Animal Models for Evaluation of Porcine Islet Products to Treat Patients with Type 1 Diabetes Bernhard J. Hering Unive

1. Animal Models for Evaluation of Porcine Islet Products to Treat Patients with Type 1 Diabetes Bernhard J. Hering University of Minnesota bhering@umn.edu FDA-CBER CTGT Advisory Committee Meeting #47 Topic II: Animal Models for Porcine Xenotransplantation Products Intended to Treat Type 1 Diabetes or Acute Liver Failure Hilton Hotel, Gaithersburg, MD; May 14, 2009

2. Outline • Rationale for developing porcine islet products for T1D • Rationale for preclinical studies in the pig-to-nonhuman primate (NHP) model • Design of informative studies in the pig-to-NHP model • Limitations of the pig-to-NHP islet xenotransplant model • Experience with porcine islet product evaluation in NHPs

3. 1. Rationale for Developing Porcine Islet Products for T1D

4. Type 1 Diabetes - Unmet Clinical Needs • Chronic complications:Microvascular and macrovascular lesionsEstimated prevalence in the US: 500.000 • Acute complications:Hypoglycemia unawareness and defective glucose counterregulationEstimated prevalence in the US: 75.000

5. No declining incidence of CAD, overt nephropathy, and proliferative retinopathy in type 1 diabetes (cumulative incidence over 30 yrs: 30 to 50%) Diabetes 2006; 55: 1463-9

6. Iatrogenic hypoglycemia causes recurrent physical and psychosocial morbidity (incl. coma, seizures, and significant social embarrassment) • The incidence of (acutely life-threatening) severe hypoglycemia in patients with T1D for >15 yrs is 320 episodes in 100 patient-years (UK Hypoglycemia Study Group 2007) • 12.5% of patients with 21-30 yr ho T1D are unaware of hypoglycemia (18% w/ T1D >30yrs) (Pedersen-Bjergaard et al., 2004) • 6% to 10% of deaths of people with T1D are the result of hypoglycemia • This problem has not abated over the 15 years since it was highlighted by the report of the DCCT in 1993 • Elimination of iatrogenic hypoglycemia will require new treatment methods that provide plasma glucose-regulated insulin replacement or secretion Diabetes 2008; 57: 3169-76

7. Decrease in glycated hemoglobin levels in subjects 25 yrs of age or older; importantly not associated with an increase in hypoglycemia • Rates of severe hypoglycemia were not different between groups • No improvements in glycated hemoglobin in subjects 5 to 24 yrs of age

8. Human Islet Products to Treat T1D Restore (near-)normoglycemia and protect from severe hypoglycemia Restore insulin independence Preliminary data suggest that: Long-term (>5 yrs) insulin independence is possible (in >60% of recipients who received depleting T cell antibodies for induction and who were insulin independent at 1 yr posttransplant - University of Minnesota, unpublished) Islet transplants slow progression of microvascular complications (see below - Warnock et al., Transplantation 2008)

9. Islet transplantation profoundly reduces the percent of recipients with one or more episodes of severe hypoglycemia Islet alone recipients with detectable C-peptide 2008 Annual Report

10. Improved HbA1c • Reduced decline in GFR • Stopped progression of retinopathy Islet Tx vs Intensive Medical Therapy

11. Rationale for developing porcine islet treatment products • Unlimited and on-demand availability of islet tissue for transplant • Quality of pig islet products prepared from suitable donor pigs is consistent and not compromised by comorbidity, senescence, brain death, and cold ischemia injury • Actual (not theoretical) risks of infectious disease transmission are lower than in human islet allotransplantation if islets are prepared from designated pathogen-free donor pigs • Porcine islet xenografts may be resistant to destruction by recurrent autoimmunity • On-demand availability of islets from known source pigs permits recipient pretreatment with donor antigen with the goal to induce donor-specific immunologic hyporesponsiveness • Genetic modification of source pigs permits stable overexpression of genes encoding cytoprotective and immunomodulatory peptides in donor islets, thereby presenting opportunities for minimizing recipient immunosuppression not available to recipients of human islet allografts Conceivably more favorable risk-benefit ratio of islet xenografts vs allografts

