AbSorber  Rejection & Graft Survival  XM-ONE ®

A Challenge in Organ Transplantation Optimizing Patient & Graft Survival

Rejection and Graft Survival  Acute rejection has become less likely to be reversed fully, and many patients develop irreversible damage after an episode of rejection *  The consequences of ARE are depending of **  Timing of ARE  Type of ARE  Severity of ARE  S-Creatinine reduction after ARE  Number of ARE’s References: *Flavio Vincenti, Clin J Am Soc Nephrol 1: 399-, 2006 ** Gerhard Opelz, Transplantation 2008;85: 661–666

Acute Rejection predicts impaired half- life Reference: Hariharan, S et al: N Engl J Med; Vol342, No 9, 2000

Pretransplant antibodies influence graft failure Reference: Gerhard Opelz for the Collaborative Transplant Study* Lancet 2005; 365: 1570–76

Degree of Immunization in HLA-identical siblings The PRA activity in HLA identical siblings is associated with poorer graft survival These findings further supports the role of non-HLA antibodies Reference: Gerhard Opelz for the Collaborative Transplant Study* Lancet 2005; 365: 1570–76

CTS Conclusions  Significant association between pretransplant HLA antibodies and outcome of kidney grafts from diseased donors.  In the study of HLA identical siblings a similar association was seen. ”When the long-term results for kidney recipients with PRA were examined over 10 years of follow- up, the influence of non-HLA-directed immunity was of similar magnitude to that of antibodies against HLA” Reference: Gerhard Opelz for the Collaborative Transplant Study* Lancet 2005; 365: 1570–76

Non-HLA antibodies associated with graft rejections 1. Sumitran-Karuppan S et al, Transpl Immunol 1997, 5: Carter V, et al: Transplant Proc 2005, 37: Jurcevic Set al Transplantation 2001, 71: Joosten SA, et al. Am J Transplant 2005, 5: Dragun D, et al; N Engl J Med 2005, 352: Lucq J, et al;Xenotransplantation 2000, 7:3. 7. Zwirner NW, et al; Human Immunol 2000, 61: Stastny P: Hum Immunol 2006, 67: Sumitran-Holgersson S et al.Transplantation 2002, 74: Jager MJ, et al; Hum Immunol 1987, 19: Reference: Suchitra Sumitran- Holgersson Current Opinion in Immunology 2008, 20:607–613 SPECIFICITIES OF NON-HLA ANTIBODIES WITH RELEVANCE IN ORGAN TRANSPLANTATION SPECIFICITYTYPE OF REJECTIONREFERENCES EC reactive abs. against vimentin, desmin-Heart txAR2, 3 EC specifi c abs. against a 100 kD antigen Kidney txHAR, AR1 EM specifi c antibodies (HMA-1 and HMA-2) Kidney txHAR, AR10 EC reactive abs. against MICA Kidney txEGL, AR7 – 9 Tissue-specifi c abs. Agrin Kidney txCR4 Abs. to Angiotensin II type-1 Receptor Kidney txVR5 Abs. to α-Gal Xeno txHAR6 AR: Acute rejections; HAR: hyperacute rejections; EGL: early graft loss; CR: chronic rejection; VR: vascular rejection.

Non-HLA Antibody Detection  There is a need for commercially available methods for the reliable and reproducible detection of non-HLA antibodies. ”As it becomes increasingly recognized that non-HLA antibodies contribute significantly to antibody- mediated processes, it will be important to find reproducible assays for these antibodies such that their prevalence and impact may be quantified better.” Reference: Kathryn J. Tinckam, and Anil Chandraker Clin J Am Soc Nephrol 1: , 2006

And so it begun …… (at Huddinge Hospital)  A girl transplanted in 1993 * - Abrupt graft failure (x3) due to Hyperacute Rejection  A boy transplanted in Abrupt graft failure (x2) due to Hyperacute Rejection  Endothelial Antibodies detected i both patients (through UVEC) *Reference: Sumitran-Karuppan S et al, Transplant Immunology 1997;5:

XM-ONE ® An Endothelial Crossmatch  Introduction & Background  Multicenter Study Data

XM-ONE® : Detects antibodies against cell bound antigens Peripheral blood endothelial cell precursor HLA I HLA II non-HLA Tie-2-r

Endothelial precursor cell Magnetic bead coated with anti- Tie-2 monoclonal antibody

The XM-ONE® kit – Product code contains the components to perform 4 tests:

The XM-ONE © testing procedure is similar to that of lymphocyte flow cutometry cross match tests. Reference: XM-ONE, package insert

Reading XM-ONE ® by flow cytometry R1 Gate on endothelial precursor cells Reference: XM-ONE, package insert

XM-ONE® prospective multicenter study Multicenter evaluation of a novel endothelial cell crossmatch test in kidney transplantation (Reference: Breimer et al; Transplantation;87(4): ,February 27,2009.) Michael E. Breimer, Lennart Rydberg, Annette M. Jackson, Donna P. Lucas, Andrea A. Zachary, Joseph K. Melancon, Jon Von Visger, Ronald Pelletier, Susan L. Saidman, Winfred W. Williams, Jr., Jan Holgersson, Gunnar Tydén, Göran K. Klintmalm, Sonnya Coultrup, Suchitra Sumitran-Holgersson and Per Grufman

