Monitoring HLA-specific antibodies In patients undergoing desensitization Matthew J. Everly, PharmD, BCPS One Lambda Inc.

Monitoring HLA-specific antibodies In all transplant patients Matthew J. Everly, PharmD, BCPS One Lambda Inc.

Preformed anti-HLA DSA+ Patient Screening De Novo anti-HLA DSA In all transplant patients Monitoring Removal of anti-HLA DSA

What we know about donor specific anti-HLA antibodies …

in transplant patients Donor Specific Anti-HLA Antibodies allograft rejection allograft failure = in transplant patients

What we do not know …

How to monitor anti-HLA DSA? How to treat anti-HLA DSA?

DSA + at Transplant Transplant Patient DSA - at Transplant

≥35% Acute Antibody Mediated Rejection Up to 15% 1- year Graft Failure Rate (non-desensitized) DSA + at Transplant “Preformed DSA” DSA - at Transplant Lefacheur et al. Am J Transplant 2008;8:324 Dunn et al. Am J Transplant 2011;11:2132

<5% De novo anti-HLA DSA in the first year Acute Antibody Mediated Rejection De novo anti-HLA DSA in the first year And beyond DSA + at Transplant “Preformed DSA” DSA - at Transplant Smith et al. Am J Transplant 2011;11:312 Dunn et al. Am J Transplant 2011;11:2132 Everly et al. Am J Transplant 2012; In submission

Anti-HLA Preformed DSA Patient

Major concern with preformed anti-HLA DSA is the associated AMR Risk

Preformed DSA Patient Amico et al. Transplantation 2009;87:1681 DSA+ at Transplant with AMR (n=37) Amico et al. Transplantation 2009;87:1681

Preformed DSA Patient DSA – (at transplant) n=194 DSA + (no AMR), n=22 DSA+ AMR+, n=21 Lefaucheur et al. Am J Transplant 2008;8:324

DSA Associated AMR risk is not uniform over the post-transplant period

119 Preformed DSA Patient Positive Crossmatch Gloor et al. Am J Transplant 2010;10:582

Preformed DSA Patient DSA- (n=12) DSA+ low MFI Sum 0 - 5,000 MFI (n=23) Preformed DSA Patient DSA+ low MFI Sum 5,000-10,000 (n=16) DSA+ high MFI Sum >10,000 MFI (n=66) Gloor et al. Am J Transplant 2010;10:582

Negative CDC Crossmatch Preformed DSA Patient 334 Negative CDC Crossmatch Aims: What is the clinical relevance of preformed DSA? What anti-HLA DSA characteristics are predictive of DSA? 67 (20%) Anti-HLA DSA + Pre-Transplant Amico et al. Transplantation 2009;87:1681

Preformed DSA Patient Amico et al. Transplantation 2009;87:1681

237 194 (82%) 43 (18%) Preformed DSA Patient Negative T- & B-Cell CDC Crossmatch Preformed DSA Patient 194 (82%) Anti-HLA DSA - Pre-Transplant 43 (18%) Anti-HLA DSA + Pre-Transplant Lefaucheur et al. Am J Transplant 2008;8:324

DSA- Preformed DSA Patient DSA+ low MFI 13/21 (62%) of AMR in first 45 days post-transplant DSA+ high MFI Lefaucheur et al. Am J Transplant 2008;8:324

CDC XM- with single antigen bead testing at transplant Preformed DSA Patient 587 patients CDC XM- with single antigen bead testing at transplant Dunn et al. Am J Transplant 2011;11:2132

Preformed DSA Patient DSA- DSA+ low MFI DSA+ high MFI DSA+ 3rd party HLA+ or Neg. DSA+ high MFI DSA+ Dunn et al. Am J Transplant 2011;11:2132

to Acute Antibody Mediated Rejection Median Time to Acute Antibody Mediated Rejection Preformed DSA Patient 7.5 days 16 days 16.5 days Gloor et al. (n=49) Lefaucheur et al. (n=21) Dunn et al. (n=46)

Can monitoring identify the preformed anti-HLA DSA patients at risk of AMR ?

