1. ”Insight into the role of non-HLA antibodies and Rejections”
XM-ONE® - A complementary approach to assessing risk in Solid Organ Transplantation
”Insight into the role of non-HLA antibodies and Rejections”

2. Content AMR, DSA and C4d Pre transplant assessment of risk
in Organ Transplant Patients
Risk Assessment using XM-ONE®

3. Antibody Detection Methods
HLA
CDC
ELISA
Flow PRA
Solid Phase Assay (Luminex)
Flow XM (using T- and/or B cells)
Non-HLA
ELISA (MICA / MICB)
Solid Phase Assay (MICA)
XM-ONE (Anti Endothelial Cell Antibodies)

4. Pre Tx antibodies influence graft failure
Objective with slide: Acknowledging HLA as a risk factor in SOT. Get customer approval
Reference:
Gerhard Opelz for the
Collaborative
Transplant Study*
Lancet 2005; 365: 1570–76

5. HLA-identical siblings also reject!
The PRA activity in HLA identical siblings is associated with poorer graft survival
Patients cannot form antibodies against their own HLA antigens; therefore they cannot form anti-HLA against lymphocytes of an HLA-identical sibling donor.
These findings further supports the role of non-HLA antibodies
Objective with slide: Get approval of non-HLA as a risk factor
Reference:
Gerhard Opelz for the
Collaborative
Transplant Study*
Lancet 2005; 365: 1570–76

6. Development of antibody awareness and diagnostics
1985:
First publications regarding endothelial alloantigens and transplantation
2005:
The Collaborative Transplant Study HLA identicals have PRA (G Opelz)
2009:
XM-ONE Multicenter study (M Breimer)
1968:
Post tx HLA ab’s associated with poor survival (Morris and co-workers)
1997:
First cases published on AECA and rejection (Heart tx, Kidney Tx)
1969:
HLA crossmatching (Patel & Terasaki)
1993:
First Olerup SSP HLA typing test on market
2002:
Introducing the LUMINEX-100
HLA
2006:
XM-ONE CE marked in Europe
2008:
XM-ONE cleared by FDA in US
non-HLA
Objective with slide: non-HLA is not new, however new tool has arrived

7. Banff Criteria for AMR C4d+ Presence of circulating DSA
Acute tissue injury
Objective with slide: Present DSA as risk factor – get acceptance
Solez et al, Am J Transpl: 2008; 8: 753–760

8. Donor Specific Antibodies are considered to be a risk factor in organ transplantation
The presence of donor-specific antibodies is associated with an increased risk of graft loss (Lachmann, N et al, Transplantation. 2009 May 27;87(10): )
The presence of preformed DSA is strongly associated with increased graft loss in kidney transplants, related to an increased risk of AMR (Lefaucheur C et al, Humoral Immunity in Kidney Transplantation. Contrib Nephrol. Basel, Karger, 2009, vol 162, pp 1-12 )
High baseline DSA patients have high rates of AMR and poor long-term allograft survival highlighting the need for improved therapy for these candidates (J. M. Gloor et al, Am J transpl American Journal of Transplantation, 2010, Vol 10 (3):582–589)
Pre-transplant Donor-Specific Antibodies Detected by Single-Antigen Bead Flow Cytometry Are Associated With Inferior Kidney Transplant Outcomes (Singh, N et al, Transplantation: 27 November Vol 90 (10): )
Objective with slide: Not only DSA but pre Tx DSA is associated with increased risk – thats why we do pre Tx DSA testing (FCXM, SPA, Luminex)

9. C4d – the footprint of DSA
”C4d is a fragment of complement component C4 released during activation of the classical complement pathway by antigen-antibody complexes”
Objective with slide: Introduce C4d into our talk – the footprint of DSA
Chakravarti DN, Campbell RD, Porter RR: Molecular Immunology 24: 1187–1197, 1987

10. C4d is associated with Graft Failure
Objective with slide: C4d + is, as well as DSA, a negactive prognostic factor
Mauiyyedi, S et al,
J Am Soc Nephrol
13: 779–787, 2002

11. C4d deposition correlates to DSA
Objective with slide: C4d and DSA is linked
Mauiyyedi, S et al,
J Am Soc Nephrol
13: 779–787, 2002

12. Antibody Detection Methods
HLA
CDC
ELISA
Flow PRA
Solid Phase Assay (Luminex)
Flow XM (using T- and/or B cells)
Objective with slide: Focus on DSA detection and ask if Luminex or Flow

