3/17/2009

Matthew J. Everly, PharmD, BCPS Terasaki Foundation Laboratory March 2009

Current Status of the Problem:
• Antibodies cause allograft loss.
– – – – – – McKenna RM, Takemoto SK, Terasaki PI. Transplantation. 2000; 69 : 319. Terasaki PI. Am J Transplant. 2003; 3 : 665 Cai J, Terasaki PI. Hum Immunol. 2005; 66 : 334 Terasaki PI, Cai J. Curr Opin Immunol. 2005; 17 : 541 Terasaki P, Lachmann N, Cai J. Clin Transpl. 2006 : 455 Terasaki PI, Cai J. Transplantation. 2008; 86 : 377

• Antibodies precede allograft dysfunction

1

3/17/2009 Antibodies Precede Allograft Loss 25000 20000 15000 10000 Failure 5000 0 0 1 2 3 4 5 Years post-Tx 6 7 8 8 7 6 Antibodies 5 4 3 2 1 0 Remaining Questions • Will a reduction/removal of antibodies improve allograft survival? • What agent(s) will lead to a durable response in antibody removal? SCR (mg/dl) MFI 2 .

5 0.3 0.0 Porportion of Allografts Surviving 0. M Ozawa. 12 Allografts Lost) 24 36 48 60 72 84 Months after Transplantation 96 3 . PG Catrou.043 (Log-Rank) DSA Reduction > 50% (N = 6.1 0. NO Allograft Loss) Log-rank p=0. KP Briley.4 0. OH Everly et al.0 0 Statistically significant at the α = 0. NO Allografts Lost) Log Rank p=0.8 0.6 0. 7 Allografts Lost) 6 12 18 24 30 36 42 48 54 60 66 72 Time from Transplantation (Months) Reduction of Donor Specific Antibody Levels Prevents Renal Allograft Loss Brody Medical School at Eastern Carolina University. and PI Terasaki Terasaki Foundation Laboratory and Brody School of Medicine at ECU 100 90 Percent Allograft Survival 80 70 60 50 40 30 20 10 0 0 12 Antibody Reduction Responders (n=7.9 0.021 0.3/17/2009 Reduction of Donor Specific Antibody Levels Prevents Renal Allograft Loss University of Cincinnati.2 p = 0. NC MJ Everly. Am J Transplant 2009.9:1-9 1. Cincinnati. CE Haisch.7 0.05 level DSA Reduction < 50% (N = 10. LM Rebellato. Greenville.033 Antibody Reduction Non-Responders (n=23.

3/17/2009 Current Treatments to Reduce Antibodies • Agents that neutralize/remove anti-HLA antibodies • Plasmapheresis (PP) • Immunoabsorption • Intravenous Immune Globulin (IVIg) • Agents that inhibit B-cell proliferation/differentiation • • • • • Splenectomy Cyclophosphamide FK506 / MMF Rituximab Thymoglobulin Current Treatments Unable to Deplete Plasma Cells w/ Thymoglobulin w/ Thymoglobulin w/ Thymoglobulin w/ Thymoglobulin Ramos et al.7:402 4 . Am J Transplant 2007.

3/17/2009 Further Evidence: Rituximab Fails to Reduce DSA Zarkhin et al. 175: 4034 Sarwal M. ATC 2006. Abstract 1012 CD20 + CD38 + /CD138+ 5 . Am J Transplant 2008.8:2607 Need to Deplete the Major Antibody Producing Cell: The Plasma Cell J Immunol 2005.

