1. Disclosure Information: Mark D. Stegall, M.D. I have the following financial relationship to disclose Research/Grant support from: Alexion and Millenium Pharmaceuticals I will discuss the following off label use and/or investigational use in my presentation: Eculizumab and Velcade

3. Acknowledgments James Gloor Suresh Raghavaiah Tayyab Diwan Cindy Groettum Jennie Wilson Justin Burns Dana Perry Walter Park Lynn Cornell James Gloor Suresh Raghavaiah Tayyab Diwan Cindy Groettum Jennie Wilson Justin Burns Dana Perry Walter Park Lynn Cornell Patrick Dean Steve DeGoey Manish Gandhi Jeff Winters Lynette Fix Kay Kosberg Surgeons, Nephrologists, Fellows and other staff

4. Actual Death-Censored 5 Year Graft Survival 70.7% vs 88.0%, p= 0.0006

5. Actual 5 Year Graft Survival T cell AHG

6. Actual 5 Year Outcomes After +XMKTx Rate of graft loss after 1 year Class I only 1.6%/yr Class II 7.0%/yr

7. What this talk is not about

8. Hyperacute Rejection Rare Very rare with a negative T cell AHG crossmatch Thus, PE or IVIG to achieve a –Tcell AHG pretransplant Very rare with anti-Class II alone

9. Combined Cellular and Antibody Mediated Rejection Most cases of AMR seen clinically in non-sensitized patients are a combination of cellular and antibody mediated rejection ?Role of non-compliance DSA levels may be transient Time of occurrence may be months to years after transplantation Most cases of AMR seen clinically in non-sensitized patients are a combination of cellular and antibody mediated rejection ?Role of non-compliance DSA levels may be transient Time of occurrence may be months to years after transplantation

11. Bortezomib and AHR 6 patients with combined cellular and humoral rejection (C4d+ and DSA by solid phase) Time of rejection episodes: POD 239, 95, 1766, 180, 2825,147 All resolved 3 of 6 had transplant glomerulopathy on follow-up

12. Two Talks Thursday Early Antibody Mediated Injury Acute Antibody Mediated (Humoral) Rejection Friday Late Antibody Mediated Injury Chronic AMR Thursday Early Antibody Mediated Injury Acute Antibody Mediated (Humoral) Rejection Friday Late Antibody Mediated Injury Chronic AMR Based on Experience with Sensitized Patients

13. Differences Between Early and Late Antibody Mediated Injury Early First 6 weeks after transplant, rarely later Incidence determined by DSA levels C4d+ Microthrombi, some cells Incidence higher with anti-Class I DSA Late Rare early, increases over years DSA levels usually low, C4d- common A cellular infiltrate termed “peritubular” capillaritis Incidence higher and outcome worse with anti-Class II DSA Early First 6 weeks after transplant, rarely later Incidence determined by DSA levels C4d+ Microthrombi, some cells Incidence higher with anti-Class I DSA Late Rare early, increases over years DSA levels usually low, C4d- common A cellular infiltrate termed “peritubular” capillaritis Incidence higher and outcome worse with anti-Class II DSA

14. Differences Between Early and Late Antibody Mediated Injury Early First 6 weeks after transplant, rarely later Incidence determined by DSA levels C4d+ Microthrombi, some cells Incidence higher with anti-Class I DSA Late Rare early, increases over years DSA levels usually low, C4d- common A cellular infiltrate termed “peritubular” capillaritis Incidence higher and outcome worse with anti-Class II DSA Early First 6 weeks after transplant, rarely later Incidence determined by DSA levels C4d+ Microthrombi, some cells Incidence higher with anti-Class I DSA Late Rare early, increases over years DSA levels usually low, C4d- common A cellular infiltrate termed “peritubular” capillaritis Incidence higher and outcome worse with anti-Class II DSA

15. Desensitization??? There is no evidence that any current therapy “desensitizes” patients— i.e. blocks alloantibody production permanently There is no evidence that any current therapy “desensitizes” patients— i.e. blocks alloantibody production permanently

