Spatial Tumour Tissue Distribution Dynamics of a Bicycle Peptide-MMAE Toxin Conjugate Profiled by Targeted Mass Spectrometry

Abstract

A highly specific-NECTIN-4 binding bicyclic peptide covalently linked to the cytotoxin monomethyl auristatin E (MMAE) via a valine-citrulline cleavable linker (zelenectide pevedotin, BT8009) is a promising modality to treat cancer patients with solid tumours. Although the pharmacokinetics and biodistribution of zelenectide pevedotin have been well characterised, a more precise view of tumour delivery and intra-tumoural distribution of its released payload is lacking. To address this, we have developed an approach to profile the spatial tumour and healthy tissue distribution of this anti-cancer agent using laser capture microdissection coupled to targeted liquid chromatography parallel reaction monitoring (LCM-LC-PRM) mass spectrometry. This LCM-assisted spatial pharmacology workflow enables the precise isolation of specific tissue regions, followed by high-resolution analysis of MMAE distribution at sub-tissue-specific levels. By identifying and optimising MS/MS-specific fragmentation ions for accurate and sensitive detection and quantitation of MMAE released from zelenectide pevedotin within tumour tissues, we observed progressive accumulation and sustained retention of MMAE in mouse tumour tissues over a period of 24 h. Conversely, in plasma, liver and muscle tissues, MMAE levels generally declined following early exposure, although inter-animal variability was observed. LCM-based intra-tumour spatial profiling revealed heterogeneous MMAE distribution between tumour cell-rich and stroma-rich regions, with distinct temporal patterns across compartments. Together, our spatial targeted MS method provided valuable insights into zelenectide pevedotin's payload distribution dynamics in vivo, thereby enhancing our understanding of its therapeutic potential and guiding the development of next-generation Bicycle Drug Conjugate (BDC).