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Cell Therapies – a therapeutic strategy in the 21st century Petra Reinke Dept. Nephrology and Internal Intensive Care & Berlin-Brandenburg Center of Regenerative Therapies (BCRT) Charité, Berlin [email protected] September 2011 : „Institutsgebäude Süd“ hosting 5 Institutions (BCRT / Julius-Wolf-Institut / Institut für Genetik / Research Group Nephrology & Transplantation / Institut für medizinische Immunologie) BCRT Central Services Objectives Platform Immune System Immune system Open access Cardiovascul. system Musculo skeletal system (Head: Prof. Dr. P. Reinke) Translational Technology Polymer-based Bio-Materials BCRT Cell Differentiation/Characterization immune cell therapy - chronic infections / tumors (effector T cells) - undesired immune reactions (regulatory T cells) - ischemia/reperfusion injury (mesenchymal stem cells) biomarkers for personalized therapies - development, meth. & clin. validation delivery of methods for the other fields - crosstalk between stem cells <–> immune cells - cell sorting technologies - biomarker analyses - biomaterial <-> immune system CELLS (ATMPs) – A NEW CLASS OF THERAPEUTIC TOOLS Cells Therapeutic Tools ATMPs Gene Therapy Tissue Eng. conventional drugs (small molecules) Antibodies Biologicals Fusion proteins Cytokines BCRT Core Unit GMP BCRT Core Unit GMP cell therapy for reshaping immune response - chronic infections / tumors (protective memory/effector T cells) - undesired immune reactions (regulatory T cells) - shaping intratissue inflammation (mesenchymal stromal/stem cells) BCRT Core Unit GMP cell therapy for reshaping immune response - chronic infections / tumors (protective memory/effector T cells) - undesired immune reactions (regulatory T cells) - shaping intratissue inflammation (mesenchymal stromal/stem cells) regulatory effector mechanisms mechanisms The medical need in transplantation and autoimmunity Problems: • lifelong immunosuppressive therapy • high morbidity and mortality and costs • no improvement of long-term results SOLUTION ? AIM: Minimizing long-term immunosuppression - supporting regulation - personalized therapy (biomarker) cell based therapeutic approach with autologous nTreg (CD 4+25+FoxP3+) IL-2 consumption ATP-degradation cytolysis by secretion of granzyme B secretion of suppressive cytokines such as TGFβ contact-dependent mechanisms favoring regulation (Treg) selective targeting pathogenic memory/effector cells Particular challenges for translation to solid organ transplantation (SOT) - high clonal size of naive alloreactive T cells - pre-existing alloreactive memory/effector T cells - pre-injured donor organ with enhanced immunogenicity (act.APC) Are Tregs able to control alloreactivity under these conditions ? How can we measure succesful regulation in order to minimize safely IS ? FP 7 EU Grant „BIO-DrIM“ 11-2012 to 11-2017 (coordinator: Petra Reinke) FP 7 EU Grant „ONE-Study“ 11-2010 to 11-2015 (coordinator: Ed Geissler; WP leader biomarker & Treg production : Petra Reinke) What are the challenges ? Pre-clinical hurdles -Appropriate animal models -Basic research -Biomarker developement and validation Clinical and technical hurdles -Isolation procedure -Expansion conditions -GMP compliance -Allogen-specific Treg activity? -Functional validation -Autologous or third party? -Single shot or repetitive? Isolation in vitro expansion Characterization in vivo analysis Clinical application What are the challenges ? Pre-clinical hurdles -Appropriate animal models -Basic research -Biomarker developement and validation anti-CD4 mab (non-depl) T-cell depletion