1 CURRENT REGIMENS AND FUTURE TRENDS IN TRANSPLANTATION Flavio Vincenti University of California, San Francisco

2 Current Trends in Kidney Transplantation  Greater use of antibody induction –Increasing: Polyclonal (rabbit ATG), alemtuzumab (Campath 1H) –Decreasing: anti CD25 (basiliximab, daclizumab)  Minimization of maintenance drugs to reduce long-term toxicities –Steroid-sparing (withdrawal within 7 days of Tx) –CNI minimization rather than CNI free

3 CNI-Sparing Regimens  Failure of CNI-Free Regimens –Anti-IL2R mAb + MMF + Steroids 1 –Anti-IL2R mAbs + Sirolimus + MMF + Steroids 2  Conversion from CNI to Tor-Inhibitors remains problematic 3 1 Vincenti F, et al. Multicenter trial exploring calcineurin inhibitors avoidance in renal transplantation. Transplantation May 15;71(9): ORION and SYMPHONY Trials. 3 Schena FP, et al. Convert Trial. Transplantation 87:

4 Sirolimus Source: 2005 OPTN/SRTR Annual Report, Table 8.6e Trends in maintenance immunosuppression prior to discharge for simultaneous kidney-pancreas transplantation,

5 Why We Need A New Paradigm in Immunosuppression Relative Risk Decreased Donor Transplants Living Transplants % AR Meier-Kriesche, H-U, et al. Amer J Transplant 2004;4: Incidence of early acute rejection episodes by era Relative risk for overall graft loss by donor type Reducing acute rejection rates has not transplanted into better long-term graft survival.

Causes of Late Kidney Allograft Loss Pascual M et al. N Engl J Med. 2002;346: Causes of late allograft loss (>1 yr after transplantation) Chronic renal allograft dysfunction leading to graft failure in 50% of cases Death of a patient with a functioning graft in 50% of cases Other diagnoses in 10-20% of cases Chronic allograft nephropathy in 30-40% of cases True chronic Rejection (immunologic injury) IF/TA of mixed origin (e.g., non- specific interstitial fibrosis and tubular atrophy) Chronic toxic effects of calcineurin inhibitors New diseases Acute rejection Recurrent diseases Chronic ABMR

7 Wish List for IS  Selective with novel mechanism to block both T and B cells  Non-nephrotoxic  Does not promote malignancy  Does not aggravate CV risk factors  Improves compliance

8 The Graveyard of Transplant Drugs FTY720FTY720 Anti- CD154 R3421R3421 Anti- CD80/86 FK778FK778 Anti- IL15 TGN1412TGN1412 Efalizumab

9 Novel Small Molecules in Clinical Trials  JAK3 Inhibitor –CP690,550  PKC Inhibitor –Sotrastaurin (AEB071)

10 Prevention of Organ Allograft Rejection by a Specific Janus Kinase 3 Inhibitor SCIENCE VOL OCTOBER 2003

11

12 Calcineurin-Inhibitor-Free Immunosuppression based on the JAK Inhibitor CP-550,690: A Pilot Study in De Novo Kidney Allograft Recipients S. Busque, J. Leventhal, D.C. Brennan, S. Steinberg, G. Klintmalm, T. Shah, S. Mulgaonkar, J. Bromberg, F. Vincenti, S. Hariharan, D. Slakey, V.R. Peddi, R.A Fisher, N. Fisher, N. Lawendy, C. Wang, G. Chan American Journal of Transplantation, in press 2009

Day Day Month Month 1.CP690, mg BID (CP15) 2.CP690, mg BID (CP30) 3.Tacrolimus (Tac) Follow-up Extension Study A Treatment Day 1 to Month 6 Surgery Screening Days: -28 to 0 All subjects receive Induction with anti-IL2R mAb (basiliximab or daclizumab) MMF 1-gm BID; up to 1.5-gm BID for African Americans (or mycophenolic acid ) Steroid taper to prednisone 5–10 mg/day by Week 12, continue through Month 6 A Study Schema Presented at: 2007 American Transplant Congress; May 9, 2007; San Francisco, CA.

14 Subjects screened N=73 CP690, mg BID N=20 Subjects enrolled N=61 CP690, mg BID N=20 Tacrolimus N=21 Discontinued N=7 Subjects completed N=17 Subjects completed N=19 Subjects completed N=13 Discontinued N=3 Discontinued N=2 Subject Disposition Presented at: 2007 American Transplant Congress; May 9, 2007; San Francisco, CA.

