Conformational and linear in allergic disease

A Pomés, PhD

HESI Symposium Prague April 2012

INDOOR Biotechnologies, Inc. Charlottesville, VA, USA Conformational and linear epitopes in allergic disease

 Which IgE epitopes are we mapping? Linear versus conformational epitopes.

 Molecular basis of cross-reactivity: relevance for assessment of potential allergenicity.

Conformational and linear epitopes in allergic disease

 Which IgE antibody epitopes are we mapping? Linear versus conformational epitopes.

 Molecular basis of cross-reactivity: relevance for assessment of potential allergenicity.

Most IgE antibody mapping studies are based on the identification of linear epitopes by design Identification of linear immunodominant IgE epitopes in Ara h 2

- 15 amino acid long peptides - offset by 8 amino acids - entire molecule - probed with a serum pool (n=15)

Stanley et al. Arch. Biochem. Biophys 1997; 342:244-53 IgE linear patterns in Ara h 2 by microarray

20-mer 2-offset Ara h 2 peptides n = 45 patients

Shreffler WG JACI 2005;116:893-9 Peptides identified as major linear epitopes show little contribution to IgE binding to Ara h 2

Inhibition of mediator release from Inhibition of IgE-binding to rAra h 2 humanized RBL stimulated with rAra h 2

Albrecht et al. JACI 2009; 124:328-36 rAra h 2: a peanut conglutin

C-148 α5

α4 α1

α2

N-28

α3 59% identity with Ara h 6

Geoff et al. 2011;66:878-5

Structure of rAra h 2 construct with Maltose Binding Protein

Ara h 2

MBP

Geoff et al. Allergy 2011;66:878-5

Two populations of peanut allergic patients by epitope diversity

Geoff et al. Allergy 2011;66:878-5

Evidence of digestion-resistant fragments in Ara h 2

Sen et al. J Immunol 2002; 169:882-7 Digestion resistant fragments in Ara h 2

Koppelman et al. Mol Nutr Food Res, 2010; 54:1711-21; Sen et al. J Immunol 2002; 169:882-7; Lehmann et al. Biochem J 2006;395:463-72 Mapping conformational epitopes in Group 1 dust mite

Der p 1/ Der f 1 Der f 1: a cysteine protease

Der f 1 Der p 1

Chruszcz et al. J Mol Biol 2009; 386:520-30 IgE binding structures of Der p 1

Regions deduced to account for IgE reactivity

Greene and Thomas, Mol Immunol 1992; 29:257-62 Antigenic determinants in Der p 1 and Der f 1

80-90% human IgE is directed 4C1* against cross-reacting sites 6A8 88% (16/18) in Der p 1 and Der f 1 10B9*

Der p 1 Der f 1

Only ~3% (2/53) mAb * Partial inhibition of 5H8* were cross-reactive IgE ab binding (38%) 69% (24/35) Chapman et al. JI 1987; 139:1479-84 Antigenic structure of Group 1 mite allergens

• Solve the structure of mAb 4C1 fragment with Group 1 mite allergens by X-ray crystallography. Fab

Papain Fab Fc + 4C1 mAb -Fab

Allergen

• Site-directed mutagenesis analysis of protein interactions.

Heavy Chain

nDer p 1

Light Chain Mutagenesis analysis of Der p 1

4C1 mAb bound to Polyclonal anti-Dpt bound to Der p 1 presented by 5H8 mAb Der p 1 presented by 5H8 mAb

Chruszcz et al. J Biol Chem 2012; 287:7388-98 Inhibition of IgE antibody binding to proDer p 1 by epitope mutants

Sera pool (n=4)

10x

Chruszcz et al. J Biol Chem 2012; 287:7388-98 Common epitope in Der f 1 and Der p 1

nDer f 1 : 4C1 mAb Fab

nDer p 1 : 4C1 mAb Fab 1.90 Å resolution 1.95 Å resolution

Chruszcz et al. J Biol Chem 2012; 287:7388-98 G1 mite allergens: mAb 4C1 epitope? Cathepsin Der p 1 Der f 1 Eur m 1 Blo t 1 K 100% 83.2% 84.4% 38.5% 30.3% Glu 13   Asn Ser Arg 17     Arg 156   Gly Gly Ile 158    Val Ala 180 Thr Thr Gln Gln Tyr 185    His Asp 198    Asn Tyr 203   Phe Leu What is known about the structure of conformational epitopes in food allergens? Structures of allergen-antibody complexes