13. Rejections in HLA DSA negative patients in Heart, Lung or Kidney Transplantation
Intro,

14. Objective with slide: Present the study shortly (heart, Italy, C4d and DSA)

15. 985 biopsies from 107 heart transplant recipients were evaluated
AMR in Heart Tx
985 biopsies from 107 heart transplant recipients were evaluated
C4d positive staining was found in 36 patients (34%)
HLA DSA identified by LUMINEX was present in 14 patients (13%) at the time of rejection
AMR was diagnosed in 8 patients (7%) according to ISHLT recommendations
Objective with slide: Background but focus on C4d in every third pt but only DSA in less then half of those
Ref: Fedrigo et al,
Transplantation,
Vol 90 (7)
Oct 15, 2010

16. C4d positive staining can occur without DSA?
No Graft Dysfunction = asAMR,
Graft Dysfunction = symptomatic AMR
107 HTx
Control
(n=71)
C4d+, DSA-, no GD
(n=22)
Questions:
C4d positive staining can occur without DSA?
C4d positive staining can only occur in the presence of AB’s?
The above AB’s were not detected by LUMINEX:
due to lack of sensitivity?
since the AB’s responsible are non-HLA AB’s?
C4d+, DSA+, no GD
(n=6)
C4d+, DSA+, GD
(n=8)
Objective with slide: Focus on C4d without DSA and the ”lack” of DSA. Is not DSA a necessity?
Ref: Fedrigo et al,
Transplantation,
Vol 90 (7)
Oct 15, 2010

17. Objective with slide: AMR in lung – not HLA DSA, what then?
25 Lung Transplant recipients that had experience 1 or more episode of acute rejection.
Flow cytometric alloantibody detection. As outlined by Pelletier et al. (7–10), a commercially available pool of microparticle beads coated with various purified MHC antigens of known specificity were used according to manufacturer’s instructions (FlowPRA, OneLambda, Canoga Park, CA).
Anti-donor MHC Class I/Class II ELISA: Donor spleen cells were used as a source of lysate, which was added to microtiter wells precoated with murine monoclonal antibody (capture antibody) with specificity to either MHC Class I antigens or MHC Class II antigens.
Endothelium is likely an important if not the critical target, and it is very possible that the antigenic targets are those that fall under the general
rubric of the “vascular endothelial cell system”.

18. Objective with slide: C4d but no PRA against Class I or II implies that other DSA’s are involved
Pre Tx testing: As shown in Table 1, PRA testing demonstrated an absence of both MHC Class I and MHC Class II circulating allo-antibodies in this patient population during the pre transplant period, excluding patient 8.
Post Tx testing: As shown in Table 1, PRA testing demonstrated a remarkable absence of both MHC Class I and MHC Class II circulating allo-antibodies in this patient population at all of the time points tested during the post transplant period. PRA values were less than the 6% value our program employs as an indicator of sensitization in all cases with one exception. No MHC Class II specificity could be assigned when the one sample which demonstrated Class II sensitization (PRA = 12%) was further tested for MHC Class II specificity.
Magro et al:
Am J of Transpl
2003; 3:
1264–1272

19. Objective with slide: HAR or very early rejections in Ktx

20. Early (<7 days) AMR in KTx
Objective of slide: Improvement in HLA DSA aviodance but still non-HLA at same range
In the Berlin study early AMR was defined as allograft dysfunction within the first 7 days post-transplant and an allograft biopsy result consistent with AMR. DSA data was aquired pre Tx.
Data presented from JHU at this years ASHI meeting (Sep 2010) showed that the incidence of pre transplant XM-ONE positivity (non-HLA AB’s) was appr 25% and in these patients about 36% had an ARE within the first 100 days post Tx (XM-ONE neg 17%).
Amico et al:
Transplantation
2008;85:
1557–1563

21. Objective with slide: Non-HLA abys are a known attributor of rejection but in this study they were not found to be MIC-A or AT1R ab’s. Rejections very early indicating complement fixing ab’s
In the patients that developed AMR without any presence of pre transplant HLA DSA one patient had DSA MIC-A but none of the patients had AT1R AB’s. No endothelial crossmatches were performed. My conclusion (?): The AB’s that occur in HLA DSA negative patients experiencing AMR are (to the vast majority) not MIC-A or AT1R AB’s but of other specificity (AECA?).
All 10 patients experiencing early AMR without measurable HLA-DSA developed rejection within a median of 2 days post-transplant (range, 1–7 days) indicating the presence of significant amounts of preformed DSA, which should be detectable by sensitive assays in the sera obtained at the time of transplantation.
Amico et al:
Transplantation
2008;85:
1557–1563

