T cell therapy of inflammation with regulatory cells Arnaud Foussat, VP Research & New Products ISCT Annual Meeting- 23-26 April 2014, Paris 1
Cellular immunotherapy Stimulate immunity Induce immune tolerance Dendritic cells NK cells Effector T cells CD8+ CD4+ gd+ CAR-T cells Activated PBMCs Provenge Regulatory T cells Tolerogenic Dendritic cells Cancer & infectious diseases 2
The Treg world Thymic derived Treg (ntreg/ttreg) CD25 + Foxp3 + CD25 + CD127 neg Specific for self Autoimmunity & transplant tolerance CTLA-4 + GITR + PD-1 + CD62L + Naïve CD45RA + vs Memory CD45RO + Peripheral derived Treg (itreg/ptreg) Specific for self & non-self Autoimmunity & transplant tolerance Chronic inflammation & allergy Ex Vivo IL2 + TGF-b Induced Treg TGF-b producing Th3 Treg IL-10 producing Type 1 Treg Multiple sub-populations with different biology, Repertoire, MoAs, Migratory & Proliferative capacity.. 3
and conventional activat ed CD4 + T cells (mice and humans) CD25 CD127 low GITR CTLA4 CD45RB low (mice only) CD45RO (humans only) Folate receptor 4 (mice only) activat ed mouse FOXP3 + T Reg cells FOXP3 Latent TGF CD103 Subpopulations of human FOXP3 + T Reg cells CD45RA + FOXP3 low (naive) CD45RA FOXP3 hi (activat ed) CD45RA FOXP3 low (cytokine-secreting) activat ed human FOXP3 + T Reg cells FOXP3 hi Latent TGF GARP CD121a (IL-1R1) CD121b (IL-1R2) Treg cells: Multi-target, multi-moa approach Treg cells displays multiple inhibitory pathways Sub-population differs in inhibitory pathways (Cytokine driven inhibition, contact molecules, ICOS, PD-1, GITR, Granzyme A/B, CD39) Antigen-specific activation but bystander activity Suppressive cytokines and secreted molecules Membranetethered TGF T Reg cell T Reg cell Galectin 1 Apoptosis Metabolic disruption IL-2 CD45 CD80 or CD86 CTLA4 FOXP3 + FOXP3 + IL-2R (CD25) CD43 CD39 CD73 IL-35 Contact-dependent inhibition DC IDO IL-10 TGF IL-10 Adenosine CD4 + or CD8 + effector T cell Tryptophan catabolites Tryptophan depletion A 2A R Apoptosis owing to cytokine deprivation Cell cycle arrest Co-stimulation blockade Apoptosis Inhibition of DC maturation and decreased T cell activation Cytolysis FOXP3 + T Reg cell Targeting DCs by other mechanisms Increased interaction with DCs Granzyme A or granzyme B T Reg cell Perforin pore CD4 + or CD8 + effector T cell Trogocytosis? MHC CD80 or class II CD86 TCR CTLA4 Neuropilin 1 Granzymes and perforin FOXP3 + LAG3 Co-stimulation blockade Apoptosis Transendocytosis of CD80/ CD86 MHC class II molecule Inhibition of DC maturation From Nature Immunology review, Ethan Shevach &Todd Davidson Affiliations Is there in vivo a dominant mechanism of action? Is a multi-moa STEMCELL required Technologies for Treg activity in vivo? Ethan Shevach and Todd Davidson are at the Regulatory T cells (Tregs) comprise only a small fraction of total MoA might be dependent on disease, Laboratory disease of Immunology, state, National disease Institute of history, CD4 + T cells previous in human peripheral treatments. blood and mouse spleen, and Allergy and Infectious Diseases, National Institutes therefore must be highly enriched to evaluate their suppressive Difference in MoA can lead to differences of Health, Bethesda, in main Maryland cellular 20892, USA. and molecular function and therapeutic targets potential. Since Tregs lack a unique cell e-mail: eshevach@niaid.nih.gov surface marker and often share phenotypic similarities with How to set-up a potency assay with The a authors Treg declare cell no competing product financial? interests. activated T cells, isolation of highly purified Tregs is typically difficult and time consuming, often requiring multiple steps. Potential less patient refractoriness Edited to multiple by Lucy Bird and target Kirsty Minton; approaches. To meet the needs of Treg researchers, STEMCELL Technologies copyedited by Gemma Ryan; designed by Simon Bradbrook. (2010) Macmillan Publishers Ltd. All rights reserved. http://www.nature.com/nri/posters/tregcells 4 has developed a full range of optimized Treg isolation kits that addresses these specific challenges. Our Complete Kits for the isolation of Tregs provide the fastest and easiest method to isolate highly purified human Tregs directly from whole blood or buffy coat in just two steps. CD4 +, CD4 + CD127 low or CD4 + CD127 low CD49d - T cells are pre-enr which combines a normal Ficoll TM density cen antibody-mediated specific cell enrichment T cells are then positively selected from the our column-free immunomagnetic cell sepa or our fully automated cell separator, Robo working with human PBMCs or mouse sple kits provide a fast, easy and gentle method Tregs. Positively selected cells express high suitable for immediate downstream experi cytometry, in vitro expansion or suppressio For more information about our complet products, or to request a free sample, pleas
