This was a single center, open-label, phase 1b study evaluating the safety of zilovertamab when used in combination with paclitaxel for treatment of patients with locally advanced, unresectable, or metastatic Her2− breast cancer. The study protocol was approved by the Human Research Protections Program (HRPP) at the University of California, San Diego (IRB #160178, NCT02776917). We obtained written informed consent from each patient prior to study enrollment.

The primary objective was to determine the safety of zilovertamab and weekly paclitaxel in patients with advanced breast cancer, evaluating for dose limiting toxicities (DLTs) of the combination in the first 28-day cycle. Secondary objectives were to assess overall safety, pharmacokinetics, and clinical activity. An exploratory objective was to compare PET/CT to standard cross-sectional imaging in a subset of patients.

Patients aged 18 years or older with Eastern Cooperative Group (ECOG) performance status 0–2 and adequate organ function were eligible if they had biopsy-confirmed, locally advanced, unresectable, or metastatic HERer2 breast cancer with no maximum number of prior lines of therapy. Patients were required to have measurable disease according to Response Evaluation Criteria for Solid Tumors (RECIST) version 1.1 [15]. Patients were excluded if they had prior taxane therapy in the advanced, unresectable, or metastatic setting or known resistance to taxane therapy, defined as refractory to paclitaxel in the neoadjuvant setting and/or developed metastatic breast cancer within 6 months of neoadjuvant or adjuvant taxane chemotherapy. Patients were excluded if they had a concurrent, uncontrolled serious illness, another primary cancer, or had uncontrolled brain metastases or leptomeningeal disease. We excluded patients with preexisting neuropathy of greater than grade 1.

Eligible patients received zilovertamab at a fixed dose of 600 mg IV on Day 1 and Day 15 for the first cycle and then Day 1 of each subsequent 28-day cycle. Paclitaxel was given following zilovertamab at a dose of 80 mg/m2 IV weekly. Treatment continued until disease progression or until patients experienced unacceptable treatment-related toxicity. If toxicity was deemed to be related to paclitaxel, patients were allowed to continue single agent zilovertamab. Toxicity and efficacy assessments were performed according to Common Terminology Criteria for Adverse Events (CTCAE) version 4.03 and RECIST version 1.1, respectively [15, 16]. Response was assessed every 8 weeks by cross-sectional imaging with computed tomography (CT) or magnetic resonance imaging (MRI). 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) scans were obtained on some patients and correlated with cross-sectional imaging.

Available baseline formalin-fixed, paraffin-embedded breast cancer samples were assessed for ROR1 expression via immunohistochemical (IHC) staining, as reported [14]. Optional tissue or malignant fluid was obtained at baseline, between Cycle 3 Day 1 and Day 15, at the time of progression from consenting patients, and when malignant pleural or ascitic fluid was drained for palliation.

Peripheral blood samples for pharmacokinetic analysis were obtained on Days 1 and 15 of Cycle 1, prior to zilovertamab infusion, at 30 min post-completion of zilovertamab infusion, and then immediately after infusion of paclitaxel. On Days 8 and 22 of Cycle 1, blood was obtained prior to the infusion of paclitaxel and during all subsequent cycles, blood was obtained for pharmacokinetic analysis only prior to the infusion of zilovertamab. We determined zilovertamab levels as reported using an ELISA to determine the plasma concentrations of human IgG that was able to bind immobilized ROR1 [13].

DLTs were defined as clinically significant adverse events considered by the investigator to be possibly, probably, or definitely related to zilovertamab or the combination of zilovertamab with paclitaxel within 28 days of investigational treatment initiation. DLTs included Grade 4 hematologic toxicity lasting more than 7 days and non-hematologic toxicity of grade 3 or higher.

Patients were enrolled in cohorts of five, and each cohort was assessed for DLTs prior to enrollment of the next cohort. If two or more patients in the first cohort experienced DLT attributed to zilovertamab at full dose, then the next cohort would be enrolled at a 50% dose reduction (300 mg flat dose). If two or more patients in the second cohort experienced DLT attributed to zilovertamab at a 50% dose reduction, then the study would be stopped. If fewer than two patients in the first cohort experienced DLT attributed to zilovertamab at full dose, but two or more in the second cohort experienced DLT at full dose, then the third cohort would be treated with 50% dose-reduced zilovertamab. For adverse events other than DLTs attributed to study treatment, dose and schedule changes were specified in the protocol. The protocol also specified that for Grade > 2 rash, allergy, or infusion reaction, nab-paclitaxel may be substituted at investigator discretion.

The intent-to-treat population includes all patients who started at least one dose of zilovertamab. Descriptive statistics are used to characterize demographics, safety, toxicities, and anti-tumor activity. Best tumor responses are shown in a waterfall plot, and a swimmer plot is used to show tumor responses for each patient while on study treatment. Confidence intervals of the median progression-free survival (PFS) time are estimated based on Kaplan–Meier estimates, with PFS defined as weeks from the first day of study treatment to first disease progression or death. The duration of partial response (PR) was defined as the time from the first PR assessment to the time of recurrence, progression, or death.