CTLA-4Ig LEW DA life-supporting kidney Tx tolerance -> secondary Treg induction in vivo Treg no tolerance in patients What was wrong with our model ? Challenges in humans: - marginal donor organs - co-infections of recipients - immunological experience Need for preclinical models adapted to clinical challenges Pascher et al. 2006, Pratschke et al. 2009, Siepert et al. 2012 Need for advanced preclinical models that are more predicting for clinical challenges ? Treg in vitro Treg in conventional SOT models Treg in advanced SOT models ? Treg in SOT patients Need for advanced preclinical models that are more predicting for clinical challenges DA anti-CD mAb (non-depl) tolerance CTLA4Ig T-cell depletion LEW life-supporting kidney Tx Treg models adapted to the clinical challenges: tolerance protocols failed brain prolonged memory co-infection death ischemia T cells (CMV) DA anti-CD4 mAb (non-depl.) CTLA4Ig T-cell depletion LEW life-supporting kidney Tx no or reduced tolerance ! Treg Pascher et al. 2006, Pratschke et al. 2009, Siepert et al. 2012 Can Treg mediate tolerance in the challenging „memory“ model ? A DA LEW >150d T cell depletion (>90%) B DA LEW <10d T cell depletion (>90%) DA-specific effector/memory 1/1000, d-7 or d-150 C DA LEW T cell depletion (>90%) DA-specific effector/memory 1/1000, d-7 or d-150 D DA 35d Treg d3 or d20 LEW T cell depletion (>90%) DA-specific effector/memory 1/1000, d-7 or d-150 >150d Treg d3 or d20 CNI up to day 10 Siepert et al.; Am J Transplant 2012 Treg block most effectively LIP of GFP+ alloreactive Tmem Frequency of GFP+ Memory T cells in peripheral blood 2,0 1,8 Tmem cells + T-cell depletion (GST: 13.7 d) GFP+ Memory T cells/ MNC [%] 1,6 Tmem + depletion + CNI d0-9 (GST: 57d) 1,4 Tmem + depletion + CNI d0-150 (GST: >150d) 1,2 Tmem + depletion + CNI d0-9 + Treg d4 (GST: >150d) 1,0 0,8 0,6 0,4 0,2 0,0 pre- Tx 5 21 49 70 100 150 Time post Transplantation [d] Treg induce long-lasting intragraft “tolerance signature” : high Toag1; high Foxp3 ; low IL-6; intermediate CD3 d 150 post-Tx (intragraft RT-PCR) 1 CD3 (infiltrates) 0,1 TOAG1 (immune silence) Expression/ b-Actin 0,1 0,01 0,01 0,001 0,001 0,1 IL6 (innate immunity) 0,01 0,01 Foxp3 (regulation) 0,001 0,001 0,0001 0,0001 0,00001 0,00001 Tmem + depletion + mTOR d3-150 Tmem + depletion + CNI d0-150 Tmem + depletion + CNI d0-9 + Treg d4 What are the challenges ? Pre-clinical hurdles -Appropriate animal models -Basic research -Biomarker developement and validation Clinical and technical hurdles -Isolation procedure -Expansion conditions -GMP compliance -Allogen-specific Treg activity? -Functional validation -Autologous or third party? -Single shot or repetitive? Isolation in vitro expansion Characterization in vivo analysis Clinical application What are the challenges ? Pre-clinical hurdles -Appropriate animal models -Basic research -Biomarker developement and validation Clinical and technical hurdles -Isolation procedure -Expansion conditions -GMP compliance -Allogen-specific Treg activity? -Functional validation -Autologous or third party? -Single shot or repetitive? Isolation in vitro expansion Characterization in vivo analysis Clinical application Manufacturing Approach starting material 35 – 50 ml blood separation of cell subpopulation starting cell nb. 1x106 nTregs depletion of CD8+ T cells enrichment CD4+25++ cell expansion max. 2x108 nTregs for application anti-CD3/28 beads + IL-2 + mTOR-inhibitor blood PBMC isolated Treg step 1 (3-4 hrs): Isolation of Treg expanded Treg step 2 (2-3 wks): Expansion of Treg > 100 fold depletion of CD8+ T cells enrichment CD4+25++ by CliniMacs anti-CD3/28 