Pathways to Protecting from Autoimmunity the Resurrected/Regenerated Beta Cells Jeffrey Bluestone, PhD Diabetes Center at UCSF
Number of Transplants Is islet replacement a viable option? 2000 1800 1600 1400 1200 Total: n = 26,660 Non USA: n = 6,548 USA: n = 20,014 1000 800 600 400 200 0 1/07
UCSF Evolution in Immunosuppression for Pancreas Transplantation 1989-Current (n=462) ERA 1-3 : OKT3 Induction ERA 4 : Thymoglobulin Induction Maintenance Incidence of pancreas rejection ERA 1: OKT3/CSA/AZA/PRED 80% ERA 2: OKT3/CSA/MMF/PRED 50% ERA 3: OKT3/TACROLIMUS/MMF/PRED 15-20% ERA 4: STEROID AVOIDANCE 10-15% Thymoglobulin Induction low dose tacrolimus/ sirolimus/mmf
Why isn t pancreas only more common? Difficult operation Immunosuppression is still intense Difficulty in recognizing rejection
Is islets transplantation feasible? Safer, Simpler Procedure than Pancreas Transplant Better quality of life? Lower requirement of immunosuppression?
Graft Survival (insulin independence) Islet Transplant Registry Edmonton 87% Shapiro and colleagues, NEJM, 2000
Ryan EA Diabetes 54:2060-2069, 2005
The Islet Transplant Experiment: Time for a Reassessment J. S. Bromberg, B. Kaplan, P. F. Halloran and R. P. Robertson Amer J Transplant 2007; 7: 2217 2218 Publication of the initial results of the Edmonton protocol in 2000 (1) raised hopes that many of the technical and immunologic hurdles of islet transplantation had finally been solved and that a new era for the treatment and cure of type 1 diabetes had arrived. Unfortunately, while short term results utilizing this specific protocol were repeated by other groups around the globe, long-term follow-up revealed that islet transplantation with this particular protocol is far less successful than originally hoped (2,3). Thus, although 5 years after transplantation 85% of recipients had measurable plasma C-peptide, wellcontrolled HbA1c levels, significant diminution in amount of daily insulin required, and virtually no clinical hypoglycemia (3), only 10% of patients experienced freedom from exogenous insulin use. While this still may represent partial success in alleviating the debilitating symptoms that brought them to islet transplant in the first place, such a claim needs to ultimately be established in a controlled trial, like other medical advances. Moreover, toxicities from the calcineurin inhibitors combined with sirolimus used for immunosuppression produced worrisome trends in renal function (4). Given continued insulin dependence, the shortage of donor organs, the complications of immunosuppression, and the great expense of this procedure, sober reassessment of the clinical applicability of this protocol and particular experiment is needed.
Why a decay in islet function? Rejection? Autoimmunity? Drug toxicity? No precursor cells? Extensive studies by Shapiro, Nepom, Alejandro and others suggest that autoimmune destruction is a major cause of graft loss.
Costimulation/Adhesion Blockade LEA29Y (Belatacept) Efalizumab (Raptiva) Vincenti, AJT 2002
CTLA4Ig is tolerogenic in mice blocks alloimmune and autoimmunity in some settings Primary Graft Second Graft Anti-ICAM CTLA4Ig CTLA4Ig Anti-ICAM Lenschow et al. Science, 1992
