1. Management of sensitized patients in kidney transplantation

2. Introduction  Between 5-10% of patients waiting for a renal transplant are classified as highly sensitised (Panel Reactive Activity ≥85% IgG)  Highly sensitised renal dialysis patients wait longer for a suitable crossmatch negative donor compared to non-sensitised patients  Identification of acceptable HLA mismatches increases the likelihood of transplantation

3. Evolution of HLA Antibody Detection Cytotoxicity Enhanced Cytotoxicity Flow Cytometry Ly C1 Dye Membrane Attack Complex Ly Anti-HLA Antibody Ly C1 Ly Membrane Attack Complex Dye Anti-Human Globulin Flow Cytometer Ly CD19 (B cell) CD3 (T cell) or Fluorescenated Anti-Human Globulin Bray et al Immunol Res. 29:41, 2004

4. METHODS FOR ANTIBODY EVALUATION Complement-dependent Cytotoxicity (CDC): - Direct CDC (Standard CDC) - Modifications Washes Extended Incubation Anti-human globulin (AHG-CDC) DTT / DTE Flow Cytometry (cells): - T cell / B cell - Pronase Complement-dependent Cytotoxicity (CDC): - Direct CDC (Standard CDC) - Modifications Washes Extended Incubation Anti-human globulin (AHG-CDC) DTT / DTE Flow Cytometry (cells): - T cell / B cell - Pronase ELISA - Yes / No - PRA % (I & II) - Specificity (I & II) “FlowPRA” Flow cytometry using microparticles (“beads”) - PRA % (I and II ) - Specificity (I & II) Multi-plex - Suspension Arrays - Protein Chips ELISA - Yes / No - PRA % (I & II) - Specificity (I & II) “FlowPRA” Flow cytometry using microparticles (“beads”) - PRA % (I and II ) - Specificity (I & II) Multi-plex - Suspension Arrays - Protein Chips Antigen Non-Specific Antigen Specific

5. Sources of HLA Sensitization Blood transfusion Blood transfusion Prior transplantation Prior transplantation Pregnancy Pregnancy Others? Among patients receiving their first kidney transplant with no known history of blood transfusions, approximately 20% of nulliparous women and 13% of men were sensitized (PRA>10%). ?unreported or unrecognized pregnancies or transfusions, ?antigenic stimulation of sperm. ?cross-reacting environmental allergens Others? Among patients receiving their first kidney transplant with no known history of blood transfusions, approximately 20% of nulliparous women and 13% of men were sensitized (PRA>10%). ?unreported or unrecognized pregnancies or transfusions, ?antigenic stimulation of sperm. ?cross-reacting environmental allergens

6. Prevalence of Sensitization UNOS: 30% of patients on the transplant wait list have a PRA of > 20% UNOS: 30% of patients on the transplant wait list have a PRA of > 20% New England Organ Bank: 56% with PRA>50%, 28% with PRA 90-100%. New England Organ Bank: 56% with PRA>50%, 28% with PRA 90-100%. Accumulation phenomenon: the mean waiting time for prospective recipients with PRA > 50% is 26.2 months, five times longer than that for unsensitized recipients with a PRA below 10 percent. Accumulation phenomenon: the mean waiting time for prospective recipients with PRA > 50% is 26.2 months, five times longer than that for unsensitized recipients with a PRA below 10 percent.

7. Why Believe Desensitization Should Succeed? Two cases for cadaver transplant Two cases for cadaver transplant -Two recipients with known marked sensitization presented for transplants; two samples for cross matching drawn approximately one week apart; first positive AHG cross match, second negative -Transplants done successfully with 10 day T cell depletion with OKT3 Hypothesis: with newer anti-T cell depleting agents, perhaps Ab depletion will work since only need to be cross match negative currently, even for just one day Hypothesis: with newer anti-T cell depleting agents, perhaps Ab depletion will work since only need to be cross match negative currently, even for just one day

8. Pros and Cons of PP/IVIG Advantages Advantages High efficacy High efficacy Somewhat predictable response Somewhat predictable response Monitoring DSA possible Monitoring DSA possible Double-incompatibility possible Double-incompatibility possible Disadvantages Disadvantages Expensive Labor intensive Only for LD Rebound possible Adverse effects

9. University of Maryland Protocol Overview Tx 0123-2-3-4-5-6-7-8-9-10 PP/Ig Anti-CD20 FK 506 MPA Steroids Thymoglobulin PP/Ig Time in days DSA Testing 4

10. UMM Protocol Immunosuppression MMF 2 g/d, started 3 days prior to 1 st PP MMF 2 g/d, started 3 days prior to 1 st PP FK506 started on 1 st day of PP, target level 15 ng/ml FK506 started on 1 st day of PP, target level 15 ng/ml Prednisone 0.25 mg/kg BID with FK506 Prednisone 0.25 mg/kg BID with FK506 Induction with OKT3, 5 mg/d x 10 days with target CD3 <5% Induction with OKT3, 5 mg/d x 10 days with target CD3 <5% Transplantation 2000; 70:1531

12. Mayo Clinic Experience-2006 AJT 2006, 6:346 p < 0.05 IVIG vs. PP groups

13. Present scenario  Demand for donor kidneys continues to outpace supply  A +ve crossmatch (+CMX) indicates presence of DSA in the recipient  Associated with graft-loss in excess of 80%

