BT1769 is a Bicycle® drug conjugate (BDC®) comprising a constrained bicyclic peptide that binds specifically to membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP14), attached via a valine-citrulline cleavable linker to the cytotoxic anti-tubulin agent maleimidocaprolyl-monomethyl auristatin E (MMAE). MT1-MMP is highly expressed in several solid tumors and is associated with poor prognosis (e.g. non-small cell lung cancer). BT1769 has low nanomolar affinity to MT1-MMP and, like an antibody drug conjugate (ADC), is designed to release its cytotoxic payload in the tumor microenvironment. Unlike ADCs, BDC molecules have a low molecular weight (~4.5 kDa), facilitating more efficient tumor penetration, and a shorter systemic half-life via renal clearance to help minimize toxicity. Therefore, BT1769 serves as a valuable pharmacological tool to investigate the potential of MT1-MMP as a therapeutic target in oncology.

Bicycle® molecules binding to MT1-MMP were identified via phage display and optimized through chemical modification to generate the BDC® tool, BT1769. We studied target affinity, selectivity and species cross-reactivity by surface plasmon resonance (SPR). Flow cytometry was used to study cell binding of a fluorescent analogue of BT1769 across several human cancer cell lines. Internalisation was evaluated via confocal microscopy. in vitro cytotoxicity was assessed in 2D and 3D assays using Cell Titer-Glo®. Balb/c nude mice or SRG rats were implanted with cell-derived or patient-derived tumor cells. Animals were dosed intravenously, on weekly or twice weekly schedules. Efficacy was determined by tumor volume measurements taken three times weekly. Blood and tissue were taken to study residual compound concentration by LC-MS/MS, and immunohistochemical evaluation of MT1-MMP expression in tumor.

BT1769 bound to MT1-MMP with high affinity and demonstrated species cross-reactivity to mouse and dog, and selectivity over MMP homologues. A fluorescent analogue of BT1769 bound and was rapidly internalized into MT1-MMP-expressing cells. BT1769 induced potent and target-specific cytotoxicity in both 2D and 3D in vitro models. Following dosing to tumor-bearing mice, BT1769 localized to tumor and delivered the payload, MMAE, to tumor cells, where it was retained. Parent drug and free MMAE were rapidly cleared from plasma and normal tissues. BT1769 showed dose-related efficacy in a range of cell and patient-derived tumor models.

BT1769 showed efficient, target-mediated, payload delivery to tumor cells, coupled with rapid systemic clearance of parent conjugate. Anti-tumor efficacy was seen across a range of cell- and patient-derived tumor models in mice and rats. This work highlights the use of Bicycle conjugates to target tumors associated with MT1-MMP expression and offer potential novel therapeutic opportunities.

All authors are employees of BicycleTx Ltd.