Immunosuppressive Protocols SIROLIMUS (Target trough 8-12 ng/l) (substitute mycophenolate if not tolerated) EFALIZUMAB 1 mg/kg/wk 0.5mg/kg/wk Drug withdrawn in all pts on May, 2009 ATG * Solumedrol Efalizumab -2 0 +2 +4 +7 10 28 90 180 27 0 365 1yr +90 1yr +180 1 yr +270 2 yr 2yrs +90 2 yrs +180 2 yrs +270 3 yrs Txp Days relative to Transplant SIROLIMUS (Target trough 8-12 ng/l) (substitute mycophenolate if not tolerated) ATG Belatacept (10mg/kg/mo) 5mg/kg/mo 5mg/kg/2mos Belatacept * Solumedrol -2 0 +2 +4 +7 10 28 90 180 27 365 1yr 0 +90 Days relative to Transplant Txp 1yr +180 1 yr +270 2 yr 2yrs +90 2 yrs +180 2 yrs +270 3 yrs
Graft Function BELA-1 BELA-3 * Insulin Dependent Insulin Independent Intermittent Use BELA-5 EFA-1 EFA-3 EFA-5 * Tx # 2 (day 445) Tx # 2 (day 442) * * * Tx # 2 (day 400) 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 Time from initial transplant (days) Discontinued Efalizumab *
C - Peptide (ng/ml) C - Peptide (ng/ml) HbA1c (%) HbA1c (%) HbA1c and c-peptide levels after Islet Transplantation Belatacept Efalizumab 10.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 BELA-1 BELA-2 BELA-3 BELA-4 BELA-5 9.0 8.0 7.0 6.0 5.0 EFA-1 EFA-2 EFA-3 EFA-4 EFA-5 3.0 0 30 75 120 180 270 365 455 545 730 Time from Transplant (d) 4.0 0 30 75 120 180 270 365 455 545 730 Time from Transplant (d) 10 9 8 7 6 5 4 3 2 BELA 1 BELA 2 BELA 3 BELA 4 BELA 5 9 8 7 6 5 4 3 2 EFA 1 EFA 2 EFA 3 EFA 4 EFA 5 1 1 0-10.0-5.0 0.0 15.0 30.0 60.0 90.0 120.0 0-10.0-5.0 0.0 15.0 30.0 60.0 90.0 120.0 Time (minutes) Time (minutes)
5-year Graft Survival Rates in Islet and Pancreas Recipients Islet Pancreas 50% % 80 70 60 50 40 30 20 10 0 50%! PAK PTA SPK Px SPK Kd 1988/89 1990/91 1992/93 1994/95 1996/97 1998/99 2000/01 2002/03 2004/05 2006/07
Islet transplantation works but is not a CURE High quality islets can be produced from cadaveric donors Immunosuppressant cocktail prevents rejection >200 patients (>300 procedures) ~90% 1 year 1. There will not be enough cadaveric islets to go around 2. Current immunosuppression regimens require life-long treatment that can result in severe complications including increased infections, cancer and organ toxicity
Renewable Islet Cell Source: Pancreatic Progenitors (PE) ViaCyte has optimized the protocol for making PE from human embryonic stem cells (hesc) In vitro protocol Nkx6.1 PDX1 Insulin Pancreatic Progenitor Kroon et al. Nature Biotechnology 26, 443-452 2008 Endocrine precursor Acini recapitulates natural embryonic development of the human pancreas basal apical lumen lumen Islet Duct Endoderm sheet Pancreas buds from the developing gut tube Branching morphogenesis. Endocrine precursor specification. Adult pancreas Adapted from JM Wells, Diabetes Metab Res Rev, 2003
Controls d246 d365 d30 d44 d71 d94 d30 d44 d71 d94 d30 d44 d71 d94 d30 d44 d71 d94 d30 d44 d71 d94 d30 d44 d71 d94 Serum C-peptide (pm) Function of hesc-pancreatic Epithelium After Implantation into Immunodeficient Mice hesc-islet Graft Implant days : 377 Human Islet Graft Implant days : 360 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 Fasting 30 min. glucose 60 min. glucose 14 12 10 8 6 4 2 0 i1 i2 36 39 40 41 42 44 Adult islets hes cell derived pancreatic endoderm Glucagon Somatostatin Insulin
hes-derived PE cells are immunogenic Day 14 x20 C57BL/6 SCID PDX Ins GCG/SST/Ins
hes PE transplant using co-stimulation blockade Anti-mThymocyte (500 mg/dose) or Anti-CD154 (500 mg/dose) Histology hes Cell Derived PE Stage: 1 2 3 4 plus CTLA4Ig (500 mg/dose) Glucose Stimulation -12-9 -6-3 0 2 4 6 40 60 90 150 Day of Transplant
Combination Immuno-therapy promotes short-term graft survival: Day 3 through Day 14. Post-Transplant: Day 3 Day 7 Day 14 C57BL/6 No Treatment Immunocompetent C57BL/6 hctla+anti-cd40l Immunosuppressed NOD.SCID Immunodeficient H&E 20x