• IgG-Fab or Fab’: • Api m 2 Padavattan et al. J Mol Biol. 2007; 368:742-52. • Bet v 1 Mirza et al. J.Immunol. 2000; 165: 331-338. • Bla g 2 (x2) Li et al. J Immunol. 2011;186:333-40. Li et al. J Biol Chem. 2008;283:22806-14. • Der p 1 (x3) Chruszcz et al. J Biol Chem. 2012; 287:7388-98. • Der f 1 Chruszcz et al. J Biol Chem. 2012; 287:7388-98. • Gal d 4 (x4) Fischmann et al. J Biol Chem. 1991; 266:12915-20; Braden et al. J Mol Biol. 1994; 243:767-81; Vocadlo et al. Nature 2001; 412: 835-8; Cohen et al. Acta Crystallogr D Biol Crystallogr. 2005; 61:628-33. • IgE-Fab: • Bos d 5 Niemi et al. Structure. 2007; 15:1413-21. • Phl p 2 Padavattan et al. J Immunol. 2009; 182:2141-51.

Bla g 2 complexes with fragments of two mAb that inhibit IgE ab binding

Bla g 2

Fab’-7C11- mAb Fab-4C3 mAb Li et al. JBC 2008 Li et al. J Immunol 2011 N-terminal lobe C-terminal lobe

Glesner et al. PLoS One 2011; 6(7):e22223 Allergen-antibody interactions

mAb 7C11 mAb 4C3

Glesner et al. PLoS One 2011; 6(7):e22223 Allergen-antibody structures

Bos d 5 (β-Lactoglobulin)/ IgE-Fab Gal d 4 (Lysozyme)/ IgG-Fab

Niemi et al. Structure 2007; 15:1413–1421

Api m 2 (Hyaluronidase)/ IgG-Fab Bet v 1 (PR-10)/ IgG-Fab Conformational and linear epitopes in allergic disease

 Which IgE antibody epitopes are we mapping? Linear versus conformational epitopes.

 Molecular basis of cross-reactivity: relevance for assessment of potential allergenicity.

Primary structure is the basis of cross-reactivity…

• <50%: Rare cross-reactivity For proteins with similar structure (same “frame-work”), the molecular surface is different. • 50-70%: Moderate risk of cross-reactivity • >70%: High risk of cross-reactivity Similarity of exposed surfaces: higher probability of cross- reactivity.

Aalberse. JACI 2000;106:228-38 Structural basis for Group 2 allergenic cross-reactivity

Der p 2 Eur m 2

100% 83%

37% 40%

Lep d 2 Tyr p 2 Gly d 2 41% Smith AM et al. JACI 2001; 107:977-84 Lipocalins: low sequence identity Most mammalian allergens, Bla g 4 and Bos d 5 are lipocalins

Bos d 2 10.2% Can f 2 15.7% Equ c 1 16.8%

Fel d 4 15.5% Mus m 1 16.6% Rat n 1 18.7%

Pomés A et al. Protein Peptide Lett 2007; 14:960-9; Identity with Bla g 4 Lipocalins: low sequence identity Most mammalian allergens, Bla g 4 and Bos d 5 are lipocalins

Can f 1 26%

Inhibition IgE to Fel d 4 by extracts from: 46% Cow (26% Bos d 2) > Bos d 2 10.2% Can f 2 15.7% Equ c 1 16.8% Horse (67% Equ c 1 > Dog (21% Can f 1)

Fel d 4 15.5% Mus m 1 16.6% Rat n 1 18.7%

Pomés A et al. Protein Peptide Lett 2007; 14:960-9; Identity with Bla g 4 … and tertiary structure is also the basis of cross-reactivity

Conformational epitope in Group 1 mite allergens

nDer f 1 : 4C1 mAb Fab nDer p 1 : 4C1 mAb Fab

Chruszcz et al. J Biol Chem 2012; 287:7388-98 Guidelines for allergenicity assessment of GM crops 1996: International Food Biotechnology Council (IFBC) and International Life Sciences Institute (ILSI).

Decision tree: 8 aa match to a known allergen as risk of cross-reactivity; pepsin digestion; in vitro IgE binding; in vivo clinical testing; double-blind placebo-controlled food challenges.

2001: UN Food and Agriculture Organization (FAO)/ World Health Organization (WHO).

Decision tree: 6 aa match to a known allergen as risk of cross-reactivity; 35% identity over an 80-aa window; pepsin digestion; target serum screening; animal model screening.

