The study employed a modification of the standard cohort of three design.29
Three patients were treated at each dose level with expansion to six patients per cohort if DLT was observed in any one of the three first patients at each dose level. Maximum tolerated dose was defined as the highest safely tolerated dose, where ≤1 patient experienced DLT, with the next higher dose level having at least two patients who experienced DLT. DLT was defined as any grade 3, 4, or 5 adverse event considered possibly, probably, or definitely related to the study drug, excluding grade 3 absolute neutrophil count lasting <72 hours, grade 3 alopecia, or any grade 3 or worse nausea, vomiting, or diarrhea where the patient did not receive maximal supportive care.29
Each cohort of three was also expanded to six or seven patients if significant biologic activity was noted.
Adaptive trial design. A phase II efficacy component was incorporated in the phase I/II study by allowing additional treatment cycles to be given if the patient had less than or equal to grade 1 toxicity. Further, across the board, dose escalations were allowed up to dose level 2 for patients with less than or equal to grade 1 toxicity when safety at the specified dose level was documented. The principal investigator was also allowed to recommend surgical resection/debulking, and Rexin-G was continued if residual disease was found by histological examination or PET-CT scan.
Clinical objectives/end points. The primary objective of this study is to determine the clinical toxicity of escalating doses of Rexin-G as defined by patient performance status, toxicity assessment score, and hematologic and metabolic profiles. Secondary objectives include (i) evaluation of the potential of Rexin-G for evoking an immune response, recombination events and/or unwanted vector integration in nontarget organs, and (ii) identification of an antitumor response to Rexin-G.
Patient population. The present phase I/II trial enrolled 13 patients with a pathologic diagnosis of pancreatic adenocarcinoma that was locally advanced or metastatic that had failed gemcitabine or a gemcitabine-containing regimen. Histologic or cytologic confirmation at diagnosis or recurrence was required. Inclusion criteria consisted of an Eastern Cooperative Oncology Group performance score of 0–1 and adequate hematologic, hepatic, and kidney function. Exclusion criteria included human immunodeficiency virus, hepatitis B virus, or hepatitis C virus positivity, clinically significant ascites, medical or psychiatric conditions that could compromise successful adherence to the protocol, and unwillingness to employ effective contraception during treatment with Rexin-G and for 6 weeks following treatment completion. An amendment to the New Investigational Drug application was approved by the FDA (BB-IND#11586) in July 2007, and the clinical protocol (C07-105) was reviewed and approved by the Western Institutional Review Board, Olympia, WA.
Patient recruitment and assignment.
The phase I/II clinical trial using Rexin-G for pancreatic cancer was registered on http://www.clinicaltrials.gov
(NCT00504998) within 1 week of study initiation, and patients were recruited on a first-come first-serve basis after appropriate screening procedures were conducted. Written informed consent was obtained from each patient at the time of enrollment. This is an open label study using escalating doses of Rexin-G. Six patients were enrolled at dose level 0–1 followed by a dose escalation in six patients at dose level 2 when safety/toxicity data in at least three patients at dose level 1 had been recorded, and no DLT was encountered. One patient was included in the dose level 2 cohort because he received an intrapatient dose escalation from dose level 0 to dose level 2.
Rexin-G is a nonreplicative “pathotropic” or disease-seeking nanoparticle bearing a functional collagen-binding motif on its envelope protein18
and encoding an N-terminal deletion mutant construct of human cyclin G1 (ref. 18
) under the control of a hybrid Moloney murine leukemia virus long-terminal repeat promoter. The gene expression vector also contains the neomycin phosphotransferase gene driven by the SV40 early promoter, which was used for precise vector titer assays. The Rexin-G vector is produced by transient co-transfection of three separate plasmids in 293T cells (human kidney 293 cells transformed with the SV40 large T antigen) maintained as a fully validated master cell bank.21,24,27
The final product exhibits a vector titer of 5 × 109
cfu/ml, a biologic potency of 50–70% growth inhibitory activity in target cancer cells, <550 base-pair residual DNA, no detectable E1A or SV40 large T antigen, and no detectable RCR.30
The clinical vector is stored in volumes of 23 ml in 30 ml vials or 40 ml in 150 ml cryobags at −80 °C. Preparation of the Rexin-G vector for patient administration consisted of rapid thawing of the vector in the vial in a 34 °C water bath. The vector was thawed 15–30 minutes prior to infusion into the patient, and given intravenously over 5–10 minutes. All personnel who handled and disposed of the vector observed Biosafety Level 2 compliance in accordance with the National Institutes of Health Guidelines for Research Involving Recombinant DNA molecules.
