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

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

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

4. What we know about donor specific anti-HLA antibodies …

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

6. What we do not know …

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

8. DSA + at Transplant
Transplant Patient
DSA - at Transplant

9. ≥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

10. <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

11. Anti-HLA
Preformed DSA Patient

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

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

14. 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

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

16. 119 Preformed DSA Patient Positive Crossmatch
Gloor et al. Am J Transplant 2010;10:582

17. 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

18. 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

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

20. 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

21. 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

22. 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

23. 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

24. 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)

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

26. 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

27. 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

28. 116 Flow Negative Crossmatch, but DSA Positive
Preformed DSA Patient
116 Flow Negative Crossmatch, but DSA Positive
Clinical Transplants In Press

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

30. ? 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

31. 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

32. 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

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

34. 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.

35. 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

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

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

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

39. 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

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

41. 62
DSA +
De Novo
Anti-HLA
27%
Everly et al. Am J Transplant. 2012; In Submission
Rebellato et al. Clinical Transplants 2011; In Press

42. 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 In Submission

43. 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

44. 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

45. 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 In Submission

46. 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 =
earliest observed entry t =
last observed exit t =
. sts list, at( )
failure _d: statuscavrip == 1
analysis time _t: modsatocavrip
Beg Survivor Std.
Time Total Fail Function Error [95% Conf. Int.]
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

47. 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

48. 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

49. In Acute Antibody Mediated Rejection
Removing
DSA
In Acute Antibody Mediated Rejection

50. Statistically significant at the α = 0.05 level
Reduction of Donor Specific Antibody Levels Prevents Renal Allograft Loss
p = (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.

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

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

53. In patients with preformed DSA
Removing
DSA
In patients with preformed DSA

54. 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

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

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

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

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

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

60. 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)

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

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

63. 2 6 All DSA positive All preemtive treatment
Patients in 2011
All DSA positive
All preemtive treatment
All with no dysfunction at DSA appearance

64. 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

65. 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

66. 85 days (median time to DSA – range from 6 - 536) Txp DSA
(MFI > 1000)
SCr ± 0.22
SCr ± 0.27

67. 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

68. 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)

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

70. Removing
DSA
Improves Outcomes

71. Problem with Treating anti-HLA DSA
Remission
Refractory
Relapse

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

73. 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 ?

74. Summary

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

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

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

78. 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

79. 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

80. 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 In Submission

81. 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

82. DSA Relapse
DSA Relapse
Removing
DSA
Improves Outcomes

83. 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 ?

84. Thank
You