16. Desensitization??? Alternate Explanations Remove DSA by plasma exchange or block with IVIG—prevents hyperacute rejection Kidney absorbs the antibody after transplant—DSA reduced Memory response may or may not occur— early AMR Chronic injury is common, but not detected Alternate Explanations Remove DSA by plasma exchange or block with IVIG—prevents hyperacute rejection Kidney absorbs the antibody after transplant—DSA reduced Memory response may or may not occur— early AMR Chronic injury is common, but not detected

17. 1 o Sensitization Naïve B Cell Plasmablast T-helper cell APC Low-affinity Abs Ү ү Activated B Cell Germinal Center Reaction Ag cytokines Proliferation Hypermutation Affinity maturation Memory Compartment High-affinity Abs Ү ү Memory B Cell High-affinity Abs Ү ү Secondary Stimulation by Ag or CpG oligos,Bystander T cells ? CD20+, CD27- CD138-, CD38- CD20-, CD27- CD138-, CD38+ CD20-, CD27+- CD138-, CD38- CD20-, CD27- CD138+, CD38+ Pathways to Antibody Production Long-lived PC ?longevity ү Ag Stegall et al Am J Transplant 2009; 9:998-1005.

19. Plasma cell Only “B” Cells actually secreting antibody Short and long-lived Pre-existing PCs source of DSA in sensitized patients Newly-emerging PCs post-transplant (converted from naïve and memory B cells)

20. 2. Positive Selection of CD 138+ cells 6 x 10 8 Mononuclear cells 6 x 10 6 CD 138 + cells 1. Bone Marrow Aspiration in Sensitized Renal Allograft Candidate ASCs 60 ml Bone Marrow

21. PC ELISpot - Frequency of Tetanus and Allospecific PCs Non-Sensitized Tetanus Alloantibody Sensitized Non-Sensitized Perry et al Am J Transplant 2008; 8:133-143

23. Methods Patients Very high alloantibody levels DSA that was deemed too high (BFXM > 450 or MFI>10,000) for our current desensitization protocols. Monotherapy Endpoint: Reduction in DSA-specific PC number Reduction on DSA/response to Plasma exchange Transplantation 2011; 91:536-541

24. Study Design. Bone marrow Bortezomib Bone marrow Bortezomib therapy (1.3 mg / m2). a) Phase 1 – 4 doses (n=4) b) Phase 2 – 16 doses (n=5) Transplantation 2011; 91:536-541

25. Results - ELISPOT

26. Effect of Bortezomib on PC *One patient’s baseline marrow clotted and hence was excluded from analysis. Category (per ml marrow) Baseline (Mean + SD) Post Bortezomib (Mean + SD) (n=8) p value Paired T test Allo spots (X 10 2 ) 16.7 + 14.56.2 + 3.6 0.048 TT spots (X 10 2 ) 25.2 + 15.713.2 + 8.1 0.032 Total Cells (X 10 6 ) 14.3 + 5.111.6 + 3.90.27 PC no (X 10 3 ) 21.5 + 8.615.5 + 12.10.21 Transplantation 2011; 91:536-541

27. Effect Of PE. Bortezomib + PE PE only

28. Effect of PE Category Bortezomib + PE (n=5) PE only (n=8) p value No of PE (Mean + SD) 11.4 + 2.711.6 + 3.9 0.9 Baseline – Post PE BFXM (Mean + SD) 272.6 + 92.195.4 + 72.2 0.008 % Change in BFXM CS (Mean + SD) 49.1 + 14.917.7 + 12.5 0.005 BFXM < 300 Post PE n (%) 3 (60)0 (0) 0.035 Transplantation 2011; 91:536-541

29. Conclusions Proteasome inhibition depletes normal human plasma cells in vivo. Depletion is significant, but not complete -- ? increases safety and tolerability, but may limit effectiveness New PCs replace old ones quickly