70 41 29 Preformed DSA Patient Positive Crossmatch High anti-HLA DSA (Flow Channel Shift >300) 29 Low anti-HLA DSA (Flow Channel Shift <300) Burns et al. Am J Transplant 2008;10:2684

Preformed DSA Patient Low DSA, NO AMR Low DSA, AMR+ High DSA, NO AMR High DSA, AMR+ Burns et al. Am J Transplant 2008;8:2684

116 Flow Negative Crossmatch, but DSA Positive Preformed DSA Patient 116 Flow Negative Crossmatch, but DSA Positive Clinical Transplants 2011. In Press

Monitoring Time Points Possible DSA Monitoring Time Points Pre-Transplant, Day of Transplant 1 week 2 weeks 4 weeks 8 weeks Preformed DSA Patient

? Preformed DSA Patient DSA- (n=12) DSA- DSA+ low MFI Sum 0 - 5,000 MFI (n=23) Preformed DSA Patient DSA+ low MFI DSA+ low MFI Sum 5,000-10,000 (n=16) DSA+ high MFI DSA+ high MFI Sum >10,000 MFI (n=66) ? Gloor et al. Am J Transplant 2010;10:582

47% of XM+ AMR+ Patients 41% of XM+ AMR- Patients Transplant Glomerulopathy Risk 47% of XM+ AMR+ Patients 41% of XM+ AMR- Patients Gloor et al. Am J Transplant 2010;10:582

Flow Positive Crossmatch Preformed DSA Patient 69 patients Flow Positive Crossmatch 11 excluded no DSA, or not clear group 33 Group 1 Convert to Flow XM - 15 Group 2 Remain Flow XM + Kidney Int 2011;10:582

Preformed DSA Patient Group 1 Group 2 Group 2 Group 1 Kimball et al. Kidney Int 2011;10:582

Monitoring Time Points Pre-Transplant, Day of Transplant 1 week 2 weeks 4 weeks 8 weeks 6 months 12 months After 12 months ? Preformed DSA Patient With for cause biopsy * If patient’s DSA remain persistently positive – treatment may be useful to improve outcomes.

Rationale to monitor DSA Identify a patient at risk of AMR at risk of Chronic Antibody Damage who should receive treatment for Antibodies Preformed DSA

Screening for De Novo DSA Pre-Transplant DSA - Patient Screening for De Novo DSA

Early Acute Antibody Mediated Rejection Preformed DSA De Novo DSA >35% <5%

Screening for De Novo DSA DSA+ low MFI DSA+ high MFI Gill et al. Transplantation 2010;89:178

Rationale to monitor DSA Identify a patient at risk of AMR at risk of Chronic Antibody Damage who should receive treatment for Antibodies De Novo DSA

East Carolina University (Rebellato et al.) Everly et al. Am J Transplant. 2012; In Submission Rebellato et al. Clinical Transplants 2011; In Press

62 DSA + De Novo Anti-HLA 27% Everly et al. Am J Transplant. 2012; In Submission Rebellato et al. Clinical Transplants 2011; In Press

10% 3-4% Year Post-transplant De Novo DSA Incidence 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1 2 3 4 5 6 7 8 9 10 11 12 Year Post-transplant 10% 3-4% De Novo DSA Incidence Everly et al. Presented at American Transplant Congress 2011, Philadelphia Everly et al. Am J Transplant 2012. In Submission

patients transplanted between 1995-2004 who had survived more than one year 243 224 19 patients excluded DSA positive at the time of transplant 173 HLA Abs 51 patients with no HLA Abs Coming back to our patient set, of the 224 patients, 173 patients developed HLA antibodies and 51 patients had no detectable HLA antibodies. Of the 173, 117 patients (or 52% of 224) develop non-donor specific antibodies and 56 or (25%) developed de novo DSA. What we’re going to focus on are these 56 patients producing de novo DSA. 74 pre- and post-tx 43 post-tx 56 de novo DSA (25%) 117 NDSA (52%) NDSA= non donor-specific antibody Smith J et al. Am J Transplant. 2011;11:312-9

When do HEART TRANSPLANT patients develop de-novo DSA? 72% N=57 22 13 6 6 5 3 1 1 Smith J et al. Am J Transplant. 2011;11:312-9

