Abstract 1325: Preclinical assessment of BT5528 anti-tumor activity in cell-line-derived xenograft (CDX) models of head and neck squamous cell carcinoma (HNSCC).

Abstract Background: Erythropoietin-producing hepatocellular receptor A2 (EphA2) is a receptor tyrosine kinase critical for cell development; it is highly expressed in a range of solid tumors, and its expression correlates with higher grade, later stage disease, and poor prognosis. There is high unmet need for patients with EphA2-expressing tumors, including HNSCC, which has one of the highest EphA2 expression levels across solid tumors. BT5528 is a Bicycle® Drug Conjugate (BDC®), comprising a highly selective EphA2-targeting bicyclic peptide conjugated to the cytotoxin MMAE via a stable valine-citrulline cleavable linker. BT5528 has low molecular weight (4.4 kDa), enabling rapid and efficient delivery of the BDC® to the tumor and subsequent payload release, with minimal systemic exposure to the conjugate. Here we evaluate anti-tumor activity of BT5528 in murine CDX models of HNSCC. Methods: Three HNSCC CDX models were generated with female NOD SCID (SCC-9 [tongue]) or Balb/c nude (Fadu [hypopharynx] and Cal27 [tongue]) mice by subcutaneous inoculation of cell-line-derived HNSCC tumor cells into the right abdominal flank. Tumor-bearing mice were treated once weekly with either vehicle or BT5528 at 1, 3, or 5 mg/kg IV (n=6 per treatment group per CDX model) once average tumor volume reached ∼160 mm3. EphA2 expression in cell lines was assessed by quantitative FACS. Tumor growth was monitored by caliper measurements and tumor volume calculated as (width2 x length)/2. Average tumor growth inhibition (TGI) was calculated twice weekly for each treatment group per model for up to 28 days. Results: Average EphA2 expression (receptors/cell) was 29,000 (Fadu, low expression), 46,000 (SCC-9, medium expression), and 135,000 (Cal27, high expression), which are comparable to expression levels observed in previous preclinical work in CDX and patient-derived xenograft models, and which correlated with BT5528 anti-tumor activity. Compared with vehicle treated mice, significant anti-tumor activity was observed for all three models. For Cal27, TGI reached 53% and 92% at 3 and 5 mg/kg IV, respectively, on Day 28 (p<0.001 and p<0.0001). For SCC-9 and Fadu, the day of measurement was the last day all vehicle-treated mice survived. TGI reached 101% and 107%, respectively, for SCC-9 on Day 21 (p<0.0001); and 95% and 107%, respectively, for Fadu on Day 25 (p<0.0001). Assessment of tumor volume over time demonstrated a clear dose response for all models tested, with Fadu and SCC-9 being the most sensitive. BT5528 was generally tolerable in line with previous studies.1 Conclusions: BT5528 has shown potent preclinical anti-tumor activity in EphA2-expressing CDX models of HNSCC at clinically relevant doses. These data support the potential to further evaluate BT5528 in HNSCC. 1. Bennett et al. Mol Cancer Ther. 2020;19(7):1385-1394. Citation Format: Lukas Stanczuk, Daniel A. Peterson, Inma Rioja, Michael Method, Michael Skynner, Gavin Bennett. Preclinical assessment of BT5528 anti-tumor activity in cell-line-derived xenograft (CDX) models of head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1325.