1. Antibodies Against MICA Epitopes HLA Epitopes in Clinical Transplantation Pittsburgh, PA June 15-16, 2011 Justin W. Mostecki, Ph.D.

2. MICA Major histocompatibility complex (MHC) class I chain-related protein A MICA is a ligand for NKG2D 33 polymorphic amino acid positions in the extracellular domain (subset shown above) MICA antibodies are associated with graft failure Duquesnoy RJ et al. Hum Immunol 69(12):826, 2008

3. MICA Epitopes El-Awar N, Terasaki P, Cai J, Deng, C, Ozawa M, Nguyen A: Epitopes of the HLA-A, B, C, DR,DQ and MICA Antigens. Clin Transpl. 175, 2007 Duquesnoy RJ, Mostecki J, Hariharan J, Balazs I: Structurally based epitope analysis of major histocompatibility complex class I-related chain A (MICA) antibody specificity patterns. Hum Immunol 69(12):826, 2008 Zou Y, Qin Z, Silveus A, Fan Y, Stastny P: Polymorphisms of MICA recognized by human alloantibodies. Immunogenetics 61(2):91, 2009 Suarez-Alvarez B, Alonso-Arias R, Bravo-Mendoza C, Lopez-Vazquez A, Ortega T, Baltar JM, Coto E, Ortega F, Lopez-Larrea C: Identification of epitopes and immunodominant regions on the MICA protein defined by alloantibodies from kidney transplant patients. Transplantation 88(3 Suppl):S68, 2009.

4. Alignment of Polymorphic Extracellular Positions of LSA-MIC Antigens

5. Eplet Positions 38 Eplets12 Monomorphic at MICB Duquesnoy RJ et al. Hum Immunol 69(12):826, 2008 Not covered with LSA-MIC panel

6. Polymorphisms Shared By Groups of MICA Antigens a (1-Ep freq ) x Ep freqmix Ep freqmix represents eplet frequency in 2/3 EA &1/3 AA population mixture Duquesnoy RJ et al. Hum Immunol 69(12):826, 2008: Zou Y et al. Immunogenetics 61(2):91, 2009: MICA-G1: 36C, 173K, 206G, 210W and 215S (MICA*001, *002, *007, *012, *017, *018) MICA-G2: 206S, 210R and 215T (MICA*004, *006, *008, *009, *019) MICA*002 & MICA*008 hybrid recombinants, MICA*008 V129M & MICA*002 K173E site-directed mutants to map epitopes Monomorphic with MICB and predicted not to illicit MICA-specific allo- antibodies Demonstrated CMGWS and AYVE patterns of reactivity El-Awar N et al. Clin Transpl. 175, 2007: Antibody eluted from MICA*004 cell line reacted with MICA*004, *009, *027 (defined by either 36Y, 129V or 173E)

7. Supereplets CMGWS and AYVE AYVE CMGWS

8. Do MICA Positive Sera Demonstrate Patterns Consistent With Monomorphic Eplets? AYVE CMGWS

9. Sample reacts with CMGWS antigens, but not AYVE antigens Sample reactivity to MICA*036 consistent with 36C eplet pattern as determined by MICA*036 absorption & elution (data not shown) MICA*005 Absorption & elution MICA*008,*008 typing MFI Sample 1: MICA*005-Eluted Antibody Demonstrates Reactivity Pattern Consistent with 173K Monomorphic Eplet

10. Sample 1: Antibody Demonstrates 173K Pattern AYVE CMKGWS CMGWS supereplet re-classified as CMKGWS to include 173K eplet Sample reactivity to MICA*036 consistent with 36C eplet pattern as determined by MICA*036 absorption & elution (data not shown)

11. Sample 2: Shows Pattern Consistent with 206SR/215T Monomorphic Eplets

12. Sample 2: Antibody Demonstrates 206SR/215T Pattern of Reactivity No MICA antigens to resolve 206SR and 215T eplets Fewer antigens with this Beta lot CMKGWS AYVESRT

13. Updated CMKGWS and AYVESRT AYVESRT CMKGWS

14. Sample 3: AYVESRT MICA-reactive sera often contain mixtures of antibody that demonstrate different patterns of reactivity

15. Sample 3: No MICA*036 & *037 Reactivity With MICA*051-eluted Antibody MICA*051 absorption & elution

16. Sample 3: MICA*051-eluted Antibody Demonstrates 25AY Reactivity Pattern

17. Sample 3: MICA*051-eluted Antibody Shows Pattern Consistent With 25AY Eplet CMKGWS AYVESRT

18. Sample 4: AYVESRT With Additional Reactivity Positive antigens shown in red contain 125E MICA*001 shown in green is 125K 25AY-containing antigens ranked at top with manual eplet analysis Next steps: - Absorption & elution performed with MICA*007 - Absorption & elution performed on MICA*007- cleared serum with MICA*036 and MICA*051

19. Sample 4: MICA*051-eluted Antibody From MICA*007- Cleared Serum Demonstrates 25AY Reactivity Pattern MICA*036 absorption & elutionMICA*051 absorption & elution MICA*007 absorbed and eluted antibody shows 125E pattern (data not shown)

20. CMKGWS AYVESRT Sample 4: MICA*051-eluted Antibody From MICA*007- Cleared Serum Demonstrates 25AY Reactivity Pattern

21. Conclusions MICA-eluted alloantibody demonstrates 173K eplet pattern to update supereplet to CMKGWS (similar to MICA-G1, but with 129M) Identified samples that demonstrate 206SR/215T pattern to update supereplet to AYVESRT (similar to MICA-G2, but with 25AY, 129V and 173E) CMKGWS and AYVESRT often contain mixtures of antibody that demonstrate patterns consistent with a single eplet following absorption and elution MICA*005, *028, *036, *037, *042 and *051 are effective at resolving CMKGWS and AYVESRT patterns when samples react with a single eplet

22. Acknowledgements University of PittsburgUniversity of Texas SW Medical Center Rene Duquesnoy, Ph.D.Yizhou Zou, M.D. Marilyn MarrariPeter Stastny, M.D. Gen-Probe Transplant Diagnostics, Inc. Ivan Balazs, Ph.D. Christine Heylen, Ph.D. Jayasree Hariharan Lioudmila Zaslavskaia Robert Vorhaben Clínic Barcelona Guadalupe, Ercilla, Ph.D.