1. Acceptable mismatches based on structural epitopes on HLA molecules Toulouse, April 2, 2008

2. 2 Highly sensitized patients Highly sensitized patients: antibodies against the HLA antigens of more than 85% of the panel. Difficult to transplant because cross-match with most donors is positive. Obvious solution: HLA identical or compatible donor but only available for a small proportion of the patients. How to find a suitable donor for these patients?

3. 3 Approaches to enhance transplantation of highly sensitized patients Do not accept that the patient is sensitized and try to remove the antibodies by: * plasmapheresis * intravenous immunoglobulins (IVIg) * anti-CD20 antibodies Accept that the patient is sensitized and try to stimulate the allocation of cross-match negative donor kidneys to these patients.

4. 4 Eurotransplant Kidney Allocation System. Point system based on different parameters: HLA match Match prognostic index (extra points for sensitized patients) Waiting time Regional donor (cold ischemia time) Country balance.

5. Still a low chance for a higly sensitized patient to be transplanted. % patients transplanted Waiting time (months)

6. 6 Need for an acceptable mismatch program Policy in Eurotransplant is the registration of the non- acceptable HLA mismatches for sensitized patients to prevent selection of donors with HLA mismatches towards which the patient has preformed antibodies. Problem: it is impossible to determine all antibody specificities in highly sensitized patients

7. 7 Acceptable Mismatch Program Basis: definition of those HLA antigens toward which the patient did never form antibodies and use this knowledge for donor selection. Original method: look at HLA type of negative panel donors in screening and extensive antibody screening against a patient specific panel (donors with a single HLA-A or –B mismatch), taking advantage of a pool of 20,000 HLA typed blood donors.

8. 8 Antibody screening Serum of patient (HLA: A1, A2, B7, B8) is tested against a panel of HLA typed blood donors. positive negative PRA is 92%

9. 9 Antibody screening Serum of patient (HLA: A1, A2, B7, B8) is tested against a panel of HLA typed blood donors. positive negative PRA is 92% HLA type: A1, A24, B7, B8 acceptable mismatch is A24

10. 10 Non-inherited maternal HLA antigens are often acceptable mismatches. NIMA NIPA Antibodies 46 72 A.M 43 6 P< 0.001 Analysis of sera from highly sensitized patients

11. 11 Very difficult patients AM are difficult to determine for highly immunized patients with rare HLA phenotypes. For these patients no suitable blood donors are available to determine acceptable mismatches or cross-matches with the few available donors are positive. Main problem: most target cells express several mismatched HLA antigens.

12. 12 A1 A2 B7 B8 Cw3 Cw6 Difficult to identify acceptable mismatches Is HLA-A2 an acceptable mismatch?

13. 13 A2 SAL: Single antigen expressing cell line

14. 14 SALs validated and shown to be useful for determination of acceptable mismatches. HLA-A HLA-B HLA-C A*0101 B*0702 B*4403 Cw*0102 A*0201 B*1402 B*4501 Cw*0303 A*0301 B* 1501 B*4601 Cw*0304 A*1101 B*2705 B*4901 Cw*0401 A*2402 B*3501 B*5501 Cw*0602 A*2601 B*3801 Cw*0801 A*3101 B*3901 Cw*1202 A*3201 B*4001 Cw*1402 A*3303 B*4002 Cw*1502 A*6901 B*4402

15. 15 Alternative approach Commercial assays including the use of single antigen beads (Luminex) although the conformation of these molecules may be different than that of membrane bound HLA molecules. HLA-A1 HLA-A2 HLA-A3 HLA-B7

16. 16 Definition of acceptable mismatches is difficult and is often based on trial and error because our interpretation of the humoral immune response to HLA is too simple. HLA-A1 HLA-A2 anti-HLA-A2

17. 17 HLA-A2 HLA-B44HLA-B51HLA-B35 HLA-A68HLA-B27 Many polymorphic sites, some of them shared between HLA alleles.

18. 18 Polymorphic Residues on B51 Structural Immunogenicity of HLA-B51

19. 19 Polymorphic Residues on B51 Structural Immunogenicity of HLA-B51 This polymorphism should be considered in the context of self HLA epitopes of the antibody producer

20. 20 For A2,A68; B27,B44 Polymorphic Residues on B51 Structural Immunogenicity of HLA-B51

21. 21 For A2,A68; B27,B44 For A2,A68; B35,B44 Polymorphic Residues on B51 Structural Immunogenicity of HLA-B51

22. 22 For A2,A68; B27,B44 For A2,A24; B7,B8 For A2,A68; B35,B44 Polymorphic Residues on B51 Structural Immunogenicity of HLA-B51

23. 23 a computer algorithm developed by René Duquesnoy HLAMatchmaker is based on this principle a computer algorithm developed by René Duquesnoy Donor HLA-A,B mismatches are defined by triplets of amino acid residues (epitopes) on antibody triplets of amino acid residues (epitopes) on antibody accessible sites of HLA molecules accessible sites of HLA molecules HLA-A1

24. 24 HLA matching at the triplet level Donor m.m B18 Patient: B7 Immune system of the recipient recognizes: A single HLA mismatch or 11 triplet mismatches

25. 25 HLA matching at the triplet level Donor m.m B18 Patient: B7 B52 A33. Immune system of the recipient recognizes: No triplet mismatches!

26. 26 No foreign antibody epitopes on HLA-B18 mismatch for patient, with HLA type HLA-A33, B51, B7.  2M 11 22 33 22 11 peptide Top viewSide view

27. 27 The number of triplet mismatches predicts HLA antibody production after renal allograft rejection triplet mismatches % of patients with donor specific HLA antibodies Dankers et al. 2005

28. 28 Validation of HLA matchmaker for the identification of acceptable mismatches in highly sensitized patients. Mismatch HLA-A HLA-B tested AM tested AM zero-triplet 18 18 54 54 CDC cross-matches confirm theoretical approach

29. 29 Additional value of HLAMatchmaker Also HLA antigens with triplet differences can be identified as acceptable mismatches. This information can be used for identification of additional acceptable mismatches. Self-triplet mismatches on basis of own HLA type A1 A2 B7 B8

30. 30 Additional value of HLAMatchmaker Also HLA antigens with triplet differences can be identified as acceptable mismatches. This information can be used for identification of additional acceptable mismatches. More self-triplet mismatches on basis of combination of acceptable mismatches and own HLA type: Consequence: more acceptable mismatches can be found A1 A2 B7 B8 AM: A3 AM: B14

31. 31 Donor selection on basis of acceptable mismatches. Patient:A24, A31, B27, B51, DR4 (highly sensitized) AM:A25, A26, B44 Suitable donors: A25, A31; B27, B51; DR4 A26, A31; B27, B51; DR4 A24, A25; B27, B51; DR4 A24, A26; B27, B51; DR4 A24, A31; B44, B51; DR4 A24, A31; B27, B44; DR4 A25, A31; B44, B51; DR4 A26, A31; B44, B51; DR4 A25, A31; B27, B44; DR4 A26, A31; B27, B44; DR4 A24, A25; B44, B51; DR4 A24, A26; B44, B51; DR4 etc. If such donor becomes available within Eurotransplant: mandatory shipment of the kidney to this highly sensitized patient

32. 32 Conclusions: HLAMatchmaker is of benefit for the identification of acceptable mismatches for highly sensitized patients. Increased chance to be transplanted: Excellent graft survival: % patients transplanted

33. 33 Acknowledgements Marian Witvliet Ilias Doxiadis Arend Mulder Jon van Rood Guido Persijn Marlies Dankers René Duquesnoy and the Eurotransplant community.