Description

Protein engineering usually means swapping one of the 20 amino acids for another and hoping the fold still behaves. ncAAs change the game. They let you add new purposeful chemistry to a protein. A neat example comes from a recent colon cancer immunotherapy paper that uses p-acetylphenylalanine (pAcF) to build an anti–PD-L1 bispecific nanobody with controlled geometry.

Here, by using pAcF to control exactly how two nanobody domains are linked, the authors avoid floppy fusions and wrong orientations. In vivo, this translates into stronger immune activation within the tumour, increased CD8⁺ T-cell infiltration, and delayed tumour growth in colorectal cancer models. Here ncAAs bring control, a new bond type, a defined connection, a reproducible architecture, for properties of homogeneity, stability, affinity, and cleaner translation from bench to manufacturing.

Citation: Hu et al., 2025

The spatial arrangement of protein domains, their architecture, determines biological function. In bispecific antibodies, how two binding domains are oriented relative to each other affects potency, selectivity, and manufacturing feasibility. Genetic fusions fix this geometry at the DNA level, offering limited flexibility.

Using p-acetylphenylalanine (pAcF) as a site-specific conjugation handle, researchers constructed anti-PD-L1 bispecific nanobodies with precisely controlled domain orientation. The defined architecture produced stronger immune activation within tumours, increased CD8+ T-cell infiltration, and delayed tumour growth in colorectal cancer models, compared to conventional fusion approaches (Hu et al., 2025).

The advantage is reproducibility: each molecule has the same connection point, the same geometry, the same stoichiometry. This translates directly to manufacturing, where homogeneous products simplify analytics, reduce batch variability, and streamline regulatory characterisation. ncAA-mediated architecture control is relevant wherever multi-domain proteins are used therapeutically.

Source publication