Treg cell therapy manufacturing Isolation of Natural pre-existing Tregs - Mandatory cell surface markers, - Polyclonal Treg populations - Expansion prior to administration - Functionality of cells required Ex vivo differentiation of Tregs - Regimen of stimulation is key - % of Treg differentiation to control effective standard doses - Polyclonal & antigen-specific Treg populations 5
Treg clinical studies 25 Treg clinical studies referenced 13 in HSCT 8 autoimmunity & chronic inflammation Diabetes Crohn s Disease Asthma Uveitis 4 in transplantation Liver transplantation Kidney transplantation 5 completed 7 Industry sponsored All Phase I & Phase II Sponsors/Collaborators Phase Status Conditions Sponsors/Collaborators Nanjing Medical UniversityUniversity of Nanjing Medical UniversityUniversity of I/II Recruiting Acute Rejection of Liver Transplant Minnesota Minnesota University Hospital of Liege II Recruiting cgvhd / acute University Hospital of Liege Dana-Farber Cancer Institute Miltenyi Biotec GmbH Prometheus Laboratories National Cancer Institute (NCI) Stanford University Laura Johnston National Institutes of Health (NIH) Stanford University Ginna Laport National Institutes of Health (NIH) University of California, San Francisco Juvenile Diabetes Research Foundation National Institute of Allergy and Infectious Diseases (NIAID) (Neostem) Massachusetts General Hospital Dana-Farber Cancer Institute The University of Regensberg 6 I Recruiting cgvhd I Recruiting GVHD, ahct I/II I I Recruiting Active, not recruiting Dana-Farber Cancer Institute Miltenyi Biotec GmbH Prometheus Laboratories National Cancer Institute (NCI) Stanford University Laura Johnston National Institutes of Health (NIH) Myeloid Leukemia, Chronic Acute Myelogenous Stanford University Ginna Laport National Leukemia Myelodysplastic Syndromes Institutes of Health (NIH) (MDS) Lymphoma, Non-Hodgkin University of California, San Francisco Juvenile Diabetes Research Foundation National Type 1 Diabetes Mellitus Institute of Allergy and Infectious Diseases (NIAID) (Neostem) Not yet recruiting Kidney Failure, Kidney Transplant Massachusetts General Hospital Dana-Farber Cancer Institute The University of Regensberg Masonic Cancer Center, University of Minnesota I Recruiting Leukemia Lymphoma Multiple Myeloma Plasma Cell Neoplasm Myelodysplastic Syndromes Masonic Cancer Center, University of Minnesota University of California, San Francisco I Not yet recruiting Late Complication From Kidney Transplant University of California, San Francisco Graft Versus Host Disease Leukemia Lymphoma Multiple Masonic Cancer Center, University of Minnesota I Completed Myeloma and Plasma Cell Neoplasm Myelodysplastic Syndromes Secondary Myelofibrosis Masonic Cancer Center, University of Minnesota H. Lee Moffitt Cancer Center and Research Institute Masonic Cancer Center, University of Minnesota I/II The Russian State Medical University Russian Academy of Medical Sciences Masonic Cancer Center, University of Minnesota I Masonic Cancer Center, University of Minnesota I Stanford University Ginna Laport Doris Duke Charitable Foundation I Not yet recruiting GVHD Withdrawn I/II Recruiting End-Stage Renal Disease Kidney Failure Recruiting Terminated Active, not recruiting H. Lee Moffitt Cancer Center and Research Institute Hematologic Malignancy Acute Myeloid Masonic Cancer Center, University of Minnesota Leukemia Acute Lymphocytic Leukemia The Russian State Medical University Russian Academy of Medical Sciences Acute Myelogenous Leukemia Acute Lymphocytic Leukemia Chronic Myelogenous Leukemia Non-Hodgkin Lymphoma Hodgkin Masonic Cancer Center, University of Minnesota Lymphoma Chronic Lymphocytic Leukemia Multiple Myeloma