22. AMR and DSA Patients receive an allograft
based on a negative complement-
dependent cytotoxicity (CDC) crossmatch
At time of transplantation many patients display donor-specific antibodies (DSA) by sensitive methods (solid-phase assays, FCXM)
Study comparing different crossmatch techniques (PRA, SPA, FCXM) in detecting DSA correlated to “AMR” (defined according to Banff minus DSA)
Objective of slide: Acceptance of AMR without defined HLA DSA
Ref: Vlad et al;
Human Imm 70
(2009) 589–594

23. Demographics n Patients 325 Primary Graft 260 Secondary Graft 65 (20%)
AMR*
29 (9%)
PRA
129 (40%)
Pos DSA SPA
27 (8%)
Pos DSA FCXM (T/B)
47 (14%)
Objective of slide: Accept the figure of 9% ”non-DSA AMR”
*AMR was diagnosed if C4d+ and morphologic tissue injury
Ref: Vlad et al;
Human Imm 70
(2009) 589–594

24. AMR* occur in 9% of CDC negative patients
*AMR=Banff without DSA
Objective of slide: AMR can occur without HLA DSA. Almost all had PRA but few had DSA
Ref: Vlad et al;
Human Imm 70
(2009) 589–594

25. Negative in test (SPA, FCXM)
Graft Survival
Negative in test (SPA, FCXM)
but ”AMR” diagnosed

26. Discussion
”Although the Luminex SPA method is excellent for identifying anti-HLA antibodies, non-HLA antibodies represent a “blind-spot” for this type of testing. Essentially, the SPA will always return a false-negative result if the target is not an HLA antigen”
”Cell-based methods are less susceptible to this type of problem, because the donor cells used for testing emulate the comprehensive antigenic makeup of the prospective graft with much greater fidelity”
In this study from Univ of Columbia 325 DD KTx patients were retrospectively analyzed. Among a number of findings I find it interesting to see that in patients having a ”diagnosed” AMR (here DSA was removed as part of the diagnosis) in patients being negative in either FCXM, SPA (luminex) or PRA pre transplant had the worst prognosis. Furthermore this difference seems to appear quite soon after Tx. Non-HLA AB’s were not discussed in the article.
Ref: Vlad et al;
Human Imm 70
(2009) 589–594

27. Background summary Assessing risk of post transplant complications
DSA, Donor Specific Antibodies, are associated with poorer outcome
C4d – ”the footprint of DSA” – can occur without detected HLA DSA
PRA does not correlate with HLA DSA – evidence of non-HLA DSA
Would assessing non-HLA DSA improve the risk assessment?

28. XM-ONE® : Detects antibodies against cell bound antigens
HLA I
HLA II
non-HLA
Snyggare bild som visar detta
Tie-2-r
Peripheral blood endothelial cell precursor 28

29. XM-ONE identifies more than HLA
HLA I
non-HLA
HLA II
The sensitivity of XM-ONE as compared to the Flow PRA Single Antigen bead assay is 35 (positive in XM-ONE) / 37 (positive in Flow PRA), 95%.
Reference:
AbSorber, Data on file

30. Expression of surface markers on Tie2 isolated cells from PBMC
MHC I
Yes
MHC II
CD11b
CD14
CD31
CD32
CD34
Low
CD68
CD133
CD144
VEGFR1
VEGFR2
Green indicates a higher expresion when compared to total non-isolated cells
Ref:
AbSorber , Data on File

31. Organ Donor Recipient Analyse by Flow Cytometry
Collect blood in BD CPT™ tubes
Organ Donor
Centrifuge to isolate PBMC
Retrieve PBMCs into 50 ml tube and wash cells
Recipient
Collect blood in serum tube
Centrifuge to
obtain sera
Incubate isolated EPCs with recipient sera and control sera.
Wash three times
Incubate PBMCs with
magnetic beads
to isolate EPC
Anti-Tie-2
Incubate with secondary
FITC conjugated antibodies
against IgG and IgM
Analyse by Flow Cytometry

32. Reading XM-ONE® by flow cytometry

33. Picture shows CE marked version;
for US no CD3 and CD19 antibodies are included

34. XM-ONE Background
Developed to detect antibodies causing Hyper Acute Rejections (HAR) in patients at Karolinska Institute, Sweden
Multicenter study designed to assess risk of HAR from pre transplant testing for non-HLA DSA (8% XM-ONE positivity, 25% HAR)
Study sample size was calculated to be 280 patients
Ethics Committee (EC) decided to have interim analysis for every 100 patients enrolled

35. XM-ONE® prospective multicenter study
Baylor University Med Center, Dallas, TX, Johns Hopkins University, Baltimore, MD, Ohio State University Med Center, Columbus, OH, Massachusetts General Hospital, Boston, MA, USA
Karolinska Institute, Huddinge Hospital, Stockholm, Sahlgrenska University Hospital, Gothenburg, Sweden
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

