Antibodies Against MICA Epitopes

HLA Epitopes in Clinical TransplantationPittsburgh, PA

June 15-16, 2011

Justin W. Mostecki, Ph.D.

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

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.

Alignment of Polymorphic Extracellular Positionsof LSA-MIC Antigens

Eplet PositionsEplet Surface Polymorphic

Position Domain Visibility Positions6 α1 + 6

14 α1 ++ 1424 α1 ± 2425 α1 ± 24 3626 α1 + 2636 α1 ± 3691 α2 + 91114 α2 ++ 114122 α2 + 122123 α2 + 122 125125 α2 ++ 125129 α2 ++ 129151 α2 ++ 151156 α2 ++ 156173 α2 ++ 173174 α2 ++ 173 175175 α2 + 175181 α2 + 181206 α3 ++ 206 210208 α3 ++ 208213 α3 ++ 213214 α3 ++ 213 215215 α3 + 215221 α3 + 221251 α3 ++ 251271 α3 ++ 271

213T251Q129M36C

206GW215S

174EG213I251R25AC25AY129V173E14G175G175S

214TT122V14W

123LE181R24A

25TC6P

271A151V114R156L156H221L125K91R125E208C251E156R208Y26V

38 Eplets 12 Monomorphic at MICB6R

26G91Q114G122L151M173K181T

206SR215T221V271P

Duquesnoy RJ et al. Hum Immunol 69(12):826, 2008

Not covered with LSA-MIC panel

Polymorphisms Shared By Groups of MICA Antigens

a(1-Epfreq) x Epfreqmix

Epfreqmix 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)

Supereplets CMGWS and AYVEA

YV

EC

MG

WS

Do MICA Positive Sera Demonstrate Patterns Consistent With Monomorphic Eplets?

AY

VE

CM

GW

S

• 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,*008typing

MF

IM

FI

Sample 1: MICA*005-Eluted Antibody Demonstrates Reactivity Pattern Consistent with 173K Monomorphic Eplet

Sample 1: Antibody Demonstrates 173K PatternA

YV

EC

MK

GW

S

• 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)

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

Sample 2: Antibody Demonstrates 206SR/215T Pattern of Reactivity

• No MICA antigens to resolve 206SR and 215T eplets• Fewer antigens with this Beta lot

CM

KG

WS

AY

VE

SR

T

Updated CMKGWS and AYVESRTA

YV

ES

RT

CM

KG

WS

Sample 3: AYVESRT

• MICA-reactive sera often contain mixtures of antibody that demonstratedifferent patterns of reactivity

Sample 3: No MICA*036 & *037 Reactivity With MICA*051-eluted Antibody

MICA*051 absorption & elution

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

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

CM

KG

WS

AY

VE

SR

T

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

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

MICA*036 absorption & elution MICA*051 absorption & elution

• MICA*007 absorbed and eluted antibody shows 125E pattern (data not shown)

CM

KG

WS

AY

VE

SR

TSample 4: MICA*051-eluted Antibody From MICA*007-Cleared Serum Demonstrates 25AY Reactivity Pattern

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

Acknowledgements

University of Pittsburg University of Texas SW Medical Center

Rene Duquesnoy, Ph.D. Yizhou Zou, M.D.Marilyn Marrari Peter Stastny, M.D.

Gen-Probe Transplant Diagnostics, Inc.

Ivan Balazs, Ph.D.Christine Heylen, Ph.D.Jayasree HariharanLioudmila ZaslavskaiaRobert Vorhaben

Clínic Barcelona

Guadalupe, Ercilla, Ph.D.