ALBERT

Beschreibung: Following a vascular injury, factor VIII (FVIII) is rapidly activated by thrombin cleavage at arginine (Arg) 372, 740 and 1689. Activated FVIII, an heterotrimer composed by the association of A1, A2 and A3-C1-C2 domains, is rapidly degraded to limit thrombosis risk. Two main phenomenons that account for the disappearence of FVIIIa consist of an intrinsic dissociation of the trimer due to the loss of A2 domain, and the cleavage of the molecule by activated protein C (APC). APC cleaves FVIIIa at Arg 336 and 562. Mutant FVIII molecules were already generated, with one or two arginines substituted, and a subsequent APC cleavage diminished. Since the thrombotic potential of high plasma levels of FVIII has been described, we aimed to generate new factor VIII molecules where the APC cleavage was only modulated. The ultimate goal being to increase in vivo the FVIIIa half-life. Among the two APC cleavage sites, the sequence around the site 562 was the most conserved between species. This region was therefore chosen to be modified with a prior verification that the amino-acids to be modified were not described in any hemophilic phenotype. Subsequently, the six following mutations were realized in a BDD-FVIII cDNA: Q561N, G563A, N564D, N564A, N564Q and I566M as well as the controls R336I, R562K and R336I+R562K. The constructs were transiently expressed in BHK cells to determine the specific activities of the corresponding molecules. The mutant specific activities, as determined by one- and two-stage clotting assays, ranged from 40 to 94 % of the wild-type except for G563A that was almost inactive. CHO clones expressing FVIII molecules were obtained. The mutants and control FVIII molecules were produced, partially purified on heparin column and further analyzed. The comparative specific activities in a two-stage clotting assay were the following (+/− SD; n=6): BDD-FVIII 100 %, Q561N 105 % +/− 45, G563A 6 % +/− 6, N564D 50 % +/− 23, N564A 45 % +/− 21, N564Q 80 % +/− 26, I566M 100 % +/− 41. The one-stage clotting assay gave identical results than the two-stage assay for each mutant. The mutants were then activated by thrombin (1:1) and the occurrence of FVIII activity was monitored. A 20- to 25-fold increase in FVIII activity was measured within 2 minutes following the addition of thrombin for all mutants. The mutants, except for the inactive G563A, were then analyzed for their resistance to APC by three different assays: an APC resistance kit (Coatest, Chromogenix), an in vitro assay that measured APC sensitivity of FVIIIa and an immunoblot assay that visualized the cleavage efficiency of the A2 fragment. These three assays confirmed the APC resistance of the previously published R336I, R562K and R336I+R562K, as compared with wild-type FVIII. They also revealed that the mutants N564D, N564A and I566M behaved similarly to the wild-type FVIII whereas Q561N and N564Q mutants were partially resistant to APC. The APC resistance ratio were the following 2.3 +/− 0.3 for BDD-FVIII, 2.1 +/− 0.4 for N564Q, 1.7 +/− 0,1 for Q561N, 1.6 +/− 0.3 for R562K and 1.3 +/− 0.3 for R336I+R562K. The N564Q and Q561N mutants exhibited a profile intermediate between wild type FVIII and R562K regarding of the loss of FVIIIa activity that was confirmed on the immunoblot profile. In conclusion we have generated new factor VIII molecules that retained their full procoagulant function while possessing a reduced sensitivity to APC cleavage.

Beschreibung: Abstract 4492 Patients undergoing allogeneic haematopoietic stem cell transplants frequently develop infections that are responsible for a high rate of morbidity and mortality during first 6 months post transplantation. The occurrence of CMV infection might reach 70% to 80% of transplanted patients; this rate is increased in HSC transplantations conditioned by in vivo and/or in vitro immunosuppressive treatment. Prophylactic treatment of CMV infection by ganciclovir or foscarnet, though proven to be efficient, faces complications such as myelotoxicity, nephrotoxicity and/or the development of virus resistance. The control of CMV reactivation is achieved in healthy individuals by CD4 +and CD8 +T lymphocytes specifically directed against CMV. After allograft, it has been demonstrated that restoration of anti-CMV immune responses is efficient but all the more delayed when conditioning is heavier. Furthermore, it has been demonstrated that the transfer of donor T lymphocytes specifically directed against CMV was able to prevent and even more to control viral reactivation. Several strategies have been described to generate anti-CMV CD8+ cytotoxic T lymphocytes (CTL) in vitro, by using as antigen-presenting cells (APCs) CMV-infected fibroblasts (Walter, NEJM 1995, 333: 1038), or EBV-transformed B cells retrovirally transduced to express CMV pp65 protein (Sun, Blood 1999, 94: 3242). However, the length of process and the presence of potentially dangerous viral particles in these trials limit their clinical utilisation. Recent progresses in technology resulting from the HLA tetramer technique have enabled to develop direct and fast enrichments of anti-CMV CTL sufficient to inject one or several doses of these lymphocytes (Cobbod abstract 155, ASH 2004). In order to reach the regulatory requirements for ancillary products to be used in an anti CMV immunotherapy protocol, a tetramer selection system and its associated production process compatible with cGMP has been developed. The developed manufacturing process (Protein Expert) is based on: - the E. coli production of the different components, HLA201 heavy chain and β2 microglobulin as well as streptavidin, the chemical synthesis of the pp65 peptide; - the production and biotinylation of the monomers-the production of the tetramers using recombinant streptavidin covalently linked to magnetic beads (Miltenyi Biotec ®) compatible with clinical applications. Seven immunomagnetical selections on MS column of HLA A2-pp65 positive tetramer cells from healthy blood donors were performed with these GMP reagents. Respective means of CD8 tetramer +cells in starting and selected fractions were 2.5 %(+/-3.1) and 99 %(+/-0.6). Expt Start Selected CD8 tetramer+ cells (%) CD8 tetramer+ cells (10e5)) CD3 + cells (%) CD8 tetramer+ cells (%) CD8 tetramer+ cells (10e5) CD8 tetramer+ cells yield (%)) 1 0,54 1,31 89 99 1,73 117 2 8,45 7,46 99 99 12,2 162 3 0,32 0,57 93 99 0,64 105 4 0,39 1,37 91 99 1,68 112 5 3,35 8,59 98 99,5 5,84 67 6 4,47 2,7 98 98 2,4 86 7 0,14 0,2 79 99,8 0,29 116 Mean 2,5 3,2 92,4 99,0 3,5 109,3 SD 3,1 3,4 7,1 0,6 4,2 29,6 Starting and selected cells were restimulated with CMV or irrelevant peptide and evaluated for interferon gamma secretion (respectively 5.4%+/5.2 and 71.8%+/-14.7) and CD107a expression (6.1%+/-2.9 and 56.2%+/-32.7). The GMP reagents used for selection demonstrated a very good efficacy for enrichment of HLA A2-pp65 positive tetramer CD8 cells with high purity and yield. Selected cells were shown to be antigen specific and cytotoxic. This procedure scaled up on Clinimacs device could provide in a very short time, sufficient CD8 tetramer positive cells for very selective adoptive immunotherapy after stem cells transplant. Disclosures: No relevant conflicts of interest to declare.