Essén Mattias, Franciskovic Eric, Sele Céleste, Godzwon Magdalena, Ohlin Mats
Department of Immunotechnology, Lund University, Lund, Sweden.
Lund Protein Production Platform LP3, Department of Biology, Lund University, Lund, Sweden.
Front Immunol. 2025 Jul 1;16:1600778. doi: 10.3389/fimmu.2025.1600778. eCollection 2025.
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.
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.
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, N40T 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.
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 IGHV3-4803 and IGKV3-2001. 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 N40T 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.
第5组变应原，如梯牧草的Phl p 5，是草花粉过敏的主要诱因。最近，通过对一名草花粉过敏受试者骨髓B细胞进行单细胞测序，分离出了针对这种变应原的212597抗体。该抗体虽然仅经历了低水平的超突变，导致重链和轻链可变区有6个氨基酸替换，但对变应原的亲和力已达到亚纳摩尔水平，这表明针对这一主要变应原组的抗体即使在未成熟、未突变阶段也可能具有高亲和力。本研究旨在评估该抗体及其推断的未突变祖先以及其他中间和等位基因变体的亲和力和生物物理特性。
采用定点诱变来逆转212579抗体的替换。对包括其推断的未突变共同祖先在内的突变体进行变应原亲和力、热稳定性和流体动力学半径方面的表征。
我们证明，即使是该抗体推断的未突变共同祖先对变应原也表现出低纳摩尔范围内的高亲和力。重链位置38处的谷氨酸是IGHV3 - 4803等位基因特有的残基，IGHV3 - 4803是212579抗体重链的种系基因起源，对高亲和力结合至关重要。在IGHV3 - 48的其他等位基因中发现的丝氨酸替换使亲和力降低了约20倍。通过体细胞超突变引入抗体重链/轻链可变域界面的N40T替换，不影响其对变应原的亲和力，但降低了其热稳定性并增加了其流体动力学半径。
针对主要变应原（Phl p 5）的未突变、高亲和力（低纳摩尔）抗体可直接在未成熟B细胞中产生，并且在适当重排的情况下，通过涉及免疫球蛋白种系基因等位基因IGHV3 - 4803和IGKV3 - 2001的重排印记到库中。这种特异性取决于免疫球蛋白种系库编码的等位基因独特残基。重链/轻链可变域界面的替换，如重链可变域中的N40T，可能会对抗体的生物物理特性产生负面影响，如果它们对抗体的可开发性特性产生负面影响，则应将其视为进一步进化或逆转的靶点。