Defective ribosomal products (DRiPs) Plasma cell DRiPs Unfolded Protein Response Activation REGULATION 26S Proteasome NFκB Gene κ Transcription PLASMA CELL SURVIVAL Enzymes & cell cycle regulators Anti-apoptotic factors Cell adhesion molecules Cytokines IĸBα Degraded Ub Ub Ub Ub P Ub P I κ B kinase Receptor signaling NF-ĸB bound to inhibitor IĸBα 6 . large amounts of Ig 2.3/17/2009 What is Bortezomib? • Bortezomib (Velcade) • Proteasome Inhibitor • FDA approved agent for the treatment of multiple myeloma (plasma cell neoplasia) • Mechanisms of Action .and B.lymphocytes and dendritic cells Reduces class I MHC expression in lymphocytes and dendritic cells Inhibit antigen processing Targets plasma cells in the bone marrow compartment Plasma Cell Survival in Hyper-Proliferative Response Ig Synthesizing 1.Pleotropic: • • • • • Blocks degradation of IκBα thereby inhibiting NF-κB gene transcription Induce apoptosis on activated and mature T.

Transplantation 2008. May 27: In press. Idica et al.specific antibody levels. Clin Transplant 2008.86:1754 • Proteasome Inhibition Causes Apoptosis of Normal Human Plasma Cells Preventing Alloantibody Production. 7 . Everly et al. Everly et al. In Press.9:201 • Proteasome Inhibition reduces donor. Trivedi et al. Perry et al. Transplantation 2009. • Elimination of Post-Transplant Donor Specific HLA Antibodies with Bortezomib. Transplant Proc 2009: 105 • Abrogation of Anti-HLA Antibodies via Proteasome Inhibition. Am J Transplant 2009.3/17/2009 Plasma Cells Deletion via Proteasome Inhibition Ig IN ENDOPLASMIC RECTICULUM Plasma cell DRiPs Unfolded Protein Response Activation Accumulation of Unfolded Proteins and DRiPs 26S Proteasome Gene Transcription Ub Ub Ub Ub P Ub Enzymes & cell cycle regulators Anti-apoptotic factors Cell adhesion molecules Cytokines PLASMA CELL APOPTOSIS P I κ B kinase Receptor signaling NF-ĸB bound to inhibitor IĸBα Current Use of Plasma Cell Depleting Therapy with Bortezomib in Solid Organ Transplant • Bortezomib Provides Effective Therapy for Antibody – and Cell – Mediated Acute Rejection.

7 ± 9.2558 0.5 64. Australia 8 . 2007 • De novo donor specific antibodies present at the time of rejection • 6/7 patients had concomitant ACR and AMR (Mixed rejection) refractory to other therapy • 1 patient with an isolated chronic rejection was also treated • Overall Follow-up from 1st course of bortezomib • 3 -12 months with mean of 140 days • 2 patients ≈ 11 month follow-up Everly et al. Rochester.5 ± 3.0995 0.0001 P-value Perry et al. Sydney.86:1754 Presented at the XXII International Congress of the Transplantation Society.7 33. MN Plasma Cell Apoptosis Treatment Untreated rATG Rituximab Bortezomib % Apoptotic CD138+ Cells 26. Cincinnati.8 ± 7. Am J Transplant 2009.9:201 Bortezomib University of Cincinnati.5 0.8 ± 10. Transplantation 2008.4 22.3/17/2009 Proteasome Inhibition Inhibits Plasma Cells In Transplant Patients Mayo Clinic. OH • • 8 Rejections episodes for 7 patients treated with Bortezomib 1st Patient was treated on July 24.

30. 14.3/17/2009 Bortezomib Regimen University of Cincinnati. Cincinnati.86:1754 Presented at the XXII International Congress of the Transplantation Society. Everly et al. 90. Australia Bortezomib’s Effect on DSA University of Cincinnati. Sydney. and 360. OH Bortezomib 1. Cincinnati. Transplantation 2008. OH Everly et al. 180. Day 7.3 mg/m2 IVP EVERY 72 HOURS X 4 1 CYCLE Plasmapheresis given 72 hours after last dose of bortezomib X 3 sessions DSA samples were conducted at Day 0 (Prior to 1st Bortezomib dose). Transplantation 2008.86:1754 Recipient #1A (DQ7) Recipient #1B (DR53) Recipient #2A (A1) Recipient #3 (DQ6) Recipient #4 (DQ9) Recipient #5 (DR16) Patient #6 (DQ6) 1100000 1000000 Immunodominant DSA MESF 900000 800000 700000 600000 500000 400000 300000 200000 100000 0 Overall Follow-up from 1st Course of bortezomib • 3-12 Months with mean of 140 days • 2 patients with 11 months follow-up 1 DA Y 7 DA Y 14 DA Y 30 DA Y 60 DA Y 9 DA 0 Y 12 DA 0 Y 18 DA 0 Y 36 0 DA Y Days Following Treatment With Bortezomib 9 .