Thirteen patients were treated with escalating doses of Rexin-G. Briefly, each treatment cycle was 6 weeks, consisting of 4 weeks treatment and 2 weeks rest period. The following two dose levels were employed: dose level 0 = 1 × 1011 cfu two times a week for 4 weeks (dose per 4-week cycle: 8 × 1011 cfu); dose level 1 = 1 × 1011 cfu three times a week for 4 weeks (dose per 4-week cycle: 12 × 1011 cfu); dose level 2 = 2 × 1011 cfu three times a week for 4 weeks (dose per 4-week cycle: 24 × 1011 cfu). Treatment was continued if there was less than or equal to grade 1 toxicity.
Safety and efficacy evaluation. Pretreatment evaluation included history, physical exam, complete blood count with differential and platelet count, a serum chemistry panel including aspartate transaminase, alanine transaminase, alkaline phosphatase, creatinine, and total bilirubin, assessment of coagulation status including prothrombin time, international normalized ratio, and activated partial thromboplastin time, testing for human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, imaging evaluation to include a whole body FDG/PET-CT scan, electrocardiography, and chest X-ray. All patients had a complete blood count and serum chemistry panel performed weekly during treatment.
Toxicity was assessed before each vector infusion, and before beginning an additional treatment cycle. Toxicity was graded using NCI CT-CAE version 3 (ref. 31
). Patients had serum collected for vector-specific antibody detection and peripheral blood mononuclear cells collected for assessment of vector DNA integration and RCR at the end of 4 weeks, at 6 weeks, or before the start of a treatment cycle.
Vector-related studies were performed in the Epeius Biotechnologies Quality Control Unit, San Marino, CA using standard operating procedures in compliance with good laboratory practices. Detection of antivector antibodies in serum, testing for presence of RCR and vector DNA integration studies in patient's peripheral blood lymphocytes, were performed as previously described.20,21
Efficacy assessment with FDG PET-CT scan was performed at the end of 4 weeks, at the end of 6 weeks, or before starting an additional treatment cycle up to 12 weeks, and every 12 weeks thereafter. All PET-CT images were performed and reviewed by independent radiologists of the Medical Imaging Center of Southern California, Santa Monica, CA, who are experts at nuclear and PET imaging, and who were blinded to the Rexin-G dose levels. Tumor responses [complete response (CR); PR; or SD] were evaluated using the NCI RECIST criteria.32
Tumor responses were also assessed using modifications of the International PET criteria33
and the CHOI criteria.34
The modified International PET Criteria defines a CR as disappearance of FDG avid uptake in target and nontarget lesions with no new lesions; PR as a decrease in maximum standard uptake value of >25% from baseline with no new lesions and no obvious progression of nontarget lesions; SD as not meeting the criteria for CR, PR, or PD, and no symptomatic deterioration attributed to tumor progression; and PD as an increase in maximum standard uptake value of >25% from baseline, any new lesions, and obvious progression of nontarget lesions.
The modified CHOI criteria defines CR as the disappearance of all disease and no new lesions; PR as a decrease in size of ≥10% or a decrease in CT density (Hounsfeld units) ≥15% with no new lesions and no obvious progression of nonmeasurable disease; SD as not meeting the criteria for CR, PR, or PD, and no symptomatic deterioration attributed to tumor progression; and PD as an increase in unidimensional tumor size of >10% and did not meet criteria for PR by CT density, any new lesions, including new tumor nodules in a previously cystic tumor.
Overall evaluation of toxicity/tumor responses was conducted by the principal investigator and associate, Sarcoma Oncology Center, Santa Monica, CA (S.P.C. and V.S.C., respectively).
Frequency tables, graphs, and summary statistics were used to describe patient characteristics and outcome data. Follow-up data from October 2007 to 13 January 2009 were analyzed. Kaplan–Meier methodology35
was used to describe graphically the distribution of OS. OS time was calculated in days and divided by 30.4 to convert to months. PFS time was approximated, using the times of patient evaluations. OS and PFS times were compared in groups of patients treated at different dose levels, using permutation tests on the logrank statistic with at least 10,000 replications. Tumor-response data by different specific criteria (RECIST, PET, and CHOI criteria) were reported. Reported P
values are two-sided, and P
< 0.05 was considered statistically significant. Analysis was done using NCSS software (Number Cruncher Statistical Systems, Kaysville, UT). Statistical analysis was performed by a biostatistician not otherwise involved in the study (W.C.B.).