30. Conclusions: Clinical Improves efficacy of PE and increases the transplant rate in patients with extremely high levels of DSA Problems with bioavailability and irreversible binding May need more prolonged treatment or combine with other agents to increase efficacy Newer agents in development Transplantation 2011; 91:536-541

31. Bortezomib Weak impact with only 1-4 “cycles” Extend to 8 cycles now Weak impact with only 1-4 “cycles” Extend to 8 cycles now

32. Prevention of Early Antibody Mediated Rejection

33. Early Acute Antibody Mediated Rejection Common in +XMKTx (20-40%) Best correlated with DSA post- transplantation (BFXM >360, MFI >8000) ~ 90% incidence Baseline DSA also somewhat predictive

34. Associated with high DSA levels post-transplant Early AMR: May cause early graft loss Associated with shortened graft survival Expensive and increases morbidity

35. Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Low baseline DSA levels (B-FXM) without AHR High baseline DSA levels (B-FXM) without AHR Low baseline DSA levels (B-FXM) with AHR High baseline DSA levels (B-FXM) with AHR Burns et al Am J Transplant 2008; 8:2684-2694

36. Early AMR (Burns et al) Time to rejection 11.3+5.9 d (range 4- 21) All except 1 C4d+ peritubular capillaries at time of diagnosis 6 C4d+/-AHR  3 progressed to AHR Time to rejection 11.3+5.9 d (range 4- 21) All except 1 C4d+ peritubular capillaries at time of diagnosis 6 C4d+/-AHR  3 progressed to AHR

37. MCR: Actual 5 Year Outcomes after +XMKTx All +XMKTx 2000-2006 (n= 154) Retrospective LABscreen DSA (beads) 52 excluded 8 lost to f/u 11 no serum for DSA testing 12 CDC+ pre  failed desensitization 16 no DSA (+XM) 5 MFI <1000 102 included in the study & compared to 204 –XMKTx matched by age/gender All +XMKTx 2000-2006 (n= 154) Retrospective LABscreen DSA (beads) 52 excluded 8 lost to f/u 11 no serum for DSA testing 12 CDC+ pre  failed desensitization 16 no DSA (+XM) 5 MFI <1000 102 included in the study & compared to 204 –XMKTx matched by age/gender 100% 5 yr graft survival

38. DSA Specificity (MFI >1000) SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II 2045.7% 8849 7711 Class II alone460% 0 8922 SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II 2045.7% 8849 7711 Class II alone460% 0 8922

39. DSA Specificity (MFI >1000) SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II2045.7% 8849 7711 Class II alone460% 0 8922 SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II2045.7% 8849 7711 Class II alone460% 0 8922

40. DSA Specificity (MFI >1000) SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II2045.7% 8849 7711 Class II alone460% 0 8922 SpecificitynCDC+ MFI-I II__ Class I alone3655.6% 9545 0 Class I and II2045.7% 8849 7711 Class II alone460% 0 8922

41. Rates of Early AMR and DSA Type Anti-Class I only 38.9% Anti-Class I and II 45.7% Anti-Class II only15% Anti-Class I only 38.9% Anti-Class I and II 45.7% Anti-Class II only15% Explains why the T cell AHG crossmatch has worked well historically Problem with Class II is late injury

42. Prevention vs Treatment?

43. Acute Humoral Rejection Plasmapheresis IVIG Lefaucheur C, Nochy D, Andrade J, et al. Comparison of combination plasmapheresis/IVIg/anti-CD20 versus high-dose IVIg in the treatment of antibody- mediated rejection. Am J Transplant. 2009;9:1099-1107. Splenectomy Rituximab Bortezomib (proteasome inhibitor) Eculizumab (C-5 inhibitor) Plasmapheresis IVIG Lefaucheur C, Nochy D, Andrade J, et al. Comparison of combination plasmapheresis/IVIg/anti-CD20 versus high-dose IVIg in the treatment of antibody- mediated rejection. Am J Transplant. 2009;9:1099-1107. Splenectomy Rituximab Bortezomib (proteasome inhibitor) Eculizumab (C-5 inhibitor)