12. 2. Rationale for preclinical studies in the pig-to-NHP model

13. Numerous animal models • Donor • Fish • Chicken • Mouse • Rat • Hamster • Rabbit • Dog • Pig • Cow • Monkey • Baboon • Human • Recipient • Immuno-incompetent rodents (e.g., athymic mice and rats, SCID mice) • Non-diabetic and STZ-diabetic rodents • Spontaneously diabetic rodents (NOD mice, BB rats) • Spontaneously diabetic large animals (dogs and monkeys) • STZ-diabetic and pancreatectomized dogs and pigs • ‘Humanized mice’ (e.g.; NOD/SCID reconstituted w/ human fetal thymus/liver and CD34+ cells or NOD-Rag1null IL2rnull mice reconstituted w/ umbilical cord blood) • STZ-diabetic and pancreatectomized NHPs

14. Animal Models: Tiered Approach • The potency of new and optimized pig islet products is typically first studied in immuno-incompetent mice. • Technical and safety questions related to porcine islet implantation and engraftment can initially be assessed in mice and pigs. • Insights gained into the mechanisms of immune function and novel immunotherapeutic strategies in mice form the foundation for subsequent studies in preclinical models; however, small animals do not allow to study the complexity of the immune response that is known to be present in human recipients. • Therefore, while animal models other than the preclinical pig-to-NHP model provide useful and supportive data, alone they are unlikely to adequately assess the safety and clinical activity of porcine islet products intended to be studied in T1D recipients.

15. Rationale for evaluating xenogeneic islet products in the pig-to-NHP model (1 of 2) • A favorable benefit-over-harm determination is a fundamental prerequisite for initiating clinical research. Thus, clinical research is only permissible if the probable benefits of the research for both individual subjects and society outweigh the possible risks. • Subjects participating in clinical research on pig islet cell therapy products are at risk of experiencing adverse events related to the xenograft product, transplant procedure, and immunosuppressive therapy. Because xenografts may additionally expose patients, and possibly society at large, to unknown potential infectious risks, (and because recipients must commit to lifelong monitoring), one of the guiding principles of the Ethics Committee of the International Xenotransplantation Association and the Changsha Communiqué recently released by the WHO is that there should be a relatively high expectation of benefit, based on sound preclinical data, before such risks can be considered acceptable in clinical trials. The IXA and WHO Perspective

16. Rationale for evaluating xenogeneic islet products in the pig-to-NHP model (2 of 2) • For the benefit-over-harm determination to be favorable in view of known and unknown risks, the anticipated benefits must be substantial such as the restoration and maintenance of normoglycemia and insulin independence after islet xenotransplantation. • A high expectation of these benefits calls for their previous documentation in a preclinical study that mimics the intended clinical scenario. • Old World monkeys (rhesus and cynomolgus macaques) and baboons are the only suitable recipient animals in which • many immunotherapeutics designed for human trials cross-react • the efficacy of immunosuppression and encapsulation strategies can be assessed with rigor in the presence of a complex and redundant immune system replete with outbred diversity, heterologous immunity, and MHC expression patterns known (or at least expected) to present barriers to long-term islet xenograft graft survival in humans.

17. 3. Design of informative studies in the pig-to-NHP model

18. Design of informative studies in the pig-to-NHP model (1 of 7) • The design of the preclinical study must mimic the intended clinical investigation to the greatest extent possible and must be performed under a high regulatory standard. • Diabetic Old World monkeys or baboons should be used as recipients. • Successful diabetes induction via streptozotocin or total pancreatectomy should be confirmed by 2 or more blood glucose levels being >350 mg/dl, absence of a C-peptide response to intravenous glucose or arginine, and lack of beta cell regeneration in the native pancreas on autopsy. • Manufacturing, release criteria, and dose and route of administration of the investigational xenoislet product tested in the preclinical study and in the intended clinical trial should be similar if not identical. • All other protocol-regulated treatment products, in particular immunomodulatory agents, should be administered at dose levels and schedules that mimic the intended clinical scenario.

19. Design of informative studies in the pig-to-NHP model (2 of 7) • Safety assessments should include regular clinical observations, physical examinations, body weights, food consumption, hematology, chemistry, and urine analysis. • A complete necropsy should be conducted on each animal including detailed microscopic examination of the islet xenograft, the host organ, and the pancreas (if present). • Studies examining the xenogeneic pathogen-related infection (particularly PERV), immune responses to the xenograft and porcine insulin, and potential associated toxicities should also be included in the study design.