15 Percent of Subjects With First BPAR CP15 (n=20) CP30 (n=20) Tacrolimus (n=20) Month 6 N % with BPAR P value (vs tacrolimus) BK nephropathy 040 Cumulative risk of CMV disease (%) 10%21%0% Percent of Patients With First BPAR (Kaplan-Meier Estimates) Presented at: 2007 American Transplant Congress; May 9, 2007; San Francisco, CA. BPAR=biopsy-proven acute rejection; CMV=cytomegalovirus

16 Current CP690,550 Phase IIB Trial Dose-finding study in de novo renal transplant recipients Lower doses of CP690,550 (15 mg initially and 10 mg BID subsequentially) tested vs cyclosporine – All patients treated with basiliximab and MMF + steroids Enrollment completed MMF=mycophenolate mofetil

Sotrastaurin (AEB071) Selective Inhibitor of Protein Kinase C

18 In contrast to calcineurin inhibitors (CNIs), sotrastaurin blocks T-cell activation via a calcineurin-independent pathway; therefore, lacks the side effects of CNI – Results in inhibition of IL-2 promoter gene activity This indicates a similar inhibitory effect on early T- cell activation as compared to CNIs; therefore, a potential to be a CNI replacement in vitro Pharmacology of Sotrastaurin: Mechanism of Action Evenou J-P. Am J Transplant. 2006;6(suppl 2):1029. Vincenti F, Kirk AD. Am J Transplant. 2008;8: Sotrastaurin

19 Placebo 25 mg AEB mg AEB mg AEB mg AEB071 p= p= Baseline Day 7 Day 14 Day 21 Day 28 PASI change from baseline (%) Sotrastaurin in Psoriasis

20Sotrastaurin 3 phase-II trials in renal transplantation – US–European Trial: CNI withdrawal at 3 months  on hold. During the first 3 months, tacrolimus + sotrastaurin was comparable to tacrolimus + MPS – US Trial: CNI-free (sotrastaurin + MPS)  on hold – European Trial: CNI-free (sotrastaurin + everolimus) still ongoing Sotrastaurin will likely be tested with a CNI regimen Vincenti F, Kirk AD. Am J Transplant. 2008;8: CNI=calcineurin inhibitor; MPS=mycophenolic sodium

Biologic Agents in Clinical Trials Belatacept: costimulation blockade Alefacept: anti-CD2

22 T-Cells Require Costimulation for Full Activation CD80/86-CD28 is the most important costimulatory pathway* Cytokine production T-cell proliferation Signal 1 Antigen triggers T- cell receptor Signal 2 Costimulation between ligands *Other costimulatory pathways exist that also serve this role APC =antigen-presenting cell

23 T-Cells Require Costimulation for Full Activation TCR signal only=no activation No cytokine production No cell division Becomes anergic Undergoes apoptosis Signal 1 only No Signal 2 APC =antigen-presenting cell; TCR=T-cell receptor; MHC=major histocompatibility complex

CTLA4 Negatively Regulates T-cell Activation CTLA4 binds CD80/86 with greater avidity than CD28 CTLA4 binds CD80/86 with greater avidity than CD28 CTLA4 negatively regulates T-cell activation CTLA4 negatively regulates T-cell activation CTLA4 (CD152) expression is induced by T-cell activation CTLA4 (CD152) expression is induced by T-cell activation CTLA4 is structurally similar to CD28 CTLA4 is structurally similar to CD28

25 Early preclinical transplant studies utilized CTLA4Ig to block B7-CD28 co-stimulation

26 Belatacept Potently and Selectively Blocks T-Cell Activation No cell division No cytokine production Anergy Apoptosis Selective costimulation blocker Belatacept (LEA29Y)

27

28 All patients received basiliximab, MMF, and corticosteroid-tapering regimen Clinical endpoint (belatacept arms unblinded) Clinical endpoint Randomization 1 month 3 months6 months1 year 150–400 ng/mL150–300 ng/mL CsA (7 ± 3 mg/kg daily)* Belatacept mg/kg every 4 or 8 weeks DAY 1 5 "Less Intensive=LI" "More Intensive=MI" 10 mg/kg Vincenti F, et al. N Engl J Med. 2005;353(8): Study Design

