This translational and industrial program aims at positioning an innovative immunmodulatory agent, an improved and recombinant fusion IL-15 protein called CYP0150, to treat cancers in the best conditions. Based on a strong basis of relevant in vivo models (human immune system mouse, primates) and ex vivo tumor biopsies, we intend to validate the toxicology/pharmacology/immunogenicity ratio of this new drug to consolidate our regulatory preclinical studies. Moreover, thanks to many emerging and revolutionary concepts demonstrating the critical role of the immune system on the efficacy of many anticancer drug classes, several therapy combinations will be evaluated to leverage the pharmacological effects of RLI as best as possible and to define the best treatment combinations to be used in phase II clinical trials. Last but not least, specific molecules related to the IL-15/IL-15Ralpha transpresentation system will be monitored in cancer patient samples before and after therapy to define a relevant biomarker useful to predict and select the best-responsive patient populations for a CYP0150-based therapy as stand-alone or in combination with other anticancer drug classes. Based on the internal regulatory preclinical development of CYP0150 and this translational collaborative program, we should be able to initiate our clinical trials in cancer patients by early-2013 in optimal conditions and to consolidate our worldwide leadership position in this IL-15-related field.

Aims. Type 1 diabetes (T1D) results from the autoimmune destruction of pancreatic ß-cells. The HLA DQB1*0302 class II HLA gene carry the highest risk for T1D, but class I MHC alleles modulate the risk, in particular HLA-A*0201. Among ß-cell autoantigens, insulin has been ascribed a key role in T1D. While this knowledge has boosted efforts to prevent T1D using insulin-specific immunotherapy, these efforts have been unsuccessful when translated into clinical trials, pointing to the need for new preclinical T1D models. The aims are to: 1) characterize the immune and metabolic phenotypes of humanized mice (YES) expressing human insulin (hPPI), HLA-A*0201, DQ8 transgenes and lacking their mouse counterparts, 2) evaluate YES mice as a new preclinical model for insulitis and diabetes, 3) characterize hPPI epitopes targeted by CD4+ and CD8+ T cells and develop T-cell assays in these mice, 4) evaluate strategies to induce immune tolerance to hPPI as a therapy in T1D. Methodology. We have obtained YES mice that lack the expression of murine class I, class II and insulin genes, express HLA-A*0201, DQ8 transgenes and human insulin (hINS) transgenes. Analysis of pancreases from YES mice shows normal islet morphology, but we have preliminary evidence that YES mice develop insulitis following immunization against a human ß cell line. To characterize the immune and metabolic phenotypes of YES mice, three YES lines will be studied. As the replacement of murine class I and class II MHC molecules may interfere with the selection of class I- and class II-restricted T cells and with antigen presentation, the main immune subsets, in particular T lymphocytes, will be quantified by FACS in thymus, spleen, lymph nodes and pancreas. Cytotoxic and proliferative responses to conventional antigens will be evaluated. Insulin gene expression will be quantified in liver, pancreas, thymus, spleen, kidney, inguinal lymph node and the metabolic phenotype of YES mice further characterized and compared to that of the parental mouse lines used. To evaluate YES mice as a new preclinical model for insulitis and diabetes, attempts will be made to characterize further and optimize the induction of insulitis and immune-mediated ß cell destruction using different immunization procedures and introduction of B7.1 expression on ß-cells. To characterize hPPI epitopes targeted by autoimmune CD4+ and CD8+ T cells and apply T-cell assays to detect ß-cell autoimmunity in this model, DQ8-restricted CD4+ and A*0201-restricted CD8+ T cell responses to human insulin will be evaluated and the repertoire of epitopes recognized defined. Immunogenicity of human insulin peptides and recognition by CD4+ T cells will be studied using proliferation assays and evaluating production of cytokines. Insulin-specific CD8+ T cells will be studied – immunization against a library of insulin peptides, cytotoxicity assays, single cell PCR of tetramer-sorted cells. T cell clones will be generated and tested for recognition of P815 cells cotransfected with HLA-A*0201 and hINS. The same T cell assays will be used to study insulin-specific CD8+ and CD4+ T cells along induction of insulitis/diabetes. To evaluate the feasibility of inducing immune tolerance to insulin, and more generally ß-cells, in this model, as preventive or therapeutic strategies in T1D, we will assess whether delivery of a human insulin-Fc fusion protein can induce immune tolerance in YES mice, and identify immune correlates and mechanisms of diabetes protection. Insulin-Fc fusion proteins will be delivered to YES offsprings upon treatment of mothers during pregnancy or lactation, or to YES mice at 3 week of age or following induction of insulitis/diabetes. Mice will be evaluated for induction of T cell tolerance to human insulin and for prevention of insulitis/diabetes. Setting up a new preclinical T1D model may help the development of T-cell assays and pave the way to “vaccination” strategies in T1D.

