”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”

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

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)

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

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

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

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

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):1505-13) 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 2010 - Vol 90 (10):1079-1084) 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)

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

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

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

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

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

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

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

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

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”.

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

Objective with slide: HAR or very early rejections in Ktx

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

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

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

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

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

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

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

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?

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

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

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

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

Reading XM-ONE® by flow cytometry

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

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

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):549-556, February 27, 2009.

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):549-556, February 27, 2009.

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):549-556, February 27, 2009.

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)

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)

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

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):549-556, February 27, 2009.

XM-ONE® positive patients experienced rejections early after transplantation Ref: Breimer et al; Transplantation 87(4):549-556, February 27, 2009.

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):549-556, February 27, 2009.

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):549-556, February 27, 2009.

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):549-556, February 27, 2009.

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

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;5:321-327

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

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

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

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

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

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

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

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?

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