Scientific Abstract
Proposal No. IBD-0008
Principal Investigator: Sander J. van Deventer, Ph.D., M.D.
Applicant Organization: Academic Medical Centre Medical Research B.V. (Amsterdam, The Netherlands)
Project Title: Treatment of Crohn's disease by Lactococcus lactis secreting human IL-10
Period of Award: December 1, 2002 – November 30, 2003
Understanding the mucosal immune system, including its regulatory network of cytokines, is very important for the development of innovative therapies for inflammatory bowel disease. The importance is highlighted by the observation that using neutralizing antibodies preventing the biological activity of tumor necrosis factor-alpha (TNF-α) is the most potent therapy for Crohn’s disease yet described. However, the benefits of this therapy are temporally restricted and finding novel avenues for remission maintenance is the most important target of contemporary research into Crohn’s disease.
Interleukin-10 (IL-10) plays a key role in maintenance of mucosal tolerance as is shown in experiments with IL-10 deficient mice. IL-10 treatment of patients with Crohn’s disease is not as effective as anti-TNF antibodies in reducing remission, but was shown to be safe and without adverse events. Studies in experimental models suggest that topical application of IL-10 is especially effective for preventing disease. Murine interleukin-10 production by genetically modified Lactococcus lactis is a highly efficient approach in animals with chronic inflammatory bowel disease. The engineering of Lactococcus lactis secreting human IL-10 is thus the next step to develop a novel safe and biologically restricted way for topical IL-10 delivery. Lactococcus lactis is non-pathogenic non-colonizing antibiotic-sensitive bacterium. We have constructed a Lactococcus lactis strain in which the thyA gene is replaced by the coding sequence of human IL-10. The absence of the thyA gene makes these organisms fully dependent on the presence of thymidine and thus unable to survive in the environment. Furthermore, the IL-10 gene is not present on a plasmid but is incorporated on the chromosome of the bacterium making it unlikely to transfer into other organisms. If homologous recombination would occur, this would also confer the dependency for thymidine on the receiving bacterium.
We will investigate the therapy of patients with Crohn’s disease using a Lactococcus lactis secreting human IL-10. We plan to treat 32 patients with Lactococcus lactis, 16 patients (moderate to severe disease activity; Crohn’s disease activity index ranging 220 - 450) with the wild type bacterium and 16 patients with the genetic engineered form. Patients will receive 1x1012 bacteria per os daily, during a two-week study period, using a capsule that disintegrates in terminal ileum. Before and after treatment, patients will be subjected to ileocoloscopy and biopsies will be collected. Local IL-10 availability will be discriminated from endogenous IL-10 based on the discrepancy in glycosylation by performing Western blotting or mass spectrometry. Clinical follow up, cytokine measurements, pathological grading and histological studies will identify possible effects of the treatment on the mucosal immune system and disease activity. The study should provide important answers as to the safety and efficacy of using Lactococcus lactis secreting human IL-10.
The proposed project is clearly novel: cytokine-based therapy by itself is a new development, and using bacteria as a tool for cytokine delivery in human disease has not been attempted before. Our cutting edge method of bacterial engineering has important advantages to, for instance, ex vivo gene therapy. Conventional antibiotics can quickly eradicate Lactococcus lactis secreting IL-10 and the highly topical delivery of the immune-suppressive cytokine is mainly active when the mucosal barrier is disrupted (i.e., during active inflammation). Although important preliminary work in experimental animal models has already been performed, the absence of human data is clear evidence of the early stages of the research.