15% 28% 42% De novo anti-HLA DSA Positive (n=62) 10 20 30 40 50 60 70 80 90 100 Percent Allograft Survival 12 24 36 48 Months after DSA Appearance 15% 28% 42% Everly et al. Am J Transplant 2012. In Submission

What is the risk for heart failure after de novo anti-HLA DSA appears? 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1 2 3 4 5 n=51 20% probability of failure 24% 37% Proportion Surviving . stset modsatocavrip, failure (statuscavrip==1) enter(time 0) exit(time 60) failure event: statuscavrip == 1 obs. time interval: (0, modsatocavrip] exit on or before: time 60 ------------------------------------------------------------------------------ 56 total obs. 5 event time missing (modsatocavrip>=.) PROBABLE ERROR 51 obs. remaining, representing 18 failures in single record/single failure data 2183 total analysis time at risk, at risk from t = 0 earliest observed entry t = 0 last observed exit t = 60 . sts list, at(0 12 36 60) failure _d: statuscavrip == 1 analysis time _t: modsatocavrip Beg. Survivor Std. Time Total Fail Function Error [95% Conf. Int.] ------------------------------------------------------------------------------- 0 0 0 1.0000 . . . 12 40 10 0.7984 0.0570 0.6575 0.8861 36 37 2 0.7569 0.0612 0.6115 0.8541 60 27 6 0.6267 0.0701 0.4738 0.7465 Note: survivor function is calculated over full data and evaluated at indicated times; it is not calculated from aggregates shown at left. . 5 patients develop DSA after CAV: 4 CII Abs, 1 both classes of Abs Year from DSA to CAV or Graft Loss (GL) Smith J et al. Am J Transplant. 2011;11:312-9

Rationale to monitor DSA Identify a patient at risk of AMR at risk of Chronic Antibody Damage who should receive treatment for Antibodies De Novo DSA

Monitoring Time Points Pre-Transplant 1 week 2 weeks 4 weeks 8 weeks 6 months 12 months Biannually after 1 year Screening for De Novo DSA With for cause biopsy

In Acute Antibody Mediated Rejection Removing DSA In Acute Antibody Mediated Rejection

Statistically significant at the α = 0.05 level Reduction of Donor Specific Antibody Levels Prevents Renal Allograft Loss p = 0.043 (Log-Rank) Statistically significant at the α = 0.05 level University of Cincinnati, Cincinnati, OH Everly et al. Am J Transplant 2009;9:1-9 Log Rank p=0.021 Antibody Reduction Responders (n=7, NO Allograft Loss) Antibody Reduction Non-Responders (n=23, 12 Allografts Lost) Log-rank p=0.033 10 20 30 40 50 60 70 80 90 100 Percent Allograft Survival 12 24 36 48 72 84 96 Months after Transplantation Brody Medical School at Eastern Carolina University, Greenville, NC Everly, et al. Transplantation. Regarding the first question, I have conduction 2 studies to look at historical patients treated at the time of acute rejection, focusing on the effect of antibody reduction. These studies both have shown that antibody reduction will improve allograft survival. However, more data is needed to cement this concept.

Lefaucheur et al. Am J Transplant 2009;9:1099

Antibody removal after AMR improves outcomes Despite Histologic Improvements … Antibody removal after AMR improves outcomes

In patients with preformed DSA Removing DSA In patients with preformed DSA

Impact of proteasome inhibitor on anti-donor HLA antibody production after kidney transplantation Mayo Clinic Trial – Mark Stegall M.D. Removing DSA 2 cycles of bortezomib … wait 2 months … possible 2 more cycles of bortezomib

Removing DSA IVIg + plasmapheresis 3 times per week For 2 weeks Kimball et al. Clinical Transplants 2011; In press

Removing DSA Kimball et al. Clinical Transplants 2011; In press

Antibody removal may improve outcomes In the patient with persistent preformed anti-HLA DSA positive … Antibody removal may improve outcomes

In patients with stable allograft function Removing DSA In patients with stable allograft function

Removing DSA 61 lung transplant patients Serial single antigen bead testing after transplant Hachem et al. J Heart Lung Transplant 2010;29:973

61 All DSA positive All preemtive treatment IVIg for at least 6 months Patients in 2011 All DSA positive All preemtive treatment IVIg for at least 6 months (+/- Rituximab)

Removing DSA Hachem et al. J Heart Lung Transplant 2010;29:973

Removing DSA Everly et al. Transplantation 2012; In Press.

