Background: BT5528 is a Bicycle Toxin Conjugate (BTC), comprising a bicyclic peptide targeting the tumor antigen EphA2, linked to a cytotoxin (monomethyl auristatin E [MMAE]) via a tumor microenvironment cleavable linker. Bicycles are a novel class of chemically synthesized constrained peptides, developed by Bicycle Therapeutics. EphA2 is reported to be overexpressed in a range of solid tumors, contributes to oncogenesis, tumor-associated angiogenesis and metastasis. Intracellular EphA2 signaling converges on pathways that are integral to cell growth, proliferation, migration and invasion. Increased EphA2 expression has been identified as a resistance mechanism to EGFR Tyrosine Kinase Inhibitor based therapy. BT5528 mechanism of action is dependent on tumor penetration, target binding and release of MMAE toxin payload. BTCs offer advantages over antibody-toxin conjugates exhibiting rapid penetration of dense tumors and decreased extra-tumor exposure. BT5528 exhibited a favorable preclinical profile supporting the initiation of a first-in-human study to investigate safety and efficacy of BT5528 in indications with evidence of EphA2 expression including non-small-cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), gastric/upper gastrointestinal (GI), pancreatic and urothelial cancers. Methods: BT5528-100 (NCT04180371) is a Ph I/II study to evaluate safety and tolerability of weekly BT5528 alone and in combination with Q4W nivolumab. Each dose escalation utilizes a 3+3 design which converts to a Bayesian design to determine MTD or MAD and RP2D for BT5528 with and without nivolumab. Eligible patients must have advanced solid tumors associated with EphA2 expression which have recurred after exhausting standard treatment options. Patients must have available tumor tissue and acceptable hematologic and organ function, with exclusions for uncontrolled brain metastases, thromboembolic events, bleeding disorders, uncontrolled hypertension, CYP3A4 inhibitors/inducers or, for the nivolumab cohorts, autoimmune disease. On-study tumor and blood samples will be collected for biomarker evaluations including tumor EphA2 expression, ADA, and candidate response biomarkers for BT5528 alone and combination with nivolumab. Pharmacokinetic data will be reported for C1D1 and D15 for BT5528 and MMAE. The expansion phase will enroll specific tumor types to evaluate clinical activity of BT5528. Enrollment is ongoing. Clinical trial information: NCT04180371.