Quality and security of blood, cells and tissues in the EU legislation : is the choice of the legal instrument a challenge ?
D. Pichereau (Toulouse cedex, FR)
Medicine is booming because of the development of science and more particularly of biology and biotechnology. Health products based on genes, cells and tissues are developed and are questioning the existing legal frameworks because of their innovative character, their origin, the therapeutic hopes and the unknown risks. The European Union has an important role to play in the management of scientific advances in order to ensure a high level of protection of public health in the EU. Due to the extraordinary advances in the field, Europe has to update and evaluate the legal acts already implemented. In this regard, the proposal from the Commission is to turn the Tissues and Cells Directives into a Regulation which will be mandatory in all EU member states. This change of legal instrument is motivated by the different approaches developed by the Member States when transposing the Directives leading, then, to gaps in the national legislations. To this end we will analyse the public consultation launched on 17th January 2017 by the European Commission as part of its better Regulation Package, and the comments provided about the relevant legislation. The aim is to assess the legislation on blood, cells and tissues, so as to understand the positive and negative aspects of the current regulation. It is also a good opportunity to look at the issues under discussion during this public consultation as well as the legal nature of the acts in order to meet the initial objective of this regulation: minimize the risks in operations on blood, cells and tissues. It is a timely occasion to take stock of the legal actuality in this field and to understand the new issues which will arise in the coming months for the use therapeutic of tissues and cells.
Elimination of anti-HLA alloantibody producing B cells through the use of a Chimeric Antigen Receptor-like HLA molecule in T cells
E. Palou-Rivera (Barcelona, ES)
T cell immunotherapy using Chimeric Antigen Receptors (CARs) to generate tumor antigen-specific effector T cells has achieved excellent results in anti-leukemic clinical trials, and in a broader appraisal, promising immunotherapeutic results. A major problem in solid organ transplantation is the presence in the recipient of donor specific antibodies, which preclude the success of the transplant due to the associated high risk of antibody-mediated rejection. We hypothesize that a CAR (chimeric antigen receptor)-like molecule with a particular HLA molecule as the CAR extracellular domain will engineer T cells to kill alloimmune B cells with anti-HLA antibodies as BcR, completely eliminating alloantibodies in a specific manner. The first step was to create this chimeric receptor with the HLA-A2 antigen as the CAR-like extracellular domain: extracellular domains of the HLA-A*02:01 molecule were amplified by PCR from cDNA of an A*02:01 positive donor. A CAR comprising the extracellular domains of HLA-A2 and 4-1BB /CD3ζ signalling domain was constructed and delivered by lentiviral transduction into human T cells. The cytotoxic capacity of these transduced T cells will be assessed by co-culturing them with EBV-transformed B cells which produce anti-HLA-A2 alloantibodies. In conclusion, specific T cells directed to kill anti-HLA alloantibody producing B cells can be generated by means of the use of an HLA CAR-like receptor. This technology could open new ways of treatment and prevention of antibody-mediated rejection in solid organ transplantation.
Kynurenine as an indicator of the modifications in differentiated hepatocytes
M. Sadeghi (Heidelberg, DE)
Tryptophan (Trp) is an amino acid required for protein synthesis and metabolic functions. About 95% of Trp is metabolized via the kynurenine (Kyn) pathway. The initial step is oxidation of Trp to N-formylkynurenine, which is converted to Kyn by one of two enzymes, Trp-2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Kyn pathway metabolites act as immune regulators. Our aim is to investigate Trp metabolism via the kynurenine pathway in Primary human hepatocytes (PHH) and tumoral HepaRG cells.
TDO and IDO activity in supernatants of cultured PHH and HepaRG cells was estimated by measuring the degradation of Trp and accumulation of Kyn in the culture media using Reversed Phase- High Performance Liquid Chromatography. IDO activity was inhibited by 1-Methyl-tryptophan (1-MT). However, TDO activity was inhibited either by addition of 680C91 or by addition of LM10. TDO or IDO expression was determined by Real-time PCR. Additionally the influence of IFN-ƒ× (an IDO enhancer) on IDO and TDO activity was studied. PHH as well as HepaRG cells degrade Trp in culture media with increasing amounts of Trp. The highest level of Trp degradation was at 72 h as compared to 24 and 48 h. In contrast, PHH supernatants of HepaRG showed increased levels of Kyn. Compared to unstimulated cells stimulated PHH and HepaRG cells with IFN-ƒ× did not show any significant difference in Trp degradation and Kyn accumulation. Degradation of Trp in the supernatants by differentiated hepatocytes was inhibited by 680C91 as well as LM10 and not by 1-MT. As expected TDO but not IDO gene transcription was detected in HepaRG cells. After IFN-ƒ× stimulation, expression of IDO was extremely up-regulated but this did not enhance Trp degradation. Hepatic cells express only TDO protein which positively correlated with increased L-Trp levels in cell culture media. Enzymatic activity of TDO in HepaRG cells was only able to degrade L- but not D- isoform of tryptophan. To conclude, TDO is the key player of Trp degradation in differentiated intact primary- and tumor hepatocytes. HepaRG accumulate high levels of Kyn which can be a mechanism of tumoral immune resistance. Inhibitors of TDO activity-680C91 or LM10- could play an important role in cancer immunotherapy.