The ability to repair DNA interstrand cross-links may be an important factor contributing to mitomycin C (MMC) and cisplatin cytotoxicities. We have assessed the repair of interstrand cross-links induced by MMC in two MMC-hypersensitive hamster cell mutants and their resistant parental cell line. Using a gene-specific repair assay, we found no evidence for repair of MMC cross-links in either parental or mutant cells, suggesting that persistence of DNA interstrand cross-links is not responsible for the differential toxicity of MMC towards hypersensitive cells. Repair of cisplatin-induced interstrand cross-links was efficient in resistant as well as in mutant cells. Therefore we concluded that a defect in excision repair of interstrand cross-links was not responsible for the cytotoxic effects of MMC and cisplatin in these hypersensitive mutants.
Abstract Background: the median survival of patients diagnosed with locally advanced or metastatic pancreatic adenocarcinoma (PDA) is 4 to 6 months. Chemotherapies (gemcitabine, FOLFIRINOX) and molecular targeted therapies only offer a marginal survival benefit. Consequently, we developed new strategies based on therapeutic gene transfer to help alleviate the dismal prognosis of PDA. We demonstrated that transferring SSTR2 gene in PDA experimental models strongly inhibited tumor progression. We generated DCK::UMK fusion gene to chemosensitize tumor cells to gemcitabine to provoke tumor shrinkage in immune competent animals. Based on these promising results, we asked whether combining SSTR2 and DCK::UMK genes delivered by a non-viral vector may impede PDA growth in patients. Methods: in this first-in-man clinical trial, 22 patients (12 first line, 10 second line) were included in the CHU of Toulouse from 12/2010 to 9/2012. Four different doses (125, 250, 500 and 1000μg) of the gene therapy product (GTP) were injected twice using endoscopic ultrasound (EUS) (day 1 and day 28). Patients concomitantly received gemcitabine for 2 months. Results: the feasibility of this approach is excellent, as all the patients were successfully injected despite differences in the site and the size of the primary tumor, and pre-existing treatments (biliary stent, surgical bypass). No serious adverse events related to the GTP were recorded. We found that less than 0.001% of GTP was detected in blood, while urine was negative in all cases. We found that CA 19.19 levels decreased in 68% (15/22) of patients following gene therapy. In first line patients, we demonstrated partial tumor shrinkage in 2 cases (16%), stable disease in 9 cases (75%), and tumor progression in 1 case (8%). In these latter patients, progression-free survival and median survival following gene therapy reached 7 and 11.3 months, respectively. Conclusions: all together, we demonstrated for the first time that delivering antitumoral genes using non-viral vectors by EUS is feasible and safe in patients with advanced PDA. As our preliminary results tend to demonstrate therapeutic efficacy, we are currently designing a phase II trial based on this approach. Citation Format: Louis Buscail, Barbara Bournet, Fabienne Vernejoul, Hubert Lulka, Gilles Cambois, Naima Hanoun, Fabian Gross, Jean Tiraby, Pierre Cordelier. Development of gene therapy for pancreatic carcinoma: from experimental models to phase I clinical trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3532A. doi:10.1158/1538-7445.AM2013-3532A
Abstract To date, pancreatic adenocarcinoma (PDAC) can’t be diagnosed early. Consequently, a majority of patient (80%) display an advanced disease that results in a low resection rate leading to a dismal overall median survival of less than 6 months. We extensively demonstrated that delivering SSTR2, DCK and UMK genes using non-viral vectors strongly inhibit tumor progression and dissemination in relevant experimental models of PDAC. Consequently, we designed a GMP-grade gene therapy product, namely CYL-02, encoding for the above mentioned therapeutic proteins, delivered by a non-viral vector for the management of 24 patients diagnosed with advanced PDAC. In this phase I clinical trial, gene therapy was administered in dose-escalation in the tumors using endoscopic ultrasound. Patients received gemcitabine during this protocol. We demonstrate that the intratumoral injection of the gene therapy product is feasible, well tolerated and safe, and results in the presence and the expression of CYL-02 in the tumors. Consequently, tumor progression is inhibited during the course of the protocol. In patients with locally advanced PDAC at the time of diagnosis, tumor size and serum levels of CA 19-9 significantly decreased following gene therapy regardless of the previous lines of treatment and median progression free survival and median overall survival reaches 6.4 and 11.4 months respectively. In addition, we carried out several advanced proteomic and circulating miRNAs analyses to identify diagnostic biomarkers predictive and/or indicative of treatment response and follow-up, to select clinical trial participants and/or to tailor therapy for individual patient. We already identified several biomarkers of clinical interest using either CE-MS (capillary electrophoresis coupled with mass spectrometry) or high throughput q(RT)PCR for peptide and miRNA identification, respectively, to create a clear prescription path for gene therapy in forthcoming clinical trials. Based on these preliminary yet very encouraging results, we propose that patients may clinically benefit from this approach. Accordingly, we are now entering phase II clinical trial using CYL-02 gene therapy product in patients diagnosed with locally advanced PDAC. Citation Format: Louis Buscail, Barbara Bournet, Fabienne Vernejoul, Gilles Cambois, Hubert Lulka, Naima Hanoun, Aline Meulle, Alix Vignolle-Vidoni, Odile Barbey, Fabian Gross, Rosine Guimbaud, Philippe Ottal, Gérard Tiraby, Pierre Cordelier. Non-viral gene therapy for pancreatic cancer, from preclinical models to phase II clinical trial. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B96.
