Structure-guided computational design and mechanistic understanding of the p95HER2-targeting NAZ-mAb antibody and its variants.
5 affiliations
- 1Department of Immunology, University of Oslo and Oslo University Hospital, Oslo, Norway.
- 2Department of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital, Oslo, Norway.
- 3Department of Clinical Cancer Research, Oslo University Hospital, Oslo, Norway.
- 4Institute of Clinical Medicine, University of Oslo, Norway.
- 5Immunoquest Therapeutics AS, Oslo, Norway.
Abstract
Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase in breast cancer and other malignancies. A subset of HER2-positive tumours expresses 611-CTF-p95HER2, a tumour-specific, hyperactive truncated isoform associated with metastasis and treatment resistance that lacks most of the extracellular domain targeted by conventional HER2-directed antibodies. We previously developed NAZ-mAb (formerly known as Oslo-2), a monoclonal antibody against 611-CTF-p95HER2. Here, we describe a computational antibody-engineering workflow for designing variants of NAZ-mAb. Starting from the sequence alone, we modelled the NAZ-mAb-611-CTF-p95HER2 complex, generated a combinatorial mutational landscape using FoldX 5.0, and prioritized candidate variants using predicted interaction energy and developability criteria. Two variants representing distinct design strategies were selected for validation: an aromatic double mutant, NAZ-mAb v1 (L:S31W/L:H107W), and a conservative single mutant, NAZ-mAb v2 (L:S31M). Both variants were successfully expressed as recombinant IgGs; NAZ-mAb v2 achieved a five-fold higher recombinant expression yield than parental NAZ-mAb, while both variants retained antigen binding with a higher apparent signal than the parental antibody in indirect ELISA. However, Biacore two-state kinetic analysis revealed weaker affinities than the parental antibody (KD NAZ-mAb v1: 326.1 nM, NAZ-mAb v2: 94.5 nM vs parental NAZ-mAb: 53.3 nM). These findings show that the computational workflow can generate antigen-engaging NAZ-mAb variants, while also highlighting the limitations of fixed-backbone interaction-energy ranking as a predictor of binding affinity and yield. This study provides a practical framework and a cautionary benchmark for computationally driven, developability-aware antibody engineering in the absence of experimental structural data.
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BibTeX
@article{rawat2026structureguided,
title = {{Structure-guided computational design and mechanistic understanding of the p95HER2-targeting NAZ-mAb antibody and its variants.}},
author = {Puneet Rawat and Jon Amund Kyte and Victor Greiff and Esmaeil Dorraji},
journal = {Current research in structural biology},
year = {2026},
month = oct,
doi = {10.1016/j.crstbi.2026.100199},
url = {https://europepmc.org/article/MED/42909185},
note = {CC BY},
}
RIS
TY - JOUR
TI - Structure-guided computational design and mechanistic understanding of the p95HER2-targeting NAZ-mAb antibody and its variants.
AU - Puneet Rawat
AU - Jon Amund Kyte
AU - Victor Greiff
AU - Esmaeil Dorraji
PY - 2026
DA - 2026/10/13
JO - Current research in structural biology
DO - 10.1016/j.crstbi.2026.100199
UR - https://europepmc.org/article/MED/42909185
AB - Human epidermal growth factor receptor 2 (HER2) is an oncogenic receptor tyrosine kinase in breast cancer and other malignancies. A subset of HER2-positive tumours expresses 611-CTF-p95HER2, a tumour-specific, hyperactive truncated isoform associated with metastasis and treatment resistance that lacks most of the extracellular domain targeted by conventional HER2-directed antibodies. We previously developed NAZ-mAb (formerly known as Oslo-2), a monoclonal antibody against 611-CTF-p95HER2. Here, we describe a computational antibody-engineering workflow for designing variants of NAZ-mAb. Starting from the sequence alone, we modelled the NAZ-mAb-611-CTF-p95HER2 complex, generated a combinatorial mutational landscape using FoldX 5.0, and prioritized candidate variants using predicted interaction energy and developability criteria. Two variants representing distinct design strategies were selected for validation: an aromatic double mutant, NAZ-mAb v1 (L:S31W/L:H107W), and a conservative single mutant, NAZ-mAb v2 (L:S31M). Both variants were successfully expressed as recombinant IgGs; NAZ-mAb v2 achieved a five-fold higher recombinant expression yield than parental NAZ-mAb, while both variants retained antigen binding with a higher apparent signal than the parental antibody in indirect ELISA. However, Biacore two-state kinetic analysis revealed weaker affinities than the parental antibody (KD NAZ-mAb v1: 326.1 nM, NAZ-mAb v2: 94.5 nM vs parental NAZ-mAb: 53.3 nM). These findings show that the computational workflow can generate antigen-engaging NAZ-mAb variants, while also highlighting the limitations of fixed-backbone interaction-energy ranking as a predictor of binding affinity and yield. This study provides a practical framework and a cautionary benchmark for computationally driven, developability-aware antibody engineering in the absence of experimental structural data.
SN - 2665-928X
ER -