ORIGINAL RESEARCH article
Front. Immunol.
Sec. B Cell Biology
Volume 16 - 2025 | doi: 10.3389/fimmu.2025.1600778
This article is part of the Research TopicThe Human Antibody RepertoireView all 4 articles
Low nanomolar affinity to major grass pollen allergen Phl p 5 as achieved in an unmutated human antibody-lineage ancestor
Provisionally accepted- 1Department of Immunotechnology, Faculty of Engineering, Lund University, Lund, Sweden
- 2Department of Immunotechnology, Lund, Sweden
- 3Lund Protein Production Platform LP3, Department of Biology, Lund University, Lund, Sweden
- 4SciLifeLab, Lund University, Lund, Sweden
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Background: Group 5 allergens, such as Phl p 5 of timothy grass, are major contributors to grass pollen allergy. Antibody 212597 specific for this allergen was recently isolated by single cell sequencing of bone marrow B cells of a grass pollen-allergic subject. This antibody, although subjected only to a low level of hypermutation resulting in six amino acid substitution across the heavy and light chain variable domains, has achieved sub-nM affinity for the allergen, suggesting that antibodies specific for this major group of allergens can be of high affinity even at the naïve, unmutated stage. The present study was designed to assess affinity and biophysical characters of the antibody, its inferred unmutated ancestor, and other intermediate and allelic variants thereof.Methods: Site-directed mutagenesis was used to revert substitutions of antibody 212579. Mutants, including its inferred unmutated common ancestor were characterized with respect to allergen affinity, thermostability, and hydrodynamic radius.Results: We demonstrate that even the antibody’s inferred unmutated common ancestor shows high affinity for the allergen in the low-nM range. Glutamate at heavy chain position 38, a residue unique to allele IGHV3-48*03, the germline gene origin of the heavy chain of antibody 212579, was critical for high affinity binding. Substitution to serine as found in other alleles of IGHV3-48 reduced the affinity about 20-fold. A substitution, N40HT in the heavy/light chain variable domain interface, introduced into the antibody through somatic hypermutation, did not impact its affinity for the allergen but reduced its thermal stability and increased its hydrodynamic radius.Conclusion: Unmutated, high affinity (low-nM) antibodies specific for a major allergen (Phl p 5) can be generated directly in naïve B cells and are, given an appropriate rearrangement, imprinted into the repertoire through rearrangements involving immunoglobulin germline gene alleles IGHV3-48*03 and IGKV3-20*01. This specificity depends on an allele-unique residue encoded by the immunoglobulin germline repertoire. Substitutions in the heavy/light chain variable domain interface, such as N40HT in a heavy chain variable domain, might negatively impact biophysical properties of the antibody and should be considered as targets for further evolution or reversion if they negatively impact an antibody’s developability properties.
Keywords: Affinity, antibody, Antibody repertoire, IgE, immunoglobulin germline gene allele, molecular evolution, somatic hypermutation, Unmutated common ancestor (UCA)
Received: 26 Mar 2025; Accepted: 02 Jun 2025.
Copyright: © 2025 Essén, Frančišković, Sele, Godzwon and Ohlin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Mats Ohlin, Department of Immunotechnology, Lund, Sweden
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