44. Hypothesis Almost all cases of AMR show evidence of complement activation— C4d+ peritubular capillaries Inhibition of terminal complement activation with anti-C5 antibody (eculizumab) will prevent AMR in +XM Kidney Transplantation Almost all cases of AMR show evidence of complement activation— C4d+ peritubular capillaries Inhibition of terminal complement activation with anti-C5 antibody (eculizumab) will prevent AMR in +XM Kidney Transplantation

45. Classical Pathway Antigen/Antibody Complexes Lectin Pathway Carbohydrate Structures Alternative Pathway M/O and Mammalian Cell Membranes Activated C1 C3 C3a C4b2a C3 Convertase C3bBb C3b C5 C3bBb3b C4b2a3b C5b-9 C6 C7 C8 C9 Weak Anaphylatoxin Immune Complex Microbial Opsonization C5 Convertase C3 Convertase Potent Anaphylatoxin Chemotaxis Cell Activation C3H 2 0 Tickover Cell Activation Neisseria Clearance RBC Lysis The Complement Cascade: Targeted Inhibition Activated MBL C4+C2 Factor B+D C3b C5a C5b X Eculizumab Target

46. Am J Tx (2011) 11:2405-13

47. Pretransplant Management Same in Both Groups <300 No PE Monitor DSA post- transplant <300 No PE Monitor DSA post- transplant >300 Pretransplant PE to achieve T and B FXM <300 Baseline T/B FXM Thymoglobulin induction, Prograf, Mycophenolate mofetil, Prednisone Am J Tx (2011) 11:2405-13

49. 1 o Endpoint Incidence of AMR in first 90 days post-transplant Thrombotic microangiopathy Graft dysfunction (↑>0.3 mg/dl) Protocol biopsies at day 0, 4, 7, 10, 14, 21, 28 Serum for SABs and T/B FXM Incidence of AMR in first 90 days post-transplant Thrombotic microangiopathy Graft dysfunction (↑>0.3 mg/dl) Protocol biopsies at day 0, 4, 7, 10, 14, 21, 28 Serum for SABs and T/B FXM

50. Study Design Historical Controls N=51 1/1/05—10/1/07 PE-based “desensitization” Consecutive Patients Historical Controls N=51 1/1/05—10/1/07 PE-based “desensitization” Consecutive Patients Anti-C5 Treated N=26 6/1/08—09/27/10 Added to existing PE-based protocol BFXM 200  450 MFI 3000-12,000+ 1 patient in each group had BFXM <450, but failed to reach <300 with PE

51. Patients CategoryEculizumab Group (n=26)Control Group (n=51)p value Female Sex21 (81%)40 (78%)1.00 Age in years at time of transplantation (Mean + SD) 48.6 + 12.548.4 + 11.40.94 Ethnicity * Caucasian White24 (92%)44 (86%)0.85 Hispanic2 (7.7%)5 (9.8%) Black0 (0%)2 (3.9%) Cause of Renal Failure Diabetes1 (7.7%)9 (18%)0.52 Polycystic disease4 (15%)7 (14%) Glomerulonephritis4 (15%)7 (14%) Hypertension3 (12%)2 (3.9%) Other11 (42%)20 (39%) Unknown3 (12%)6 (12%) Prior Kidney Transplant13 (50%)13 (25%)0.043 Donor-Specific Alloantibody Levels B flow crossmatch Baseline channel shift (mean + SD) 330 + 84327 + 730.85 Anti-Donor HLA Single Antigen Bead Assay Baseline mean fluorescence index (mean + SD) 7188 + 35036473 + 49460.51 Antidonor Antibody Specificity Anti-Class 1 only 10 (38%)30 (59%)0.10 Anti-Class 2 only7 (27%)13 (25%)1.00 Anti-Class 119 (73%)38 (75%)1.0 Anti-Class 216 (62%)21 (41%)0.10 Both Anti-Class 1 and 29 (35%)8 (16%)0.082 Pretransplant Plasma Exchange ** Number of patients receiving18 (69%)35 (69%)1.00 Number of PEs (mean + SD)4.0 + 3.63.7 + 3.40.76 [WKK1] Is space does not allow keeping of both “anti-class xonly” and “andi-class x” I would keep “andi-class x” and drop “andi-class x only”. “andi-class x” better shows that the Eculizumab group has same or higher ab levels.[WKK1]