20. Design of informative studies in the pig-to-NHP model (3 of 7) • Control cohorts should be incorporated into the design of the preclinical study to delineate toxicities due to immunomodulatory drugs and immunoisolation capsules from toxicities due to the xenograft product. • These cohorts should not receive pig islet cell products and include • unmodified animals as well as • non-diabetic control animals given immunomodulatory agent(s) and/or immunoisolation capsules

21. Design of informative studies in the pig-to-NHP model (4 of 7) • Posttransplant metabolic assessments should include • frequent measurements of fasting and nonfasting blood or capillary glucose levels • monthly HbA1c or glycosylated hemoglobin levels • periodic metabolic tests to determine glucose, porcine, and nonhuman primate C-peptide responses to intravenous glucose, intravenous arginine, or mixed meals

22. Design of informative studies in the pig-to-NHP model (5 of 7) • A sufficient duration of follow-up, within the limitations of the model, is necessary to evaluate adverse side effects related to the investigational xenograft product and accompanying immunomodulatory agents, the reversibility of any abnormal findings as well as the durability of all activity/efficacy endpoints. • The limitations of the model that compromise the ability to monitor monkeys receiving maintenance immunosuppression long-term are discussed below. …

23. Design of informative studies in the pig-to-NHP model (6 of 7) • The evaluation of the safety and efficacy of re-transplants should be incorporated into the study design if the clinical development program includes the potential for more than one islet xenotransplant. • At least three scenarios may be considered: • Partial function at 1 month posttransplant (islet products from adult donors only) • Decline in function over time requiring resumption of exogenous insulin therapy • Rejection (absence of porcine C-peptide after stimulation)

24. Design of informative studies in the pig-to-NHP model (7 of 7) • Primary study endpoints • Efficacy: Proportion of recipients maintaining fasting blood glucose levels <150 mg/dl and non-fasting levels of <200 mg/dl at 6 months in the absence of exogenous insulin or in the presence of greatly reduced insulin requirements (compared to pre-tx). It is suggested that this endpoint be met in ≥5 of 8 consecutive NHPs; follow-up should be for a period of ≥6 months after the initial transplant in all cases and ideally for 12 months in at least 1 or 2 successful cases. (Please note that the blood glucose levels after islet transplantation are determined by the donor islets and that blood glucose levels in healthy source pigs are considerably higher than in monkeys. Criteria for protocols not requiring immunosuppression may be less stringent. ) • Safety: Proportion of recipients experiencing opportunistic infections, malignancies, organ failure, or other limiting toxicities. Adopted from David Cooper et al.: Preclinical Efficacy and Complications Data Required to Justify a Clinical Trial. IXA Consensus Statement on Requirements for Initiating Clinical Trials of Pig Islet Products. Xenotransplantation (in preparation)

25. 4. Limitations of the pig-to-NHP islet xenotransplant model

26. Metabolic limitations of the NHP model • Studies in NHPs do not allow to model effects of pig islet products on hypoglycemia unawareness and defective hormonal counterregulation (likely the main indication for islet xeno-tx) • The pig islet xenograft function reported in NHPs underestimates the potency of pig islet products that can be anticipated in humans • The metabolic demand of NHPs - as assessed by C-peptide levels necessary to normalize blood glucose - is much higher than that of pigs, and more importantly, higher than that of humans • The stimulated C-peptide levels in cynomolgus monkeys are 10 x higher than levels in pigs; in humans these levels are 2 times higher than in pigs- Casu A et al., Diabetologia 2008 • Chronic ‘mild hyperglycemia’ in conjunction with relative insulin deficiency in pig islet xenograft recipients may contribute to compromised health and welfare and “failure to thrive”

