First-in-man phase 1 clinical trial of gene therapy for advanced pancreatic cancer: safety, biodistribution, and preliminary clinical findings

Abstract

This phase 1 trial was aimed to determine the safety, pharmacokinetics, and preliminary clinical activity of CYL-02, a nonviral gene therapy product that sensitizes pancreatic cancer cells to chemotherapy. CYL-02 was administrated using endoscopic ultrasound in 22 patients with pancreatic cancer that concomitantly received chemotherapy (gemcitabine). The maximum-tolerated dose (MTD) exceeded the maximal feasible dose of CYL-02 and was not identified. Treatment-related toxicities were mild, without serious adverse events. Pharmacokinetic analysis revealed a dose-dependent increase in CYL-02 DNA exposure in blood and tumors, while therapeutic RNAs were detected in tumors. No objective response was observed, but nine patients showed stable disease up to 6 months following treatment and two of these patients experienced long-term survival. Panels of plasmatic microRNAs and proteins were identified as predictive of gene therapy efficacy. We demonstrate that CYL-02 nonviral gene therapy has a favorable safety profile and is well tolerated in patients. We characterize CYL-02 biodistribution and demonstrate therapeutic gene expression in tumors. Treated patients experienced stability of disease and predictive biomarkers of response to treatment were identified. These promising results warrant further evaluation in phase 2 clinical trial.

Figure 1

Clinical trial flowchart and injection of the gene therapy product in patients. (a) Flowchart of the THERGAP trial for advanced pancreatic cancer. Patients received two intratumoral injections of CYL-02 using endoscopic ultrasound (EUS) followed by gemcitabine infusions. Complete clinical examinations and biological assessments were performed during each visit and twice on the day of the CYL-02 injections (days 1 and 28, at 1 hour before and 6 hours after CYL-02 injections). Blood samples were obtained from patients during each visit (twice on the day of the CYL-02 injection: before and 6 hours after) and were processed for serum and plasma (EDTA-treated tubes) preparations. Urine was collected before and at 24 and 48 hours after CYL-02 injection. The tumor marker CA 19-9 was quantified before (visit 0 and day 1) and at 2 months (day 60) following treatment. V: visit. For intratumoral gene transfer, lyophilized CYL-02 was reconstituted by adding 2.5 ml of sterile water 10 minutes before starting EUS. Gene therapy was performed under general anesthesia. (b) Pancreatic carcinoma of the body. The tumor is delineated with a white dashed line. The biopsy needle was then positioned at the center of the tumor. (c) Needle (with arrows) using EUS guidance within the tumor (dashed arrow indicates the hyperechoic needle tip) and, after removing the stylet, CYL-02 was slowly injected using backward and forward movements, including a fanning technique of the needle within the tumor under ultrasound control. (d) Pancreatic carcinoma of the body immediately following CYL-02 injection showing a white cloud within the tumor (delineated by the arrows). At the end of the procedure, 1.5 ml of 5% glucose (w/v) solution was injected within the tumor to empty the needle. CT, computed tomography; EDTA, ethylenediaminetetraacetic acid; THERGAP, gene therapy for advanced pancreatic adenocarcinoma.

Figure 2

Biodistribution and expression of the gene therapy product. (a) CYL-02 was detected by qPCR at the time indicated in the blood of patients receiving 1,000 μg of the gene therapy product. *Indicates 6 hours postintratumoral injection of CYL-02. Data are means ± SD of four biological replicates per group with three experimental replicates and expressed as copies per ml of blood. Experimental threshold: 10 copies/ml of blood; experimental background in blood: 7.8 ± 0.2 × 10⁴ copies per ml of blood. (b) CYL-02 DNA was detected by qPCR in the tumors of patients at 1 month following gene therapy. Data are means ± SD of four (patients receiving 250 and 1,000 μg of CYL-02) or six (patients receiving 500 μg of CYL-02) biological replicates per group with three experimental replicates and expressed as copy numbers of CYL-02 per ng of tumor DNA. For statistical comparison of two experimental groups, the bilateral Student's t-test was used (*P < 0.05). Experimental threshold: 10 copies/ng of DNA; experimental background in tumors: 0 copies/ng of DNA. (c) DCK::UMK and SSTR2 genes expression were measured in tumors before and 1 month following gene therapy with 1,000 μg of CYL-02. Data are means ± SD of four (patients receiving 1,000 μg of CYL-02) biological replicates per group with three experimental replicates and expressed as arbitrary units (2^−ΔCt with ΔCt = CT(DCK::UMK or SSTR2) − CT(18S)). For statistical comparison of two experimental groups, the nonparametric Wilcoxon test was used (***P < 0.005). qPCR, quantitative PCR.

Figure 3

Radiological findings and clinical efficacy of gene therapy. (a) Representative CT-scan pictures before (V1 = baseline) and 2 months after treatment (V12). (b) CA 19.9 was measured in the blood of 13 patients with locally advanced pancreatic cancer before and at the completion of the gene therapy protocol. Data are expressed as % change from baseline. Dotted line indicates 50% inhibition threshold. *Indicates prior treatment. CT, computed tomography.

Figure 4

Biomarker discovery. (a) Score plots from results of PCA of peptide profiles from plasma patients high (n = 3) or low responders (n = 3) to treatment according to the first two principal components (Dim 1: 50.63%, Dim 2: 7.49%). Confidence ellipses at 95% around each sample from each group are also represented. Patient number is indicated. (b) Individual box plots for the circulating proteins predictive of efficacy identified in this study. Results are expressed as protein frequency in high-responder (HR) versus low-responder (LR) patients. (c) Quantification by ELISA of A2MG levels in patient plasma prior gene therapy. Data are expressed as protein level in ng/ml. The bilateral Student's t-test was used (*P < 0.05). (d) Individual box plots for the circulating miRNAs predictive of efficacy identified in this study. Results are expressed as Ct in high-responder (HR) versus low-responder (LR) patients. A2MG, α2-macroglobulin; ELISA, enzyme-linked immunosorbent assay; miRNA, microRNA; PCA, principal component analysis.