Transplantation 2009. – Serial measurements of HLA antibody were conducted weekly before. Transplantation 2009. May 27: In press. • 13 Patients Treated with Bortezomib / Plasmapheresis ± Rituximab Trivedi et al. May 27: In press. during. • • Bortezomib dosing – Given at 1. Trivedi et al.3 mg/m2 x 4 doses with a 10 day wait between cycles Endpoints – Response = reduction of MFI of primary antibody (first to appear) below 1000 MFI. • All patients were consented to the use of bortezomib “offlabel” for treatment of antibodies post transplant. • DSA Analysis – Antibody intensity was quantified by fluorescence intensity (MFI).3/17/2009 Terasaki Foundation – IKDRC-ITS Bortezomib Study • Patients transplanted at the Institute of Kidney Diseases and Research Centre Ahmedebad. Methods • Patients – Patients were treated “off-label” for elevation in antibodies post transplant – IRB approval and patient consent was obtained for all patients. – MFI >1000 was considered positive. 10 . & after treatment via single antigen bead on Luminex. India between January 2008 and December 2008.

DSA and Treatment Characteristics DSA Responders (n=9).3 +/. n (%) p-value 1.2 +/.0. • All patients were treated under clonal deletion protocol prior to antibody appearance.3/17/2009 Patient Characteristics • Mean patient age: 29.3 years • All patients were male • All patient received living donor transplants • All patients were non-sensitized based on crossmatch at time of transplant. Transplantation 2009. Transplantation 2009. 11 . Trivedi et al. May 27: In press. May 27: In press.2 5 (56) 4 (44) 2 (22) 64 1.0. n (%) Scr at DSA Appearance Class I Class II Use of > 1 cycle of Bortezomib Time to DSA Appearance (days. median) DSA Partial Responders (n=4) .1 ± 9.2 1 (25) 3 (75) 1 (25) 76 NS NS NS NS NS Trivedi et al.

CD33) DQ6 (Pt . 12 .CD79) A24 (Pt . May 27: In press.CD37) A24 (Pt . May 27: In press.CD68) DQ5 (Pt .CD15) B57 (Pt .CD72) Mean Fluoresence Intensity 12000 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 14 28 42 56 70 84 98 112 126 140 154 168 182 196 210 224 Days Post-Bortezomib Trivedi et al.CD86) 112 126 140 154 168 182 196 Days Post-Bortezomib Trivedi et al.CD39) B8 (Pt .3/17/2009 Results: Bortezomib’s Effect on DSA Responders 12000 11000 10000 Mean Fluorescent Intensity 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 0 14 28 42 56 70 84 98 A1 (Pt .CD40) DQ2 (Pt . Transplantation 2009. Transplantation 2009.CD2) DR7 (Pt-CD32) DQ7 (Pt .CD5) DR11 (Pt .CD35) DR51 (Pt . Results: Bortezomib’s Effect on DSA Partial Responders 15000 14000 13000 Cw7 (Pt .