14. Protocols to overcome sensitisation & blood group incompatibilities  2 regimes: 1) low-dose IVIG with PP – used in live donor ABO-incompatible and +CMX Tx 2) High-dose IVIG – in live +CMX Tx for highly sensitised pts. on deceased donor list

15. Desensitisation for ABO- incompatible Tx  Success after initial failures attributed to splenectomy in addition to PP, azathioprine, ALG and steroids  Ref: Transplant Proc 1987;19: 4538-42 Transplant Proc 1985;17: 138-43 Transplant Proc 1985;17: 138-43 Transplantation 1998;65: 224-8 Transplantation 1998;65: 224-8

16. Medical splenectomy  Anti-CD 20 monoclonal antibody now allows splenectomy-free protocols  John Hopkins standard PP/IVIG desensitisation protocol plus single-dose anti-CD 20 monoclonal antibody on the final pre-transplant day  Ref: Am J Transplant 2004; 4: 1315-22 Transplantation 2003; 76: 730-1 Transplantation 2003; 76: 730-1

17. New more powerful maintenance immunosuppression  Therapies with B-cell anti-proliferatives e.g. MMF. Excellent results now possible without B-cell ablative therapies after ABOi transplantations Ref: Am J Transplant 2004; 4: 561-8  At Cedars-Sinai – employs Rituximab 1 g for 1 week prior to PP, PP every other day 5 times followed by high-dose IVIG

18. Immunomodulation with IVIG: Desensitization of highly HLA-sensitized pts.  Results in reduced allosensitisation  Reduced ischemia-reperfusion injuries  Fewer acute rejection episodes  Higher successful longterm outcomes  Reductions in anti-HLA antibodies  Effective in treatment of allograft rejections  Ref: J Am Soc Nephrol 2004;15:3256-62

19. NIH – IGO 2 Study  Multi-center, controlled, double blind  IVIG vs placebo in highly sensitized pts.  IVIG was superior to placeboin reducing anti-HLA antibody levels (p = 0.004) and improving rates of transplantation without concomittantlyincreasing the risk of graft loss  Ref: J Am Soc Nephrol 2004;15:3256-62

20. Low dose IVIG (100 mg/kg) and PP  Alternative to high-dose IVIG (2 g/kg)  Limited to live donor tranplantation  PP removes circulatingDSA while IVIG inhibits the function of residual DSA and limits the production of alloantibody  Starting immunosuppression with tacrolimus and MMF. Induction on day of transplant with daclizumab and steroid bolus  Ref : Transplantation 2000;70: 887-95

21. The Mayo Clinic protocol  Single pre-transplant dose of anti-CD20  Inclusion of splenectomy  Use of rabbit anti-human T-cell polyclonal antibody (Thymoglobulin) instead of daclizumab  Suggest more aggressive induction with T-cell depleting agents provides better control of T-cell alloreactivity  Ref: Am J Transplant 2003; 3: 1017-23

22. IVIG +/- Rituximab for desensitization  For those who do not respond to IVIG alone or  Who have high-titer anti-HLA antibodies  IVIG (2 g/kg) followed by 2 weeklydoses of Rituximab (1 g). Another IVIG (2 g/kg) dose given I week after final Rituximab dose  Reduces desensitization time from 16 to 4-5 weeks – reduced costs + improved outcomes  Ref: N Engl J Med 2008; 359: 242-51

23. Complication of IVIG therapy  Highly sensitisedexcipient content patients require higher doses of IVIG (1-2 g/kg/dose)  Higher rates of infusion related complications  Adverse events could be related to differences in various IVIG products  Some common effects: ARF with sucrose- containing, thrombotic episodes with hyperosmotic and hemolysis with isoosmolar products  Ref: Clin J Am Soc Nephrol 2006; 1: 844

24. Adjunctive therapy  Splenectomy ABOi and +CMX renal transplantation is associated with a higher rate of AMR Accompanied by sudden onset of oliguria or anuria These grafts – successfully rescued by immediate slenectomy and reinitiation of PP/IVIG Ref: Transplantation 2007; 83: 99 -100

25. Conclusions  High-dose IVIG/rituximab and PP/low- dose IVIG desensitization in highly sensitized pts. are safe and viable alternatives to prolonged periods of dialysis while awaiting compatible donor organ  IVIG is critical to all the protocols

26. Conclusions – contd. Desensitization protocols in living donor transplantation -Accelerate transplantation relative to deceased donor wait listing -Results approximately equal to ECD DD transplants at 5 years -Unpredictable rejection most common complication -Infectious complications remarkably rare -Plasmapheresis carries risk in patients with cardiovascular disease

27. Conclusions – contd. High Risk Scenarios Donor specific antibody (DSA) as consequence of renal rejection – (versus transfusion) particularly early AMR Donor specific antibody (DSA) as consequence of renal rejection – (versus transfusion) particularly early AMR Repeat HLA-Ag mismatch in setting of DSA to repeat mismatch Repeat HLA-Ag mismatch in setting of DSA to repeat mismatch Prior early AMR Prior early AMR

28. Future Studies Clearer epidemiologic data on non-pathologic – “beneficial” re-appearance of DSA Clearer epidemiologic data on non-pathologic – “beneficial” re-appearance of DSA -Is all DSA harmful but with varying pace of pathologic injury? -Is accommodation present in humans? Prospective, multi-center trial with protocol biopsy to 5-8 years could answer question regarding pathogenic versus beneficial effect of DSA re-appearance Prospective, multi-center trial with protocol biopsy to 5-8 years could answer question regarding pathogenic versus beneficial effect of DSA re-appearance

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