The primary objectives of this phase I, multicenter, open-label study, were to assess the safety, tolerability, and PK of repeated dosing of DS-8895a in patients with advanced solid tumours and to determine its optimal dose for subsequent clinical studies. The secondary objectives were to explore tumor response to DS-8895a treatment and the potential biomarkers related to DS-8895a.
Inclusion criteria were as follows: advanced solid tumors in Step 1, immunohistologically confirmed EPHA2-positive gastric or esophageal cancer in Step 2, refractory to standard treatment or no standard treatment available, age ≥ 20 years old, Eastern Cooperative Oncology Group performance status ≤1, sufficient organ function within 7 days prior to registration (Additional file 2), adverse drug reaction of prior anti-cancer therapy resolved to Grade 1 or Grade 2 and assessed as clinically eligible by investigators, certain treatment-free period from the final dose/treatment of any previous therapy to the date of registration (Additional file 3), life expectancy ≥3 months, and written informed consent to the study including agreement to biomarker analysis of archival and biopsied tumor samples. A tumor was considered EPHA2-positive if ≥25% of tumor cells had weak to moderate (score 2+) or strong (3+) EPHA2 staining immunohistochemically.
Major exclusion criteria were as follows: symptomatic or treatment-required brain metastasis within 6 months of registration; positive for hepatitis B surface antigen, hepatitis C virus, or human immunodeficiency virus antibody; active gastrointestinal hemorrhage requiring blood transfusions within 2 weeks of registration; treatment with other investigational drugs within 3 weeks of registration; lactating or pregnant mothers; and unwillingness to use adequate contraception during the study and for 6 months after the final DS-8895a administration.
Study design and treatment
The study protocol, amendments, and informed consent forms were approved by the Institutional Review Boards at each study site, and the study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the International Council for Harmonisation guideline for Good Clinical Practice, and followed all other applicable regulatory requirements in Japan. Research using samples for genome/gene analysis was conducted in compliance with the Ethical Guidelines for Human Genome/Gene Analysis Research  and the Ethical Guideline for Clinical Research  as well as the above guidelines. This study was registered at clincaltrials.gov (NCT02004717).
This phase I study conducted in Japan comprised two steps: Step 1 as the dose escalation cohort in patients with advanced solid tumors, and Step 2 as the dose expansion cohort in patients with EPHA2-positive esophageal and gastric cancers (Additional file 4). In Step 1, the dose of DS-8895a was sequentially increased from Level 1 (0.1 mg/kg) to Level 6 (20 mg/kg), and intravenously administered over 2 h every 2 weeks with a 28-day period for assessment of dose-limiting toxicity (DLT). Each dose level consisted of three or six patients. If no DLT was observed in the first three patients, the dose level was escalated. If a DLT occurred in 1/3 patients, three patients were added to that dose level. If 2/6 or 3/6 patients experienced a DLT, dose escalation was discontinued and the dose level was judged as maximum tolerated dose (MTD). If the first 2/3 or 3/3 patients experienced a DLT, this dose level was also judged as MTD. In this case, further evaluation of the previous dose level was conducted, adding three or more patients. If the initial dose level was MTD, the study was stopped. Intra-patient dose escalation was not allowed. DLTs are defined in Additional file 5. Infusion-related reactions (IRRs) were excluded from the DLT evaluation.
The starting dose and administration schedule of DS-8895a were determined based on data from unpublished studies of intravenous administration of DS-8895a to cynomolgus monkeys (data on file, Daiichi Sankyo Co., Ltd.). Starting dose of 0.1 mg/kg was 260 times lower than the calculated human equivalent dose of no-observed-adverse-effect level obtained in these studies. An administration schedule of 2 weeks was chosen based on PK in cynomolgus monkeys and mice after a single intravenous or intraperitoneal administration of DS-8895a, respectively.
In Step 2, safety and PK were evaluated in up to 20 patients at the dose determined in Step 1. In both Steps 1 and 2, one cycle consisted of 4 weeks, and multiple cycles were allowed unless the discontinuation criteria for an individual patient were met.
Discontinuation criteria included evident disease progression, an adverse event (AE) making treatment continuation difficult, postponement of the study treatment > 4 weeks, deviation from the inclusion criteria after registration, patient’s request of withdrawal from study treatment, and investigator’s judgement.
Administration of DS-8895a was postponed if the patients did not meet the following criteria: ≥1000/μL neutrophil count, ≥75000/μL platelet count, non-hematological toxicity ≤ Grade 2 or improvement to the baseline level. Cancer treatment other than DS-8895a was prohibited from the day of obtaining informed consent to the day of follow-up assessment (30–45 days after final administration). Treatment for concomitant symptoms of cancer was allowed.
