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Background <p>Activatable antibodies aim to minimize on-target off-tumor toxicity by suppressing antigen binding through a masking domain that is released only upon encountering a tumor-associated stimulus. Anti-idiotypic single-domain antibodies (dAbs) raised against the complementarity-determining regions (CDRs) of a parental antibody are attractive masking modules because they engage the paratope through a native, structurally defined antibody–antibody interface. However, the high intrinsic affinity that makes such dAbs effective masks can also prevent full recovery of antigen binding after protease-mediated cleavage of the linker connecting the mask and the antibody. Here we addressed this limitation by structure-guided affinity tuning of an anti-trastuzumab dAb mask fused to the anti-HER2 4D5 single-chain variable fragment (4D5scFv).</p> Results <p>Analysis of the dAb–trastuzumab crystal structure (PDB ID 7PKL) identified Y61 and W104 of the dAb as principal anchor residues at the masking interface. An initial alanine scan showed that the wild-type construct effectively suppressed HER2 binding prior to cleavage but failed to recover binding after Tobacco etch virus (TEV) protease cleavage, whereas the single mutants Y61A and W104A restored full unmasking. The Y61A/W104A double mutant lost masking entirely. Fine-tuning at the more extensively engaged W104 position (W104A, W104L, W104F) identified W104F as the optimal variant, yielding the largest masked-to-unmasked dynamic range of approximately 22-fold, calculated as the ratio of the EC<sub>50</sub> value of uncleaved W104F to that of TEV-cleaved W104F, while preserving complete recovery of antigen binding. Replacement of the TEV site with a matrix metalloproteinase-9 (MMP-9) cleavage site preserved this behavior in a tumor-relevant context: dAb(W104F)-MMP-9-4D5scFv exhibited approximately 125-fold suppression of apparent HER2-binding potency in the absence of MMP-9 and recovered binding to within approximately 3-fold of the parental 4D5scFv after cleavage. BLI analysis further showed detectable HER2 binding of MMP-9-treated dAb(W104F)-MMP-9-4D5scFv, with an apparent <i>K</i><sub>D</sub> of 10.0 nM, whereas dAb(WT)-MMP-9-4D5scFv and uncleaved dAb(W104F)-MMP-9-4D5scFv showed no detectable binding under the same assay conditions. In a cell-based binding assay on HER2-positive BT-474 cells, only the W104F construct displayed clear MMP-9-dependent cellular binding, while the wild-type construct remained inactive under both conditions. Notably, MMP-9-dependent cleavage was also confirmed under a 10% FBS-containing condition, and anti-HER2 ELISA of the resulting reaction products showed HER2-binding recovery of the W104F construct.</p> Conclusions <p>Structure-guided affinity tuning of an anti-idiotypic dAb mask provides a tractable design principle for protease-activatable antibodies, enabling a well-defined masked–unmasked dynamic range and protease-dependent activation under tumor-relevant conditions. The framework is conceptually generalizable to other anti-idiotypic mask–antibody pairs and offers a rational route to widen the therapeutic window of antibodies whose clinical utility is currently constrained by on-target off-tumor toxicity.</p>

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Affinity-tuned anti-idiotypic masking enables protease-activatable HER2-targeting antibodies with an optimized masked–unmasked dynamic range

  • 期刊论文

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  • 发表日期：

    2026年8月5日

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  • Hyomin An,
  • Namyeong Kim,
  • Inchan Kwon

摘要

Background

Activatable antibodies aim to minimize on-target off-tumor toxicity by suppressing antigen binding through a masking domain that is released only upon encountering a tumor-associated stimulus. Anti-idiotypic single-domain antibodies (dAbs) raised against the complementarity-determining regions (CDRs) of a parental antibody are attractive masking modules because they engage the paratope through a native, structurally defined antibody–antibody interface. However, the high intrinsic affinity that makes such dAbs effective masks can also prevent full recovery of antigen binding after protease-mediated cleavage of the linker connecting the mask and the antibody. Here we addressed this limitation by structure-guided affinity tuning of an anti-trastuzumab dAb mask fused to the anti-HER2 4D5 single-chain variable fragment (4D5scFv).

Results

Analysis of the dAb–trastuzumab crystal structure (PDB ID 7PKL) identified Y61 and W104 of the dAb as principal anchor residues at the masking interface. An initial alanine scan showed that the wild-type construct effectively suppressed HER2 binding prior to cleavage but failed to recover binding after Tobacco etch virus (TEV) protease cleavage, whereas the single mutants Y61A and W104A restored full unmasking. The Y61A/W104A double mutant lost masking entirely. Fine-tuning at the more extensively engaged W104 position (W104A, W104L, W104F) identified W104F as the optimal variant, yielding the largest masked-to-unmasked dynamic range of approximately 22-fold, calculated as the ratio of the EC50 value of uncleaved W104F to that of TEV-cleaved W104F, while preserving complete recovery of antigen binding. Replacement of the TEV site with a matrix metalloproteinase-9 (MMP-9) cleavage site preserved this behavior in a tumor-relevant context: dAb(W104F)-MMP-9-4D5scFv exhibited approximately 125-fold suppression of apparent HER2-binding potency in the absence of MMP-9 and recovered binding to within approximately 3-fold of the parental 4D5scFv after cleavage. BLI analysis further showed detectable HER2 binding of MMP-9-treated dAb(W104F)-MMP-9-4D5scFv, with an apparent KD of 10.0 nM, whereas dAb(WT)-MMP-9-4D5scFv and uncleaved dAb(W104F)-MMP-9-4D5scFv showed no detectable binding under the same assay conditions. In a cell-based binding assay on HER2-positive BT-474 cells, only the W104F construct displayed clear MMP-9-dependent cellular binding, while the wild-type construct remained inactive under both conditions. Notably, MMP-9-dependent cleavage was also confirmed under a 10% FBS-containing condition, and anti-HER2 ELISA of the resulting reaction products showed HER2-binding recovery of the W104F construct.

Conclusions

Structure-guided affinity tuning of an anti-idiotypic dAb mask provides a tractable design principle for protease-activatable antibodies, enabling a well-defined masked–unmasked dynamic range and protease-dependent activation under tumor-relevant conditions. The framework is conceptually generalizable to other anti-idiotypic mask–antibody pairs and offers a rational route to widen the therapeutic window of antibodies whose clinical utility is currently constrained by on-target off-tumor toxicity.