The incidence of type 2 diabetes mellitus (T2D) in industrialized countries has increased dramatically over the past several decades. There are approximately 37 million people suffering from T2D in North America and 3 million in France. Whereas T2D has been initially considered to be mainly limited to older people, early onset diabetes is increasingly observed in young people. This increasing incidence of T2D is closely accompanied with the other components of the so-called “metabolic syndrome”, which includes cardiovascular diseases and obesity. T2D is characterized by high blood glucose in the context of insulin resistance and ultimately leading to insulin deficiency. Genetic and biochemical evidence showed that dysregulation at the level of the insulin producing pancreatic ß-cells plays a major role in T2D development. These cells express at least 20 different members of the G protein-coupled receptor (GPCR) super-family, which are privileged drug targets. In 2009, several genome-wide association studies revealed an unexpected association between the MTNR1B gene, encoding the melatonin MT2 receptor, and both fasting plasma glucose levels and T2D risk. The strongest association was observed for the common SNPs rs10830963 located in the unique intron of MTNR1B and at a non-conserved locus, raising the question of its functional consequences. The melatonin MT2 receptor belongs to the GPCR super-family and is targeted by the neurohormone melatonin. Melatonin is mainly secreted by the pineal gland in a circadian manner with high levels during the night. Its synthesis is regulated by light and the circadian clock located in the suprachiasmatic nucleus. Importantly, melatonin can also regulate circadian rhythms. Several reports in the literature link melatonin and circadian rhythms with glucose homeostasis and metabolic dysfunction. Some studies showed that melatonin may have a direct effect on pancreatic ß-cells by negatively modulating their insulin secretion, however the molecular mechanisms involved remain unclear. Recent studies suggest a regulatory role of pancreatic MT2 receptors on the circadian rhythm of the pancreas, providing an additional possible link between melatonin and pancreas physiology. Disruption of central and peripheral circadian rhythms, including in the pancreas, has indeed been shown to lead to metabolic disorders including T2D. To clarify the link between melatonin receptors and T2D risk in humans, we propose to identify rare mutants located in the coding regions of melatonin receptor genes and characterize the functional properties of the corresponding receptors.

Rationale: 45 million individuals worldwide are bilaterally blind, among them 4.9 million are suffering from corneal defects. Corneal disorders are consequences of congenital diseases, or environmental insults such as physical injury, viral infection, chemical and thermal burns. The general result is corneal neo-vascularization that leads to loss of corneal transparency and blindness. Corneal transplantation is widely used with high success at the short term but is limited by the shortage in post mortem cornea and immunorejection prevalence is significantly increasing with time within the first 5 years. Therefore, there is a major need for alternative allo- or autologous sources for corneal cell therapy. Background: Oral epithelial cells, mesenchymal stem cells, hair-follicle stem cells and embryonic stem cells were investigated for their therapeutic potential for corneal diseases. However, only partial success has been achieved by the use of these cells since they were not able to uniformly commit into corneal epithelial cells and/or no experimental data have been provided to demonstrate their ability to stratify and reconstitute in vitro a corneal tissue or displayed abnormal thickness and barrier function. Artificial cornea is an alternative acellular solution that is suggested only for patients with complex ocular diseases who are at high risk for donor graft failure. These complicated surgeries unfortunately allow limited vision improvement and are expensive. Innovative product: we propose here to design a standardized and reproducible technology to produce in large amount "ready to use" cornea from human pluripotent stem (induced or embryonic) cell lines. We will take advantage of the unlimited growth capacity of these cells and their pluripotent ability to differentiate into any cell type. As a proof of concept, we have recently developed an efficient technique to generate corneal cells expressing putative corneal-specific markers from either human embryonic stem cells or human induced pluripotent stem cells. We have shown that these corneal cells are able to stratify in vitro for the production of a corneal tissue. Potential applications of the innovative product: the finalized product will be used in two main applications: a. corneal regenerative medicine: here, we aim to produce unlimited cornea from pluripotent stem cells. Such tissue will serve as “ready-to-use” source to be transplanted immediately into injured eye as allo-graft. This product will be easily accessible for surgeons around the world, standing by for grafting into patients. In addition, for patients presenting immuno-rejection problem, autologous cornea will be produced directly from their own cells. b. cellular model for cosmetical toxicity and drug testing: the corneal tissue obtained from pluripotent stem cells should become a valuable tool to perform, in a reproducible and physiological manner, toxicity assays of cosmetic products and pharmacological tests of drugs. The EU Cosmetic Directive lead to the ban of animal testing for cosmetic ingredients (acute tests). Major efforts are still made to find reliable and relevant alternative methods to eye irritation testing. So far, quite unsuccessfully as no suitable model is yet established. Our reproducible in vitro tissue model will allow predicting the safety and efficacy profiles of actives and formulations. Specifically, it will be used in ocular irritation assays, as tests for corneal permeability and metabolism and mucin production. To be done within 2 years: our current goals are (1) to standardize the quality of iPSC/huESC-derived corneal tissues, (2) to evaluate their therapeutic potential in vivo, (3) to demonstrate that this model may serve as a useful in vitro tool for toxicity assays, thereby allowing the reduction in animal testing.