Development and In vitro antitumor evaluation of a novel anti-p16 antibody fragment-drug conjugate.

The cell cycle regulator p16 is overexpressed in multiple cancers, and its elevated expression frequently correlates with aggressive tumor behavior and poor prognosis. However, the development of therapeutic agents directly targeting p16 has remained challenging. Antibody-drug conjugates (ADCs) represent an established modality for targeted drug delivery. To explore alternative formats that may address inherent challenges of conventional ADCs-such as potentially limited tumor penetration due to their large size and the risk of immunogenicity associated with the Fc domain-we engineered a novel p16-targeting antibody fragment-drug conjugate (AFDC). This conjugate is based on a humanized single-chain variable fragment (scFv) fused to a cell-penetrating peptide, aiming to achieve efficient intracellular delivery of the cytotoxic payload doxorubicin via p16-mediated targeting. We isolated five unique p16-specific scFvs from hybridoma cells. Following humanization, each was fused with a cell-penetrating peptide (CPP) S4 to construct scFv-p16-S4 variants. Following affinity assessment, scFv-p16-S4-04 was selected as the backbone and conjugated to doxorubicin via a chemical linker to generate the AFDC. In cellular assays, the resulting AFDC exhibited a p16 expression-dependent selectivity, showing stronger cytotoxicity against p16-high cancer cells (HeLa, BT-549) than against p16-low cells (MDA-MB-231, LO2, HEK-293 T). This selective activity correlated with target-dependent internalization. Furthermore, in p16-positive triple-negative breast cancer (TNBC) organoid models, AFDC treatment markedly reduced cell viability, underscoring its potent antitumor activity. Collectively, this proof-of-concept study supports the potential therapeutic value of a p16-targeted AFDC for p16-high tumors and illustrates the viability of the AFDC platform as an emerging strategy to target intracellular proteins.