52. Results Category Eculizumab Group (n=26) Control Group (n=51) p value Follow-up (mean months + SD, range) 11.9 + 6.1 (3.0 – 27.5) 48.8 + 14.1 (7.8 – 69.8) Graft Survival at 1 year (n, %)16/16 (100%)49/51 (97%)1.00 Antibody mediated rejection < 3months (n, %) 2 (7.7%)21 (41%)0.0031 Patients developing High DSA Levels < 3 months * 13 (50%)22 (43%)0.63 High DSA Biopsies C4d+ (n, %)13 (100%)20 (90.9%)0.52 High DSA and C4d+ biopsies Showing AMR (n, %) 2 (15%)20 (100%)<0.0001 Cellular Rejection <3 months (n, %)1 (6.2%)1 (2.0%)0.42 [WKK1] Please include the graft loss data in the dataset.[WKK1] Am J Tx (2011) 11:2405-13

53. JASN 2010; 21:1398-1406 AMR % with MFI 3001-6000 = 36.4% >6000 = 51.3% AMR (n=2)No AMR (n=24) BFXMMFIBFXMMFI 2507705>200 3711133N=2<3000 N=7>3000 N=9>6000 N=6>10,000

54. Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Day 10 Day 28 BaselineDay 4 Low baseline DSA levels (B-FXM) without AHR High baseline DSA levels (B-FXM) without AHR Low baseline DSA levels (B-FXM) with AHR High baseline DSA levels (B-FXM) with AHR Burns et al Am J Transplant 2008; 8:2684-2694

55. Results Category Eculizumab Group (n=26) Control Group (n=51) p value Follow-up (mean months + SD, range) 11.9 + 6.1 (3.0 – 27.5) 48.8 + 14.1 (7.8 – 69.8) Graft Survival at 1 year (n, %)16/16 (100%)49/51 (97%)1.00 Antibody mediated rejection < 3months (n, %) 2 (7.7%)21 (41%)0.0031 Patients developing High DSA Levels < 3 months * 13 (50%)22 (43%)0.63 High DSA Biopsies C4d+ (n, %)13 (100%)20 (90.9%)0.52 High DSA and C4d+ biopsies Showing AMR (n, %) 2 (15%)20 (100%)<0.0001 Cellular Rejection <3 months (n, %)1 (6.2%)1 (2.0%)0.42 Am J Tx (2011) 11:2405-13

56. Control BFXM 550 =AMR Eculizumab BFXM 604 =Nl N=22 (43%) All AMR N=13 (50%) 2 AMR Histology with High DSA post-TX BFXM >359 post-transplant

57. Case: Early AMR with Eculizumab Elevated creatinine on POD #7 and 14 Increased DSA Biopsy—thrombotic microangiopathy Eculizumab level therapeutic with no hemolytic activity Both treated with PE, resolved Cause unclear?

58. AMR and DSA IgM Day 7 - AMR Day 14 - AMR

59. No DSA IgM High DSA IgG No AMR or TG

60. Plasma Exchange: Post Transplant Control group BFXM >300 at baseline, 7 d PE per protocol Any AMR Eculizumab group 1 patient per protocol early 21 patients no PE per protocol 2 patient with AMR Control group BFXM >300 at baseline, 7 d PE per protocol Any AMR Eculizumab group 1 patient per protocol early 21 patients no PE per protocol 2 patient with AMR

61. Category Eculizumab (n=26) Control Group (n=51) p value Post-Transplant PE3 (12.5%) 39 (76.5%) <0.0001 Splenectomy in patients with AMR 09 (17.7%) 0.025 Graft Dysfunction Δ Cr (mg/dl)=Maximum - Nadir in first month 0.45 + 0.370.93 + 1.150.0087 Other Outcomes: Morbidity

