MODIFIED PEPTIDES AS A NOVEL IMMUNOTHERAPY FOR RHEUMATOID ARTHRITIS

Background: Rheumatoid arthritis (RA) is a highly prevalent and severe systemic autoimmune disease associated with permanent disability and strong socio-economic burdens. Currently, there is no therapeutic treatment and RA patients rely on lifelong, costly treatments. Imcyse develops modified peptides eliciting antigen specific cytolytic CD4 T cells (cCD4+) that induce apoptosis of antigen presenting cells (APC) in a contact dependent manner. cCD4+ also induce apoptosis of autoantigen-specific bystander T-cells, activated by the same APC thus eliminating the risk of general immunosuppression. Peptides consist of MHC class II T cell epitopes of a target autoantigen modified in their flanking region by the addition of an amino acid sequence containing a thiol-disulphide oxidoreductase active motif1. Objectives: The goal of this study was to synthesize modified peptides from a target RA autoantigen and test their potency to generate in vitro specific and cytolytic CD4+ T cells from RA patients. Methods: We designed modified peptides from a target RA autoantigen after in silico and in vitro assessment to identify MHC II core binding region, HLA class II binding capacities and physiochemical properties. CD4+ T cells were purified from PBMC of a newly diagnosed seropositive RA patient and co-cultured with autologous APC in the presence of the modified peptide. The CD4+ T cells were restimulated periodically. Peptide’s ability to generate specific CD4+ T cells was evaluated by flow cytometric analysis of the expression of surface activation marker CD154 (CD40L). The peptide specific CD4+ T cell lines were sorted based on their surface CD154 expression. Their pro-apoptotic activity was assessed after overnight (O/N) co-culture of CD4+ T cells with fluorescent tracer labelled autologous lymphoblastoid cells lines (LCL). Flow cytometry quantification of LCL apoptosis was measured by annexin V staining. MHC II restriction of CD4+ T cells was demonstrated by the addition of blocking antibodies against HLA-DR, DP or DQ molecules. Results: CD4+ T cells were in vitro expanded after six consecutive stimulations with the peptide. We investigated their specificity by flow cytometry and showed that 69% of CD4+ T cells that were stimulated O/N in the presence of the peptide expressed the activation marker CD154 versus 29% of CD4+ T cells that were stimulated in its absence. These cells were sorted based on CD154 expression following specific stimulation. Cell enrichment was then assessed by flow cytometric analysis. Data showed that more than 91% (background 3%) were peptide specific based on CD154 expression. After co-culture of CD4+ T cells with LCL, in independent experiments, Annexin V binding was detected on peptide loaded LCL, ranging from 69% to 89%, while when LCL were kept unloaded these values were between 30% and 55%, respectively, indicating that when specifically activated, these CD4+ T cells had pro-apoptotic activity. When both the peptide and blocking antibodies against HLA-DR, DP or DQ molecules added in the co-culture the pro-apoptotic activity was inhibited by 68%, 20% and 25%, respectively. Conclusion: The preliminary but very promising data show that our modified peptide generates peptide-specific CD4+ T cells with lytic properties that lyse target APC in an HLA class II specific manner. Our plan is to show that these CD4+ T cells can also induce apoptosis in bystander T cells and to further validate our results in additional RA donors. References: [1]Carlier, V. A., Vanderelst, L., Janssens, W. \u0026 Jacquemin, M. G. Increased Synapse Formation Obtained by T Cell Epitopes Containing a CxxC Motif in Flanking Residues Convert CD4 + T Cells into Cytolytic Effectors. 7, (2012). Disclosure of Interests: Eleni Araklioti Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ludivine Herman Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Ngoc Quynh Nhu Nguyen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Roxana Roohi Ahangarani Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Milos Erak Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Bernard Lauwerys: None declared, Patrick Durez Speakers bureau: AbbVie, Bristol-Myers Squibb, Celltrion, Eli Lilly, Pfizer, Sanofi, Vincent Geenen: None declared, Aaron Winkler Shareholder of: Shareholder of Pfizer, Inc, Employee of: Full time employee of Pfizer, Inc, Marcelle Van Mechelen Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Luc Vander Elst Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region, Vincent Carlier Grant/research support from: This work was supported by Pfizer Inc. and by Walloon Region