Myelodysplastic Syndrome Graft Versus Host Disease Leukemia Lymphoma Multiple Masonic Cancer Center, University of Minnesota Myeloma and Plasma Cell Neoplasm Myelodysplastic Syndromes Leukemia Leukemia Chronic Myelogenous Leukemia (CML) Blood and Marrow Transplant Stanford University Ginna Laport Doris Duke (BMT) Myelodysplastic Syndromes Charitable Foundation (MDS) Lymphomas: Non-Hodgkin Neostem I Planned Asthma Neostem Neostem I/II Planned Steroid Resistant Asthma Neostem Neostem II Planned Transplantation Neostem Neostem II Planned Type 1 Diabetes Mellitus Neostem Neostem University of Gdańsk, Poland University of Chicago University of Perugia, Italy Weizmann Institute of Science, Immunology Department, Rehovot, Israel I Ongoing, not recruiting Completed Completed Asthma,Type 1 Diabetes (Juvenile Diabetes), Autoimmune Disorders Type 1 Diabetes Mellitus HLA-haploidentical stem cell transplantation Neostem TxCell, France I/II Completed Refractory CD TxCell, France University of Gdańsk, Poland University of Chicago University of Perugia, Italy Weizmann Institute of Science, Immunology Department, Rehovot, Israel
13 Treg in HSCT Expansion Published data Freshly isolated donor derived CD4 + CD25 + Treg (Di Anni et al, Blood, 2011) In vitro expanded third party UCB derived CD4 + CD25 + Treg Anti-CD3+anti-CD28 Beads + IL-2 (Brunstein et al, Blood, 2011) In vitro expanded family donors derived CD4 + CD25 + CD127 neg Anti-CD3+anti-CD28 Beads + IL-2 (Trzonkowski et al, clinical Immunology, 2014) 13 Phase I to phase II clinical trials referenced Allogeneic Tregs Prevention & treatment Polyclonal vs allo-specific Tolerability demonstrated No increase in infection Fast immune reconstitution Preliminary efficacy data Less high grade GVHD Alleviation of symptoms MLR IL-10 anergized allo-specific donor T cells Type 1 Treg cells induced upon MLR + IL-10 (Bachetta et al, Frontiers in Immunology, 2014) No controlled study yet Issue of Immunosupression Fine tuning of the dose 7
4 Treg in Solid Organ Transplant MLR MLR Planned or ongoing Autologous allo-ag-specific CD4 + CD25 + Treg IL-2+TGFbeta + recipient s DC (NCT01624077) Autologous allo-specific CD4 + CD25 + Treg MLR with CTLA-4 Ig (NCT02091232 ) Polyclonal autologous expanded CD4 + CD25 + CD127 neg Treg (NCT02088931) Polyclonal autologous expanded CD4 + CD25 + CD127 neg Treg (NCT01446484) 4 Phase I to phase II clinical trials referenced in liver (1) and kidney (3) transplantation Autologous Tregs No results published yet Polyclonal vs allogeneic vs Allo-Agspecific approaches Issue of immunosuppressive regimen Dose 10 6 /kg with Ag-specific Tregs Dose 2.10 8 to 10 9 with autologous 8
8 Treg in Autoimmunity & chronic inflammation Published data Expanded Ag-specific Type 1 Treg in Crohn s Disease Ova-specific, in vitro expanded (Desreumaux et al, Gastroenterology 2012) Autologous polyclonal CD4 + CD25 + CD127 neg in T1D Anti-CD3+anti-CD28 Beads + IL-2 (Marek, Diabetes care 2012) Ongoing Autologous polyclonal CD4 + CD25 + Treg in Uveitis Anti-CD3+anti-CD28 mab + IL-2 (La Pitié Salpetrière Hospital, Paris) 8 Phase I to phase II clinical trials referenced Autologous Tregs Polyclonal vs Ag-specific Tolerability demonstrated Preliminary efficacy data No controlled study yet Fine tuning of the dose Dose 10 6 to 10 9 Autologous polyclonal CD4 + CD25 + CD127 neg Treg in T1D Anti-CD3+anti-CD28 Beads + IL-2 (NCT01210664 ) 9
Manufacturing challenges Isolation of a subpopulation GMP sorting (Flow cytometry vs immuno-affinity) Single marker or gene expression profile Purity of the isolated populations (first Treg populations< 60%Foxp3+ cells) Potency assay Issue of multiple putative mechanisms of action Phenotypic evaluation of different inhibitory molecules (cytokines, surface markers, enzymes, cytolytic granules.) In vitro assays (cell contact inhibition, inhibition with soluble factors) In vivo/in vitro blocking experiments Potency can include migration/proliferative activity/cell adhesion T-cell therapy/cell therapy challenges GMP manufacturing, closed system & automation Scale-up for late stage clinical trial Batch-to-batch comparability (identity/potency/purity) Product batches Surrogate markers of Potency Main putative MoAs Extensive characterization Homogeneous expression Clinical experience Responders vs non-responders Monitoring of immune target cell populations + Non-clinical results 10