27. Immunologic limitations of the NHP model • Studies in NHPs do not allow to examine the efficacy of the immunotherapeutic strategy in preventing autoimmune destruction of pig islet products • Maintaining immunosuppressed NHPs in a healthy state long-term (more than 3 to 6 months) is a major challenge; administering immunosuppressive drugs on a daily basis affects the well-being, growth, and protective immunity of animals • Differences in the absorption and pharmacokinetics of immunosuppressants between NHPs and humans make NHPs more susceptible to drug-related toxicity • E.g., the sirolimus dose required to achieve comparable trough levels in macaques is 10 fold higher than in humans; thereby exposing the GI mucosa to much higher local concentrations than seen in humans and likely contributing to the chronic diarrhea and malabsorption invariably seen in NHPs treated for more than 6 months with sirolimus • Mouth ulcers in humans are believed to be related to local exposure rather than high blood trough levels)

28. Substantial weight gain in juvenile cynomolgus monkeys after discontinuation of immunosuppression

29. Only anecdotal experience with immunosuppression in NHPs for >90 to 180 days

30. 5. Experience with porcine islet product evaluation in NHPs

32. Preclinical studies in the pig-to-NHP islet xenotransplant model • At 15 institutions in 181 NHPs, including xenotransplants in 72 non-diabetic and 109 diabetic recipients. • Prolonged restoration of insulin independence after pig islet cell xenotransplantation for >3 months in NHPs with spontaneous, streptozotocin- (chemically-) and pancreatectomy-(surgically-) induced diabetes has independently been reported by 5 groups, with functional islet xenograft survival exceeding 6 months in several recipients. • These findings indicate the feasibility of preclinical evaluation of pig islet cell xenotransplant products in the rigorous NHP model.

33. Minnesota: BSM + -CD154 + RAD + FTY720 + LFM Emory/AB: BSM + -CD154 + Rapa + LEA29Y

34. Insulin Pig-to-NHP islet xenotransplantation

35. Porcine and Human/NHP C-Peptide Levels

36. Intravenous Glucose Tolerance Tests

37. Engraftment of neonatal porcine islets in diabetic non-human primates by blockade of the CD28/40 costimulatory pathways Russell M, Cardona K, Oliva V, Korbutt G, Cano J, Jiang W, Strobert E, Rajotte R, Pearson T, Larsen C Emory University and University of Alberta Long-term reversal of diabetes after intraportal transplantation of 25,000 to 50,000 neonatal porcine IE per kg in pancreatectomized rhesus monkeys immunosuppressed with the anti-CD25 mAb basiliximab (induction only) + antagonistic anti-CD40 mAb (induction only) + CD28 antagonist LEA29Y + rapamycin #305.8 1st Joint Conference of CTS, IPITA, and IXA – Mpls, MN, 9/15-20, 2007

38. Genetic Engineering of pigs for improved transplant outcomes Normoglycemia for >98 and >115 days after intraportal transplantation of adult islets isolated from CD46 transgenic pigs in cynomolgus monkeys treated with ATG, anti-CD154 mAb, and MMF David L. Ayares et al., David K. C. Cooper, et al. Revivicor Inc., and University of Pittsburgh #404.2 1st Joint Conference of CTS, IPITA, and IXA – Mpls, MN, 9/15-20, 2007

39. 2009

40. Encapsulation of adult pig islets in monolayer cellular device to correct STZ diabetes in primates w/o immunosuppression Pierre Gianello & Denis Dufrane Univ. of Louvain, Brussels, Belgium Correction of diabetes for 6 months #411.3 1st Joint Conference of CTS, IPITA, and IXA – Mpls, MN, 9/15-20, 2007 Bull Mem Acad R Med Belg 2007; 162: 439-449

41. Conclusions • Reports from independent groups demonstrate the feasibility of preclinical evaluation of porcine islet products in the rigorous NHP model (with the limitations noted). • Old world monkeys and baboons are considered the most suitable recipient animals; mainly because immunosuppression and encapsulation strategies can be assessed with rigor. • Yet, the pig-to-NHP model does not allow to model all important metabolic and immunological manifestations of Type 1 diabetes. • Pig islet products are more likely to restore normoglycemia in humans than in monkeys. • Only anecdotal experience exists with immunosuppression in monkeys for >180 days; infections, diarrhea, malabsorption, and weight loss are frequently encountered in animals immunosuppressed for longer periods. • Failure to show robust porcine islet function beyond day 180 may merely suggest failure of the model to accommodate the protocol.

42. Acknowledgements Melanie L. Graham Henk-Jan Schuurman JDRF PPG #21-2006-881 NIH U19 AI067151 Schulze Diabetes Institute