Rossmueller Gregor, Mirkina Irina, Thiele Michael, Puchol Tarazona Alejandro, Rueker Florian, Kerschbaumer Randolf J, Schinagl Alexander
OncoOne Research & Development GmbH, Karl-Farkas-Gasse 22, A-1030 Vienna, Austria.
Department of Biotechnology, Institute of Molecular Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.
Antibodies (Basel). 2024 Dec 20;13(4):104. doi: 10.3390/antib13040104.
Rigorous assessment of antibody developability is crucial for optimizing lead candidates before progressing to clinical studies. Recent advances in predictive tools for protein structures, surface properties, stability, and immunogenicity have streamlined the development of new biologics. However, accurate prediction of the impact of single amino acid substitutions on antibody structures remains challenging, due to the diversity of complementarity-determining regions (CDRs), particularly CDR3s.
In this study, we combined in silico tools with in vitro assessments to engineer improved antibodies against the oxidized isoform of the macrophage migration inhibitory factor (oxMIF), building on the first generation anti-oxMIF antibody imalumab.
We identified hydrophobic hotspots conferring increased self-interaction and aggregation propensity on imalumab, which unravels its unusually short half-life in humans. By introducing mutations into the variable regions, we addressed these liabilities. Structural prediction tools and molecular dynamics simulations guided the selection of mutations, which were then experimentally validated. The lead candidate antibody, C0083, demonstrated reduced hydrophobicity and self-interaction due to the restructuring of its heavy chain CDR3 loop. Despite these structural changes, C0083 retained target specificity and binding affinity to oxMIF.
Altogether, this study shows that a small number of well-selected mutations was sufficient to substantially improve the biophysicochemical properties of imalumab.
在推进到临床研究之前，对抗体可开发性进行严格评估对于优化先导候选药物至关重要。蛋白质结构、表面性质、稳定性和免疫原性预测工具的最新进展简化了新型生物制品的开发。然而，由于互补决定区（CDR）的多样性，尤其是CDR3的多样性，准确预测单个氨基酸取代对抗体结构的影响仍然具有挑战性。
在本研究中，我们将计算机模拟工具与体外评估相结合，以基于第一代抗氧化巨噬细胞迁移抑制因子（oxMIF）抗体imalumab设计出改良抗体。
我们确定了导致imalumab自身相互作用增加和聚集倾向增强的疏水热点，这揭示了其在人体内异常短的半衰期。通过在可变区引入突变，我们解决了这些问题。结构预测工具和分子动力学模拟指导了突变的选择，然后进行了实验验证。先导候选抗体C0083由于其重链CDR3环的重构，疏水性和自身相互作用降低。尽管有这些结构变化，C0083仍保留了对oxMIF的靶标特异性和结合亲和力。
总之，本研究表明，少量精心选择的突变足以显著改善imalumab的生物物理化学性质。