To understand how two structurally analogous ligand-receptor systems, the nociceptin/opioid receptor-like 1 (ORL1) and dynorphin A/κ-opioid receptor 1 (KOR1) systems, achieve selectivity, receptor chimeras were generated and analyzed. Replacing discrete domains located between the N-terminus and top of the third transmembrane helix of the KOR1 by the homologous domains of the ORL1 receptor yields hybrid receptors, which, in comparison with the parent KOR1, display up to 300-fold increased affinity but low sensitivity toward nociceptin, and unaltered (high) affinity and sensitivity toward dynorphin A. These substitutions contribute elements for binding of nociceptin but do not suppress determinants necessary for binding and potency of dynorphin A. More importantly, further replacement in these chimeras of the second extracellular loop with that of the ORL1 receptor fully restores responsiveness to nociceptin without impairing responsiveness to dynorphin A. A bifunctional hybrid receptor has thus been identified that binds and responds to both nociceptin and dynorphin A as efficiently as the ORL1 receptor does to nociceptin and the KOR1 to dynorphin A. Together, these results suggest that distinct peptide activation mechanisms operate in the two receptor systems. In particular, the second extracellular receptor loop appears to be an absolute requirement for activation of the ORL1 receptor by nociceptin, but not for activation of the KOR1 by dynorphin A.
ABSTRACT Pancreatic ductal adenocarcinoma remains one of the greatest challenges in oncology for which therapeutic intervention is urgently needed. We demonstrated that the intratumoral gene transfer of somatostatin receptor 2, to combat tumor aggressiveness, or of deoxycytidine kinase and uridylate monophosphate kinase, to sensitize to gemcitabine chemotherapy, has antitumoral potential. Here, we describe the development of CYL-02 non-viral gene-therapy product, that comprises a DNA-plasmid encoding for the three aforementioned genes complexed with PolyEthylEnimine (22 kDa). In this work, we performed preclinical toxicology, biodistribution and therapeutic activity studies of CYL-02 in experimental models of pancreatic cancer. We demonstrated the safety of CYL-02 and defined the maximal tolerated dose in two animal species. CYL-02 co-administrated with gemcitabine did not increase gemcitabine toxicity. Biodistribution studies revealed that CYL-02 is rapidly cleared from blood following intravenous administration, and sequestered in tumors following intratumoral injection. CYL-02 drives the expression of therapeutic genes in cancer cells and strongly sensitizes tumor cells to gemcitabine, with significant inhibition of tumor cells dissemination. This study was instrumental for the later use of CYL-02 in patients with advanced pancreatic cancer, demonstrating that rigorous and thorough preclinical investigations are informative for the clinical transfer of gene therapy against pancreatic cancer. GRAPHICAL ABSTRACT
The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (kappa-opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C-terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the kappa-opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C-terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5-Arg6 sequence and prefers the kappa-opioid receptor, whereas the other comprises the nociceptin Thr5-Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5-Gly6 sequence, does not discriminate between the two types of receptor.
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. 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.