62. Complications All patients receive pretransplant meningococcal vaccine No treatment related infections All patients receive pretransplant meningococcal vaccine No treatment related infections

63. DSA Levels after Transplant Levels change Specificities change Causes complex Absorption by the allograft Memory B cell response Anti-idiotypic “blocking” Abs Levels change Specificities change Causes complex Absorption by the allograft Memory B cell response Anti-idiotypic “blocking” Abs

65. Late Results?

66. Stopping Eculizumab Goal BFXM <200 No AMR after stopping eculizumab 4 weeks: 8 patients stopped 9 weeks: 6 stopped >9 weeks: 2 continued 2 stopped at 1 year No AMR after stopping eculizumab 4 weeks: 8 patients stopped 9 weeks: 6 stopped >9 weeks: 2 continued 2 stopped at 1 year Am J Tx (2011) 11:2405-13

67. Evidence of CHR in C5 inhibitor- treated patients? Caveat: Trial not designed to test prevention of chronic humoral rejection Different post-transplant treatments (some received only 1 month C5 inhibitor, most had no post-transplant plasma exchange)

68. Eculizumab vs Historical Control 3 yr Death Censored Graft Survival

69. Chronic Injury: Transplant Glomerulopathy This is the lesion of chronic AMR

70. Late Events: Chronic Injury, Graft Loss and Death Transplant Glomerulopathy at 12 months Eculizumab 6.7% (1/15) Historical Controls 36% (15/42) P=0.044 Graft loss and Death 2 graft losses at 2 years due to TG in eculizumab group 1 death due to Burkitt’s lymphoma at 2.5 years Transplant Glomerulopathy at 12 months Eculizumab 6.7% (1/15) Historical Controls 36% (15/42) P=0.044 Graft loss and Death 2 graft losses at 2 years due to TG in eculizumab group 1 death due to Burkitt’s lymphoma at 2.5 years

71. Continuous Eculizumab Pt Eculiz Rx weeks AMR3 mos cg/ci 6 mos cg/ci 1 yr cg/ci Cr*BFXM* #952No1/1 1/23.1121

72. Single Antigen Beads MFI/DSA B Flow Cytometric Crossmatch

73. Conclusions: Terminal complement blockade with eculizumab Decreases the incidence of early AMR Prevents AMR and graft dysfunction with higher DSA levels post-transplant Decreases need for PE and splenectomy Decreased TG at 1 year? Chronic changes may require additional therapy

74. Multicenter Trial 80 patients (1:1 randomization) Eculizumab vs Standard of Care (PE or IVIG) Central HLA Lab for entry criteria (BFXM >300) + DSA by flowbeads 1 o Endpoint = AMR in first 90 d Eculizumab rescue for “refractory” rejection in standard of care arm 80 patients (1:1 randomization) Eculizumab vs Standard of Care (PE or IVIG) Central HLA Lab for entry criteria (BFXM >300) + DSA by flowbeads 1 o Endpoint = AMR in first 90 d Eculizumab rescue for “refractory” rejection in standard of care arm

75. Future Studies Role of IgM in early AMR in eculizumab-treated patients? Role of more prolonged eculizumab therapy? Role of bortezomib vs eculizumab to treat new-onset AMR in patients not receiving eculizumab? Role of IgM in early AMR in eculizumab-treated patients? Role of more prolonged eculizumab therapy? Role of bortezomib vs eculizumab to treat new-onset AMR in patients not receiving eculizumab?

76. Thank You

77. Actual Death-Censored 5 Year Graft Survival 70.7% vs 88.0%, p= 0.0006

78. Actual 5 Year Outcomes After +XMKTx Rate of graft loss after 1 year Class I only 1.6%/yr Class II 7.0%/yr

79. Histology at 5 Years* 1/3 of grafts already lost 1 yr5 yr cgptctiscgptctis -XM3.57.797.8 +XM I8.420.950.047.% I/II37.844.458.363.7% *75-80% capture rate