36. Study Design Lymphocyte crossmatch (LXM) n=147 Clinical follow-up
Recruitment
Patient Information
Lymphocyte crossmatch (LXM)
n=147
Clinical follow-up
Endothelial Cell crossmatch
(XM-ONE)
> 3 month / rejection
Decisions about transplantation and immunosuppressive treatment based on results from LXM and solid phase assay
If a rejection episode occured responsible staff was informed about XM-ONE results before making decisions about immunosuppressive treatment
Patients were recruited to the study after passing normal immunological tests performed at each centre. All patients was tested with at least one lymphocyte crossmatch and the solid fase assays performed at the clinic such as Luminex and FlowPRA. Decision about transplantation and immunosuppressive treatment was made based on the results from these tests. In addition XM-One test was performed.
The study had the following objectives:
Frequency of XM-ONE positive patients in the transplanted population
Clinical outcome in XM-ONE positive vs XM-ONE negative patients
- rejections
- creatinine
3. Correlation between lymphocyte crossmatches and XM-ONE
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

37. Patient Demographics (N=147 )
Gender
Male
59%
Female
41%
Recipient Race
Caucasians
123 (84%)
Afro-Americans
12 (8%)
Hispanics
2 (1%)
Other
10 (7%)
Donors LD
83% DD
17%
Sensitizing events
Pregnancy
47%
Blood transfusion
28%
Previous transplant
14%
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

38. XM-ONE® result correlates to rejections
Total
(n=29)
XM-ONE positive (n=35)
46%
(16/35)
XM-ONE negative (n=112)
12%
(13/112)
TOTAL
(n=147)
20%
(29/147)

39. XM-ONE® result correlates to rejections, PRA levels does not
PRA>10% with ARE (n=9)
PRA<10% with ARE (n=20)
Total
(n=29)
XM-ONE positive (n=35)
46%
(6/13)
45%
(10/22)
(16/35)
XM-ONE negative (n=112)
15%
(3/20)
11%
(10/92)
12%
(13/112)
TOTAL
(n=147)
27%
(9/33)
18%
(20/114)
20%
(29/147)

40. XM-ONE® assesses risk for acute rejections in kidney transplant recipients
PRA>10% with ARE (n=9)
PRA<10% with ARE (n=20)
Total
(n=29)
XM-ONE positive (n=35)
46%
(6/13)
45%
(10/22)
(16/35)
XM-ONE negative (n=112)
15%
(3/20)
11%
(10/92)
12%
(13/112)
TOTAL
(n=147)
27%
(9/33)
18%
(20/114)
20%
(29/147)
Total
(n=29)
XM-ONE positive (n=35)
46%*
(16/35)
XM-ONE negative (n=112)
12%
(13/112)
TOTAL
(n=147)
20%
(29/147)
*p<0.0005

41. XM-ONE® positive crossmatch correlates to acute rejections
This slide shows that XM-One positive patients had a significantly higher risk of rejections that XM-One negative patients, 46% vs 11%. This difference is even larger if you extract the patients with deceased donors, 66% vs 11%, however the number of patients in this group is very small.
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

42. XM-ONE® positive patients experienced rejections early after transplantation
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

43. All C4d positive rejections had non-HLA DSA as defined by XM-ONE positivity
XM-ONE® positive (35)
XM-ONE® negative
(112)
C4d positive at first rejection episode 6
(17%)
(0%)
C4d negative at first rejection episode 10
(29%) 13
(12%)
4 AB mediated rejections. Patients with pos C4d have shorter graft survival
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

44. Creatinine levels were significantly higher in XM-ONE® positive patients
1.77
mg/dL
1.73
mg/dL
1.43
mg/dL
1.39
mg/dL
Ändra siffror
p < 0,05
p < 0,05
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

45. Study Conclusions
XM-ONE® positive patients experience significantly more rejections than XM-ONE® negative patients
XM-ONE® positive patients experienced earlier and more severe rejections than XM-ONE® negative patients
XM-ONE® positive patients have higher creatinine values at 3 and 6 months after transplantation
4 AB mediated rejections. Patients with pos C4d have shorter graft survival
Ref: Breimer et al; Transplantation 87(4): , February 27, 2009.