Safety and tolerability assessments
All AEs, clinical laboratory tests (hematology, blood chemistry, and urinalysis), vital signs (systolic and diastolic blood pressure, pulse rate, body temperature), and electrocardiogram (ECG) were assessed according to the National Cancer Institute – Common Terminology Criteria for Adverse Events, version 4.0 (Japanese version).
Initially, no premedication was used to prevent IRRs. However, after IRRs were observed at dose Levels 1 and 2, the protocol was amended to allow a premedication with antihistamines and antipyretics in Level 3, and a premedication with additional corticosteroids in Level 4 and thereafter. If no IRR occurred in previous DS-8895a administration, it could be omitted from subsequent doses.
In Cycles 1 and 2, blood samples for PK analysis were collected immediately before and after DS-8895a administration; 4, 7, 24, and 72 h after starting administration on day 1; any time point on day 8; immediately before and after the next DS-8895a administration on day 15; and immediately before subsequent DS-8895a administration on day 29. From Cycle 3, blood samples were taken immediately before and after DS-8895a administration on day 1. Blood was also collected on the day of study discontinuation and on the 30th day after the final dose.
The serum concentrations of DS-8895a in Cycles 1 and 2 were used to calculate PK parameters (maximum serum concentration [Cmax], time to reach maximum serum concentration [Tmax], area under the concentration-time curve [AUClast] up to the last quantifiable time, AUC during dosing interval, AUC up to infinity [AUCinf], and terminal elimination half-life [T1/2]) using non-compartment models and the WinNonlin® software program (Certara, Princeton, USA). The lower limit of detection was set at 1000 ng/mL.
Archival tumor samples were assessed for EPHA2, E-cadherin, human epidermal growth factor receptor 2 (HER2), and epidermal growth factor receptor (EGFR) expression (Steps 1 and 2). In Step 2, tumor biopsy samples were obtained before Cycle 1 and 2, and EPHA2, CD16, NKp46/NCRI, CD3, CD68, programmed death ligand-1 (PD-L1), E-cadherin, EGFR, and HER2 expression were assessed by immunohistochemistry. EPHA2 was detected using anti-human EPHA2 mouse monoclonal antibodies (clones 018 and 058, Daiichi Sankyo Co., Ltd.).
Blood and serum samples were collected to test circulating CD16-positive NK cells, NK activity, human leukocyte antigen (HLA)/killer cell immunoglobin-like receptor (KIR) mismatch, cytokines, and soluble EPHA2; a detailed blood sampling schedule is given in Additional file 6. HLA/KIR mismatch was assessed by typing these genes using previously described methods . Circulating CD16-positive NK cells (CD3−CD56+CD16+, in Steps 1 and 2) and the ratio of CD3−CD56+ CD137+ cells to CD3−CD56+CD16+ cells (only in Step 2) in blood samples were analyzed using flow cytometry. Circulating soluble EPHA2 (only in Step 2) in serum was analyzed by sandwich ELISA. Cytokines (granulocyte-macrophage colony-stimulating factor, interferon [IFN]γ, interleukin [IL]-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-18, macrophage inflammatory protein [MIP]-1α, MIP-1β, monocyte chemotactic protein [MCP]-1, tumor necrosis factor [TNF]α, and TNFβ) in serum samples were analyzed by multiplex assays (in Steps 1 and 2). Natural cytotoxicity (NK cell activity) of NK cells was assessed by measuring the ability of the patient’s peripheral blood mononuclear cells to lyse K562 cells in vitro before Cycle 1 in Step 2.
Tumor response to DS-8895a (best overall response, duration of best overall response, response rate, and disease control rate) was evaluated using the Response Evaluation Criteria in Solid Tumors, Version 1.1., using transverse section images of computerized tomography or magnetic resonance imaging scans. All baseline evaluations were performed using images taken within 21 days of registration. Tumor evaluation was performed every 6 weeks (± 1 week), or whenever investigators considered necessary.
The Guidelines for the Clinical Evaluation of Anti-cancer Drugs  were used to determine the sample size for Step 1. For Step 2, the sample size of up to 20 patients was estimated to be sufficient to evaluate safety and efficacy of DS-8895a.
Summary statistics were calculated for all categorical and quantitative data. The Kaplan–Meier method was used to estimate the survival distribution function for time-to-event analyses. Progression-free survival (PFS) was defined as the time from the first dose of DS-8895a to progression, relapse, or death from any cause, whichever occurred first. Pre-planned analytical populations consisted of an efficacy analysis set (patients who received at least one dose of the study drug and had at least one tumor assessment), an MTD evaluable set (patients who received at least one dose of the study drug in Step 1), a PK and pharmacodynamics analysis set (patients who received at least one dose of the study drug and from whom appropriate samples were obtained), and a safety analysis set (patients who received at least one dose of the study drug). SAS® System Release 9.2 software (SAS Institute Inc., Cary, NC) was used to perform statistical analysis.