000 4.000 0 0 1 2 3 4 5 0 1 (B57(17)) (B57(17)) (DR7) B 26 3 8 35 4 12 57(17) 35 Cw 7 - DRB1 17 7 8 8 DRB345 52 3 3 DQ 6 4 Bw 6 5 3 1 Donor 4 Bortezomib Apheresis 3 SCr 2 Months Post Transplantation Trivedi et al. 13 . Transplantation 2009.000 MFI 5.000 7. May 27: In press.000 9. Mean +/.000 6.870 55 % NS 0. Transplantation 2009. Mean +/SD Last Follow-Up MFI.3/17/2009 Results: DSA Comparison DSA Responders (n=9). Mean +/. n (%) Pre-Treatment MFI. median (range)) DSA Partial Responders (n=4) .000 3.001 - 30 (14 -143) N/A - Trivedi et al.000 1.1457 9150 +/.SD Peak MFI during Treatment. May 27: In press.004 <0.000 2.SD Percent DSA Reduction (mean) Time to DSA Removal (days.3745 11825 +/. n (%) p-value 3961 +/.3649 <1000 76 % 6450 +/.000 8.2654 5700 +/. Bortezomib’s ability to abrogate DSA A Patient 10.

000 14.000 4.000 11.000 9.000 (A1) 4 Bortezomib 3.000 1 0 0 1 2 3 4 5 6 7 0 Months Post Transplantation Trivedi et al.000 0 0 1 2 3 4 5 MFI MFI Donor 1 2 68 68 8 57 B 35 5 4 4 (DQ5(1)) (DQ6(1)) (DQ6(1)) (DQ5(1)) C 7 12 DRB1 17 17(3) 11 2 1 DQ 3 2 DRB345 52 52 6 4 Bw 5 4 Bortezomib Apheresis Rituximab 3 SCr 2 1 0 Months Post Transplantation Trivedi et al.000 1.000 6.000 Patient Donor 24 24 68 1 7 51 B 35 7 4 12 C 12 16 DRB1 14 15 13 15 DQ 1 1 4 DRB345 51 52 51 52 Bw 6 4 6 5 4. May 27: In press.000 8.000 5. High Intensity DSA Responds well but 2 . 14 .000 1. May 27: In press. Transplantation 2009.3 cycles of bortezomib may be needed A 5.3/17/2009 Monitoring with Bortezomib is Needed To Determine RIGHT TIME/ RIGHT DOSE A Patient 15.000 2.000 10.000 7.000 12.000 13. Transplantation 2009.000 3.000 MFI Apheresis 3 SCr 2 2.

mycophenolate/tacrolimus 4. Minimal Transient Toxicity with 1-2 cycles – (2 cases of generalized weakness. transient drop in platelets.000) may require multiple cycles for removal What is the Next Step… 15 . Rituximab. Higher MFI antibodies (>10. <5% Nausea/diarrhea. Bortezomib provides significant reductions/removal of DSA 2. 3. Safe following or when given concurrently with Thymoglobulin.3/17/2009 Bortezomib Summary: 1. Plasmapheresis.

3/17/2009 FIRST: MONITOR FOR ANTIBODIES LOW-RISK Pre-Transplant HIGH-RISK (XM-) Pre-Transplant 2 weeks POST-TRANSPLANT 1 month 2 months 3 months 6 months 9 months 1 year Beyond 1 year: Annually 3 months 6 months 9 months 1 year Beyond 1 year: Bi-Annually More research is needed to confirm the clinical significance of each testing time point THEN. TREAT THE ANTIBODY Study Design • • • • Multicenter 5 year study (with interim analysis annually) 300 Patients (1:1 randomization) Pharmaceutical Support 16 .

Terasaki • Drs. E. Using HLA single antigen monitoring should provide the framework for improving survival through removal of antibodies. One cycle alone of bortezomib may not be enough for all patients 4. Bortezomib shows promise as the first agent capable of removing HLA antibodies 3. Steve Woodle and Rita R.3/17/2009 Conclusions 1. Current agents Are Ineffective at removing Alloantibodies 2. Alloway • Terasaki Foundation Laboratory • University of Cincinnati • IKDRC-ITS • Brody School of Medicine Eastern Carolina University 17 . Paul I. Acknowledgements • Dr.

3/17/2009 Questions Email: meverly@terasakilab.org Phone: 310-479-6101 ext 125 18 .

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