46. Enhancing the risk assessment
Factors influencing graft survival
HLA as well as non-HLA antibodies are associated with impaired graft half-life
Patients experiencing acute rejections have shorter graft survival
S-creatinine is recognized as a surrogate marker for graft half-life
XM-ONE® identifies patients at risk*
Detects patients at increased risk of rejections and reduced kidney function
XM-ONE® provides valuable information on the expected immune respons on the transplanted organ
*Referenced to Breimer et al, Transplantation 2009

47. Karolinska Cases A boy transplanted in 1998
A girl transplanted in 1993 *
- Abrupt graft failure (x3) due to Hyperacute Rejection
A boy transplanted in 1998
- Abrupt graft failure (x2) due to Hyperacute Rejection
Endothelial Antibodies detected i both
patients (through UVEC)
Both patients have retrospectively been
confirmed as XM-ONE® positive against
the first donor
*Reference:
Sumitran-Karuppan S et al,
Transplant Immunology
1997;5:

48. A French case
Patient transplanted with a kidney from a living related donor
No HLA antibodies detected in crossmatches or solid phase screens
The kidney was lost a few days after transplantation
Pretransplant sera as well as rejection sera was sent to Karolinska for XM-ONE® tests
Pretransplant sera contained donor specific IgM antibodies. At the time of rejection the patient had class switched to IgG
Data were presented at the recent EFI meeting

49. A US Case
A patient with Negative PRA and Negative lymphocyte crossmatches experienced Graft loss due to antibody mediated rejection
The clinic contacted OSUMC that was participating in the XM-ONE® prospective study and XM-ONE tests were performed
The pretransplant sera was positive in XM-ONE tests against the donor but negative against several independent blood donors
Presented at the ATC meeting, Jon von Visger et al

50. ABOi Case Study Johns Hopkins University Hospital
Day 0
Non sensitized, AB0 incompatible KTx (AB to 0)
Anti-A titer: 256 to 8 on day of transplant
XM-ONE®, strongly positive
Immediate graft function
Day 3 post-tx
S-cr rose to 2.6 mg/dl, urine output fell
Torsion of kidney, repaired and regain of allograft function
Biopsy: g2, i0, t0, v0, ptc3,
C4d3, Suggested of AMR
No rise in anti-A titer
Day 6 post-tx,
2 x pp
XM-ONE® negative
Discharged with S-cr =1.3 mg/dl
Ref: A Jackson,
ATC Workshop,
May 1, 2010

51. Case, Karolinska Hospital, Stockholm, Sweden
22 -year-old female
FSGS since childhood and started hemodialysis in 1999
First KTx 2000;
the graft failed within hours
Negative CDC and FXM (the rejection was believed to be caused by non-HLA Abs)
2004:
transplantation was again considered, the mother was evaluated as donor
Blood group-incompatible (A1 RhD-positive and the donor A1B RhD-positive)
Anti-donor RBC titers: 1:16 for both IgM and IgG.
HLA:
HLA typed using PCR-SSP (no repeated HLA mismatches)
No anti-HLA class I or II Abs (FC-based FlowPRA, conventional T- and B-lymphocyte CDCs, T-lymphocyte FXM, all negative
Non HLA, XM-ONE®
IgM +, IgG -
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8
Immunosuppressive protocol for AB0i + IA

52. Disease Course
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8

53. Disease Course Decision on biopsy on XM-ONE result Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8

54. Post Tx Day 9 post-transplant:
S-cr from 72 to 91 μmol/L.
XM-ONE® switched to be positive for IgG (pre-Tx IgM +)
anti-A1B titers low (IgM 2, IgG 1).
Ultrasonography: slight increase in the R.I. index to 0.6–0.7
T-lymphocyte XM negative
Immunoadsorptions were re-initiated.
Day 10: Kidney biopsy showed acute vascular rejection (Banff type IIA-B) with a humoral component (C4d positive).
Day 14: S-Cr 349 μmol/L.
Following rejection treatment (0.5 g Solu-Medrol on three consecutive days) and repeated immunoadsorptions, the patient’s kidney function was normalized
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8

55. Summary Acute rejection occur in HLA negative patients
The multicenter study published in 2009 showed that, in these HLA negative patients, 24% are positive to non-HLA ab (as identified by XM-ONE®)
As shown by Breimer et al, XM-ONE® positivity is a risk factor for early rejection and subsequent impaired renal function
In patients being HLA negative in the standard assays Would you like to know more?

57. Immunosuppression from Karolinska Case (slide 51)
Rituxmab (375 mg per m2 bodysurface) 2 months pre-operative and on the day of operation;
Repeated (n = 5) protein A immunoadsorption
IvIg (25 g) after the last immunoadsorption
Conventional immunosuppressive regimen
Prograf, CellCept, Prednisolon regimen starting >1 week pre-operative
Ref: Holgerson, J
et at, Clin Transpl.
2006:535-8