Safety of Treg cell products Potential toxicity of Treg cells could be related to: Purity - Control of proinflammatory cell content (Th17 ) - Patients with inflamed conditions may have circulating pro-inflammatory cells Tumorigenicity/uncontrolled proliferation - Karyotyping, in vitro persistence, dependence to TCR stimulation & growth factors, clonogenicity assays, telomere shortening capacity - In vivo long-term studies with cell tracking Plasticity/de-differentiation - In vitro & in vivo plasticity assays (Th17, Th1) - Stability of phenotype, potency and epigenetic markers in proinflammatory promoting culture conditions - Tregs from inflammatory patients might be more prone to plasticity Indiscriminate immunosupression - In vivo toxicology studies - Patients clinical monitoring especially in patients with a depressed immune system 11
Other challenges Alteration of ntregs in chronic inflammation & autoimmunity CD25+ FoxP3+ Treg polyclonal deficiency in autoimmune & chronic inflammatory diseases Cause or consequence Treg are normalized in remitting patients Issues of CD25, Foxp3 & Treg markers also expressed by proinflammatory cells Defect in Ag-specific cell number, differentiation pathway and/or molecular signalling Alteration of CD46 mediated IL-10 Treg differentiation in MS patients Alterations in CD46-mediated Tr1 regulatory T cells in patients with multiple sclerosis (Astier AL, J Clin Invest) Alteration of Desmoglein-3 specific Tr1 cells in pemphigus vulgaris Type I regulatory T cells specific for desmoglein-3 are more frequently detected in healthy individuals than in patients with pemphigus vulgaris (Veldman C, J Immunol. 2004) Alteration of CTLA-4 intracellular signalling in Rheumatoid Arthritis patients Defects in CTLA-4 are associated with abnormal regulatory T cell function in rheumatoid arthritis. (Flores-Borja F, PNAS, 2008) Enhanced plasticity in Tregs from Rheumatoid Arthritis patients Regulatory T cells in rheumatoid arthritis showed increased plasticity toward Th17 but retained suppressive function in peripheral blood. (Wang T, Ann Rheum Dis, 2014.) Autologous re-infusion of patients un-manipulated Treg cells might not be an ideal approach 12
Tregs clinical application Understanding the Treg compartment defects in patients can help to define the better strategy Antigen specific versus polyclonal Tregs Natural versus induced Treg cells Gene engineering of Treg cells Regeneration of a defective compartment Pharmacological inhibition of inflammation Induction of tolerance Inhibition of inflammation Persistence or induction of memory Induction tolerance Persistence or induction of memory 13
Ovasave development Ovasave : autologous ovalbumin-specific type 1 regulatory cells 100 90 80 70 60 50 40 30 20 10 0 Percentage of Patient in CDAI response/remission (all doses, n=20) 40 40 15 Week 5 Response Remission 10 Week8 100 75 75 CDAI response = decrease 100, CDAI remission <150 80 60 40 20 0 Percentage of patients in CDAI Response or Remission at 10 6 (n=8) 38 25 Week 5 Week 8 Response Remission Mean (±SEM) CDAI variation at 10 6 (n=8) 0.0-50.0-100.0-150.0-200.0 0 2 4 6 8 10 12 Weeks Analysis of 8 patients receiving 10 6 cells 14
Ovalbumin-specific proliferation (Change from baseline) Proliferation change from baseline Ovasave development Biomarker of clinical response: PBMC proliferative activity in response to Ovalbumin D 10 6 cohort 0,4 0,2 0-0,2-0,4-0,6-0,8 0 5 10 Weeks post-administration E 2 1,5 1 0,5 0-0,5-1 CDAI vs Ova-response coefficient: 0.55 P=0.013-1,5-400 -300-200 -100 0 100! " #$%&' ( ) *+%,-. / %0( 1+23) +% Supports efficacy and mechanism of action of Ova-Treg cells in CD patients Phase IIb to start in H2 2014 in refractory Crohn s Disease patients 15
Conclusion Treg cells represent a novel therapeutic approach for treatment of chronic inflammation, autoimmunity and transplantation. Natural anti-inflammatory activity Multiple MoA and multiple cellular and molecular targets Early development stage Tolerability of the approach demonstrated, encouraging preliminary efficacy observed Need fine tuning of doses and comparative analysis of different sub-populations and strategies 16