2 6 All DSA positive All preemtive treatment Patients in 2011 All DSA positive All preemtive treatment All with no dysfunction at DSA appearance

2 6 27% (n=7) 23% (n=6) 50% (n=13) Both DSA Classes DSA Class I Alone DSA Class II Alone 50% (n=13) - 65% of patients had ONLY a single DSA positive at the time of treatment - 39% of class II were DQ DSA

Bortezomib on days 1, 4, 8, 11 B+P+R Plasmapheresis (n=14) on same days with bortezomib Rituximab (n=9) single dose Corticosteroid pulse – (all patients) B+P+R (n=15) Bortezomib Alone (n=11) Male Gender 24 (92) Age at Transplant 29.3 ± 9 # of HLA mismatch 2.9 ± 1.4

85 days (median time to DSA – range from 6 - 536) Txp DSA (MFI > 1000) SCr - 1.17 ± 0.22 SCr - 1.26 ± 0.27

26 Patients 24 – Greater than 50% reduction in DSAmax MFI Reduction of DSAmax MFI by at least 50% 26 Patients 24 – Greater than 50% reduction in DSAmax MFI 2 – Less than 50% reduction in DSAmax MFI Median time to 50% reduction 37 days

Serum Creatinine Change from Start of Treatment to last follow-up Median Serum Creatinine Change from Start of Treatment to last follow-up 6.5% Scr Increase Remission (n=8) Removing DSA 41% Scr Increase Relapsed (n=10) 46% Scr Increase Partial/No Response (n=8) (only a 50% reduction)

Antibody removal may improve outcomes In the patient with persistent de novo anti-HLA DSA … Antibody removal may improve outcomes

Removing DSA Improves Outcomes

Problem with Treating anti-HLA DSA Remission Refractory Relapse

Removing DSA “ A rapid partial response (50% reduction of DSAmax with 1 month of treatment was associated with a complete response”

When Removing DSA Prior to start of treatment 1 month Post-Treatment Monitoring Time Points Prior to start of treatment 1 month (possibly 2 months if not using plasmspheresis) When Removing DSA Refractory Remission Continued monitoring if continued treatment At 3 & 6 months then every 6 months Relapse ?

Summary

Nearly All transplant patients can benefit for some degree of monitoring …

AMR Appears Early Preformed DSA Patient Amico et al. Transplantation 2009;87:1681

Monitoring Early Identifies those AT RISK of AMR Preformed DSA Patient Burns et al. Am J Transplant 2008;8:2684

Monitoring Late Identifies those AT RISK of Failure Group 1 Group 2 Preformed DSA Patient Group 2 Group 1 Kimball et al. Kidney Int 2011;10:582

Monitoring Time Points Preformed DSA Patient Monitoring Time Points Pre-Transplant 1 week 2 weeks 4 weeks 8 weeks 6 months 12 months After 12 months, annually With for cause biopsy

De novo anti-HLA DSA appears at any time post transplant & Identifies those AT RISK of Failure Screening for De Novo DSA Everly et al. Presented at American Transplant Congress 2011, Philadelphia Everly et al. Am J Transplant 2012. In Submission

Monitoring Time Points DSA- Screening for De Novo DSA Monitoring Time Points Pre-Transplant 6 months 12 months Annually after 1 year With for cause biopsy

DSA Relapse DSA Relapse Removing DSA Improves Outcomes

When Removing DSA Prior to start of treatment 1 month Post-Treatment Monitoring Time Points Prior to start of treatment 1 month (possibly 2 months if not using plasmspheresis) When Removing DSA Refractory Remission Continued monitoring if continued treatment At 3 & 6 months then every 6 months Relapse ?

Thank You meverly@terasakilab.org