29 Phase II: First-Year Study Results Low rate of acute rejection, comparable across arms Comparable patient and graft survival Statistically-significant preservation of measured glomerular filtration rate Lower rates of chronic allograft nephropathy in both belatacept arms vs cyclosporine Favorable trends in cardiovascular and metabolic parameters 3 posttransplantation lymphoproliferative disorder cases in belatacept MI regimen Belatacept MI (n=74) Belatacept LI (n=71) Cyclosporine (n=73) Acute rejection at 6 months, n (%) 5 (6.8)4 (5.6)6 (8.2) Vincenti F, et al. N Engl J Med. 2005;353(8):

30 Deceased donor-4 week Deceased donor-8 week Living donor-4 week Living donor-8 week X X X = withdrawn from study = patient died Numbers represent serum creatinine mg/dL Patients Enrolled in the Extension Trial of Maintenance Belatacept Therapy at UCSF (as of October 2008)

31 Clinical Trials With Belatacept Results to be reported at ATC meeting May Phase-III trials (BENEFIT & BENEFI-EXT) have completed enrollment Conversion trial from calcineurin inhibitor to belatacept Rapid steroid withdrawal trial – 3 arms (steroid administered for 5 days) Antithymocite globulin (rabbit) + belatacept + MMF Antithymocite globulin (rabbit) + belatacept + sirolimus Antithymocite globulin (rabbit) + tacrolimus + MMF MMF=mycophenolate mofetil

32 Percentage FOXP3/CD3 Ratio of FOXP3 + to CD3 + cells in kidney biopsies in patients with rejection

33 Alefacept Alefacept is a dimeric fusion receptor protein LFA3 liked to the Fc portion of human IgG1 Available in an IV and IM formulation Approved for use for psoriasis

Alefacept: The Molecule Human LFA-3-IgG1 fusion protein LFA-3 portion binds to CD2 on T lymphocytes, blocking the interaction between LFA-3 and CD2, and interfering with T-cell activation Vincenti F, Kirk AD. Am J Transplant. 2008;8:

35 Ellis CN, et al. N Engl J Med. 2001;345:248. Mechanism of Action of Alefacept

Alefacept: Phase 2 Active Comparator Study  A phase 2, randomized, open-label, parallel group (4 arm; all active), multi-center study to assess the safety and efficacy of alefacept in de novo kidney transplant recipients  320 kidney patients Arm 1: Active comparator–tacrolimus, basiliximab, MMF, steroids Arm 2: Experimental–alefacept, tacrolimus, MMF, steroids Arm 3: Experimental–alefacept, tacrolimus, steroids Arm 4: Experimental–alefacept, tacrolimus, MMF, steroids  Expected completion in October 2009 ClinicalTrials.gov identifier: NCT

Targeting Humoral Responses Are Still An Unmet Need Current approaches with plasmapheresis, IVIg and Rituximab are not adequate

Methods Between September 2005 and May 2007, a total of 20 highly sensitized patients (with a [±SD] T-cell panel-reactive antibody level, determined by use of the complement-dependent cytotoxicity assay, of 77±19% or with donor-specific antibodies) were enrolled and received treatment with intravenous immune globulin and rituximab.

Study Design The study used an open-label design to examine whether human polyclonal intravenous immune globulin (10% formulation) given: twice (2 g per kilogram of body weight on day 0 and day 30), plus rituximab given twice (1 g on day 7 and day 22), could reduce the rate of, or eliminate, a positive cross-match in highly HLA-sensitized patients awaiting transplantation at Cedars-Sinai Medical Center.

Results At study entry, the mean time on dialysis among recipients of a transplant from a deceased donor was 144±89 months (range 60 to 324). However, the time to transplantaton after desensitization was 5±6 months (range, 2 to 18). Sixteen of the 20 patients (80%) received a transplant.

Acute Rejection Episodes Acute rejection episodes occurred in 50% of patients who received a transplant, and 31% of these episodes were C4d+ antibody-mediated rejections. Most rejection episodes occurred within the first month after transplantation and were reversible with treatment.

45 Vincenti F, Kirk AD. Am J Transplant. 2008;8: T Cell

Emerging Therapies for Humoral Rejection Eculizumab – Humanized anti-C5 mAb Proteasome Inhibitors – Bortezomib 46

47 The Proteasome

Novel Therapies for Desensitization and Humoral Rejection The Proteasome Inhibitors – 1 st generation: Brotezomib – 2 nd generation: Carfilzomib 48

Conclusion An ideal immunosuppression regimen does not yet exist Many agents, small molecules and biologics that target novel pathways are in clinical trials More effective therapies are emerging against B cells/plasma cells and anti- HLA antibodies 53