Preserved Human Nuclear staining, PDX, and Insulin through Day 14 Post-Transplant: Day 3 Day 7 Day 14 C57BL/6 No Treatment Immunocompetent C57BL/6 hctla+anti-cd40l Immunosuppressed HuN PDX INS 20X NOD.SCID Immunodeficient
Days Post Transplant- Tx CTLA4Ig+anti-CD40L Day 84 Day 109 Day 141
C-peptide production after alternative forms of immune suppression Human c-peptide (pm) 3 month IVGTT 1000 100 10 1 B6 NOD RAPA CD154+ CTLA4Ig ATG+ SCID CTLA4Ig
Therapies that work in immunocompetent animal are not working in autoimmune mouse models. db NOD (hctla4ig+anti-cd40l) Day 100 NOD.RAG1KO Day 100 db NOD (hctla4ig+atg) Day 131
Membrane System is Critical to the Encaptra Device The membrane is designed to: Exclude host immune cells Retain graft cells Facilitate vascularization immediately adjacent to graft tissue Two components: Outer membrane: Biocompatibility & vascularization Inner membrane: Cell impermeable yet open to transport of nutrients, proteins, etc. Mesh Device Lumen Membrane backing Vascularizing/cell impermeable membrane laminate
Where do we go from here? I L -15 TNF-a I L -6 I L -1b AutoAb Antigen-PBMC A c ti va t ed M ac r ophage I L -23 A T G CTLA4-Ig IFN-g t I L -12 B c ell/ APC CD20 T CR CD3 A n ti - CD3 ( t eplizumab) ++ nai v e T c ell ++ I L -10 ++ ++ T GF-b T Eff/Mem ++ CD2 - - Alefacept/ LFA-3Ig - I L -10 plasmid G - CSF (Neulasta) ++ T r eg I L -2 BH T -3021 (p r oinsulin plasmid) apop t osis GA D -alum ATG/Anti-CD3 ++ I nsulin-pbmc T r eg T he r a p y
Can we use Tregs as Therapeutics?
Blood Glucose (mg/dl) Remission (%) CD62L Adoptive Treg immunotherapy works in NOD mice Teff alone Teff+ extregs IFNg Tregs block pathogenic T cell differentiation Tregs effectively suppress diabetes 60 0 500 400 300 200 100 0 5 NOD Tregs BDC Tregs 30 55 80 105 130 days after cell transfer Ag-specific Tregs are most effective 70 60 50 40 30 20 10 Ag-specific Treg therapy untreated 0 0 5 10 15 Weeks after treatment 20 Ag-specific Tregs reverse diabetes
CD25 Isolated human CD4 + CD25 + CD127 lo T cells can be expanded efficiently from peripheral blood Absolute cell number Fold exp post day 0 10 4 CD4 + T cells 9 1.1 10 4000 10 3 10 2 10 1 10 0 5.83 10 0 10 1 10 2 10 3 10 4 CD127 30 20 10 94 0 10 0 10 1 10 2 10 3 10 4 FOXP3 ntreg: CD4 + CD25 + CD127 -/lo FOXP3 + 7 3.4 10 6 1.0 10 4 3.3 10 0 5 10 15 day post expansion 3000 2000 1000 0 (536) Putnam and Brusko, Diabetes, 2008. 750 fold (340) CD4+CD127-CD25+ v 5 billion Tregs
Expansion of donor-specific Tregs using CD40-activated allogeneic B cells
SUMMARY Islet cell replacement is a viable option Targeted immunosuppression (co-stimulation blockade and ATG induction) can lead to 5 year survival equal to pancreas transplantation Human PE cells derived from hes can develop into glucose responsive islets in mice Current immunotherapies do not block PE graft rejection in diabetic mice New immune depleting protocols coupled with Treg therapy can lead to prolonged islet survival in diabetic mice Current efforts are underway to move Treg therapy into the clinic
Collaborators Greg Szot Jiena Lang Cecilia Austin ViaCyte Team Evert Kroon Kuniko Kadoya Laura Martinson Amy Putnam Weihong Liu Michael Lee Funded by: Qizhi Tang Peter Stock Andy Posselt Tx team National Institute of Allergy & Infectious Diseases Juvenile Diabetes Research Foundation JDRF