beads + IL-2 + mTOR-inhibitor blood PBMC isolated Treg step 1 (3-4 hrs): Isolation of Treg depletion of CD8+ T cells enrichment CD4+25++ by CliniMacs expanded Treg step 2 (2-3 wks): Expansion of Treg anti-CD3/28 beads + IL-2 + mTOR-inhibitor Novel method; TCR sequencing by next-generation sequencing > 100 fold Babel N et al. submitted blood PBMC step 1 (3-4 hrs): Isolation of Treg isolated Treg expanded Treg step 2 (2-3 wks): Expansion of Treg TCR repertoire pattern before/after expansion (>100,000 sequences / line analysed) fresh expanded TCR analysis by next-generation sequencing: 1. TCR repertoire of nTreg and Tconv is completely different (almost no overlap) 2. Expansion does not alter polyclonal TCR repertoire of nTreg GMP adapted isolation/expansion of human Treg – % CD45RA+ in Tregs - feasible for healthy donors Decreasing CD45RA+ naive Treg with increasing age End-stage kidney disease (ESKD) patients have less CD45RA+CD62L+ naive Treg but more CD45RA-CD62L+ central memory Treg compared to age-matched controls More (central)memory Treg in ESKD patients Are (central)-memory Treg functionally comparable to naive Treg and useful for therapy ? NTx patients (years post Tx) (Central) memory nTreg are very potent Treg FoxP3+ Helios+ * * Naïve Treg Treg cm Treg central memory (cm) Treg em - FoxP3/Helios/CTLA4 ++ - high suppressive capacity - FoxP3 TSDR demethylated - homing receptors for tissue infiltration but also lymphnodes (CD62L) => highly potent nTreg Which protocols are suitable for testing Treg ? 3 CENTERS (GER, UK) Controls (18-24 living donations) - IL2R-Ab induction therapy - Tacrolimus, MMF, Steroid Treg DC Treatment (48 living donations) - Tacrolimus, MMF, Steroid - Treg (day 7) Tr1 Mreg Biomarker: - safety: allo-Ab, viral load, virus spec. T cell - efficacy: tolerance profile (TR-PCR) - TCR deep sequencing Clinical Trials using Tregs Target Treg Source Trial Status GvHD UBCB Phase I Completed (Italy) GvHD Peripheral 3rd party Phase I Completed (US) DM I Autologous Phase I Recruiting (US) KTx Autologous Phase I CTA Q2 2013 GvHD Ind. autologous Phase II Recruiting (US) Foxp3 How to monitor success / failure of Treg therapy R6 2 % R5 CD25 1. Toag-1 Molecular marker of „silenced“ adaptive immunity (Sawitzki et al. AJT 2007) • useful to identify pre-transplant activation state (= individual risk) • down-regulation post-Tx = predicting failure of therapy • recovery of Toag-1 post therapy = successful therapy (reprogramming tolerance) 2. Donor-specific Memory T cells (Elispot) (Nickel et al. Transplantation 2005) • useful to identify high-risk patients before Tx => high levels not suitable for strong weaning or even tolerance protocols • stably low levels post-Tx = successful control of alloreactivity 3. Molecular signature of tolerance (Sagoo P et al. JCI 2010) • signature associated with spontaneous tolerance • might be useful for indentifying patients that can be partly weaned after successful Treg therapy What are the challenges ? And where we are ? Pre-clinical hurdles - Appropriate animal models - Basic research on Treg biology - Biomarker developement and validation Clinical and technical hurdles - Isolation procedure - Expansion conditions - GMP compliance - Functional validation - Clinical Study Protocol - Clinical Study - Manufactoring Authorization acc.to EMA/FDA rules - Autologous or third party? - Single shot or repetitive? Isolation in vitro expansion Characterization in vivo analysis Clinical application I have to thank….. Support: BMFB DFG (SFB 650 & TR-SFB 36) EU FP7 (The One Study) T-cell Europe special thanks to clinicians, patients and nurses