2003: Codex Alimentarius Commission, FAO/WHO.

Weight-of-evidence approach: 35% identity over an 80-aa window; source; pepsin digestion; abundance; serum IgE tests.

Goodman RE et al. Nature Biotech 2008; 26:73-81 3D Structures of allergens in PDB

• Protein Data Bank: 79,521 Structures (Feb 21, 2012) • WHO/IUIS Allergen Database 715 allergens (www.allergen.org) • Inhaled Indoors 16 • Inhaled Outdoors 24 • Ingested Foods 24 • Injected Venoms 9 • Through Skin 3 • TOTAL 76 allergens (140 X-Ray; 32 NMR) 172 structures In complex with mAb 8 allergens (14 structures)

Solved food allergen structures: Only ~12 from 138 allergen families!

Bos d 4 Bos domesticus Alpha-lactalbumin Gal d 4 Gallus domesticus Lysozyme Tri a 28 Triticum aestivus Alpha-amylase inhibitor Bos d 5 Bos domesticus Beta-lactoglobulin Ara h 2 Arachis hypogaea 2S albumin, Conglutin Ara h 6 Arachis hypogaea Ric c 1 Ricinus communis Gly m 5 Glycine max 7S globulin; Beta-conglycinin, vicilin, cupin Ara h 1 Arachis hypogaea Ara h 3 Arachis hypogaea 11S globulin, glycinin, cupin Pru du 6 Prunus dulcis Mus a 5 Musa acuminata Glucanase Pru p 3 Prunus persica Non-specific lipid transfer protein Tri a 14-homolog Triticum aestivum Zea m 14 Zea mays Gal d 2 Gallus domesticus Ovalbumin Gal d 3 Gallus domesticus Ovotransferrin Api g 1 Apium graveolens Pathogenesis related protein (PR-10) Dau c 1 Daucus carota Gly m 4 Glycine max Pru av 1 Prunus avium Pru av 2 Prunus avium Thaumatin-like protein Structures of plant food allergens Prolamin superfamily Prolamins 2S albumins Ara h 2, Ara h 6, Ric c 1 NS-lipid transfer Pru p 3, Tri a 14-hom, Zea m 14 α-amylase/ protease inhibitors Tri a 28

Cupin superfamily 7/8S globulins Ara h 1, Gly m 5 11S globulins Ara h 3, Pru du 6, Gly m 6 Profilin Bet v 1 related proteins (PR-10) Api g 1, Dau c 1, Gly m 4, Pru av 1 Oleosins Endochitinases β-1,3-Glucanases Mus a 5 Thaumatin-like proteins Pru av 2

Structures of animal food allergens Tropomyosins EF-hand domains (parvalbumins) Caseins

Lipocalins (β-lactoglobulin) Bos d 5

C-type lysozyme/α-lactalbumin Gal d 4, Bos d 4 Kazal-type protease inhibitors

Serpin Gal d 2 (ovalbumin)

Transferrins Gal d 3

Conclusions I: Conformational and linear epitopes in allergic disease

• Most reported mapping studies are based by design on the identification of linear epitopes.

• Linear epitopes do not explain the full allergenicity of food allergens, and there is evidence that conformational epitopes play a very important role as well.

• The structural basis of cross-reactivity to conformational epitopes has been proven for Der p 1 and Der f 1 in complex with a mAb that overlaps with IgE ab binding sites.

Conclusions II: Conformational and linear epitopes in allergic disease

• The primary and tertiary structures of allergens are the basis of cross-reactivity.

• Molecular models of allergen structure alone and in complex with are useful for localizing epitopes, and understanding cross-reactivity and mechanisms of allergenicity.

• Although not requested by the actual guidelines of allergenicity assessment of GM crops, knowledge of IgE antibody binding epitopes can contribute to allergenicity prediction.

Acknowledgements INDOOR Biotechnologies, Inc., Charlottesville, VA, USA Jill Glesner Sabina Wünschmann Lisa Vailes Martin D. Chapman

National Cancer Institute, Frederick, MD, USA Macromolecular Crystallography Laboratory Mi Li Alla Gustchina Alex Wlodawer

University of Virginia, Charlottesville, VA, USA Molecular Physiology and Biological Physics Maksymilian Chruszcz Wladek Minor

National Institutes of Health, Research Triangle Park, NC Geoff Mueller Lars C Pedersen

Funded by the National Institute of Allergy and Infectious Diseases NIH Award RO1AI077653