treating Bet v1 induced allergy.