Study Design - multicenter clinical study with XM-ONE® Lymphocyte crossmatch (LXM) Endothelial Cell crossmatch (XM-ONE) Clinical follow-up Recruitment Patient Information Study Design:  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 Reference: Breimer et al; Transplantation. 87(4): , February 27, n=147 > 3 month / rejection

Baylor University Med Center Dallas, TX, USA Göran B. Klintmalm Sonnya Coultrup Johns Hopkins University Baltimore, MD, USA Donna Lucas Andrea A. Zachary Annette Jackson J.K. Melancon Karolinska Institute, Huddinge Hospital, Stockholm, Sweden Jan Holgersson Gunnar Tyden Suchitra Sumitran-Holgersson Ohio State University Med Center Columbus, OH, USA Ron M. Pelletier Jon von Visger Massachusetts General Hospital Boston, MA, USA Winfred W. Williams Jr. Susan L. Saidman Sahlgrenska University Hospital Gothenburg, Sweden Michael E. Breimer Lennart Rydberg AbSorber Per Grufman Study Centres and Investigators Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

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

XM-ONE® positive crossmatch is associated with an increased rate of rejections Patients n=147 Rejections 19,7% (29 / 147) XM-ONE® positive 46% (16 / 35) p <0,0005 XM-ONE® negative 12% (13 / 112) Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

All C4d positive rejections occured in XM-ONE ® positive patients Reference: Breimer et al; Transplantation. 87(4): , February 27, Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

Creatinine levels were significantly higher in XM-ONE ® positive patients p < 0,05 Reference: Breimer et al; Transplantation. 87(4): , February 27, Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

XM-ONE® positive patients experience rejections also in the absence of donor specific HLA antibodies Rejection Frequency XM-ONE® PositiveNegative Lymphocyte Flow Cytometry Crossmatch* Positive 4 / 7 (57%) 0 / 7 (0%) Negative 10 / 24 (42%) 11 / 75 (15%) * Not all patients were tested with Lymphocyte Flow Cytometry Crossmatch Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

 XM-ONE® positive patients experience significantly more rejections then XM-ONE® negative patients  XM-ONE® positive patients experienced earlier and more severe rejections then XM-ONE® negative patients  XM-ONE® positive patients have higher creatinine values at 3 and 6 months after transplantation  XM-ONE® positive patients experience rejection episodes also in spite of negative Lymphocyte crossmatch Conclusions Reference: Breimer et al; Transplantation. 87(4): , February 27, 2009.

Oxford Study Preliminary data from recipients of living donor renal transplants (n=22). HLA and MICA antibody screening: Luminex Standard PBL crossmatches: complement dependent cytotoxicity (CDC) and flow cytometry (FC). XM-ONE® EPC crossmatch: FC using patient serum and cells isolated with XM-ONE® Reference: J Procter, M Ray, J Agudelo, A Muthusamy, PJ Friend, KJ Wood, SV Fuggle EFI

Results Allogeneic PBL CDC and FC Crossmatch: All patients negative Allogeneic XM-ONE® Crossmatch: 6/22 (27%) patients IgM positive 3/22 (14%) patients IgG positive All IgG+ patients included in IgM+ group Reference: J Procter, M Ray, J Agudelo, A Muthusamy, PJ Friend, KJ Wood, SV Fuggle EFI

Post transplant creatinine levels Significant difference in the change in serum creatinine levels (µmol/L) between 1-3 and 1-6 months post transplant between groups Creatinine levels improved in the XM-ONE negative, but not in the XM-ONE positive patients Reference: J Procter, M Ray, J Agudelo, A Muthusamy, PJ Friend, KJ Wood, SV Fuggle EFI 2009

Conclusion In 22 LD recipients negative in CDC and FC crossmatch 6 patients (27%) were found positive with the XM-ONE test No difference was seen regarding incidence of acute rejections (1 / 6 XM-ONE positive patient, 1 / 16 XM-ONE negative patient) S-creatinine was significantly increased in the XM-ONE positive patients both at three (p<0,02) and six months (p<0,04) post transplant compared to the one month value The results from the Oxford XM-ONE study gives further support to the multicenter study published in Transplantation Feb 27, 2009, by Breimer and co-workers Reference: J Procter, M Ray, J Agudelo, A Muthusamy, PJ Friend, KJ Wood, SV Fuggle EFI

Conclusion (I)  HLA antibodies are associated to rejections  However, rejections are still present in ”HLA-alike” and HLA –identical patients  There are strong evidence that non-HLA antibodies contributes to the occurence rejections

Conclusion (II)  XM-ONE® identifies patients that have an increased risk to develop rejections early after transplantation  XM-ONE® provides the transplant team with valuable information on the patients response to the transplanted organ before it occurs

The XM-ONE® kit – Product code contains the components to perform 4 tests: