Why CDRs are not what you think they are or How to identify the real antigen binding regions Kunik et al. (2012) Structural Consensus among Antibodies Defines the Antigen Binding Site. PLoS Comput Biol. 23;8(2) Kunik et al. (2012) Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Research. Vered Kunik and Yanay Ofran Bar-ilan University, Israel

Molecular recognition Why study antibodies? Therapy Diagnosis Vaccines autoimmunity Immunity Molecular recognition

Antibody structure Fc Fab Light chains Heavy chains Constant region Variable region (Fv) Constant region Ag Ag Fc Light chains Heavy chains 1IGT

Antibody-Antigen interface Complementarity Determining Regions – CDRs 2XQB paratope L1 L2 L3 H3 H2 H1 Constant region Variable region Ag

The most commonly used CDR identification methods Sequence based Sequence & Structure Structure based CDRs are identified by searching for the regions that are most different between antibodies Kabat Chothia IMGT 1970 1987 1989 Wu TT, Kabat EA (1970). J Exp Med 132: 211–250 Kabat et al. (1983) Bethesda: NIH. 323 p. Chothia C, Lesk AM (1987). J Mol Biol 196: 901–917. Chothia et al. (1989) Nature 342: 877–883. Lefranc MP et al. (2003) Dev Comp Immunol 27: 55–77.

Antigen binding residues How well do CDR identification methods capture antigen binding interface? Antigen binding residues CDRs (Kabat,Chothia ,IMGT) Recall & precision 67 Ab-Ag complexes Recall = # of antigen binding residues in CDRs/total # of antigen binding residues Precision = # of antigen binding residues in CDRs/total # of residues in CDRs

Miss 15 - 22% of antigen binding residues Performance of CDR identification methods Miss 15 - 22% of antigen binding residues

Paratome – An automatic structure based Antigen Binding Regions identification method Ofran et al. (2008) . The journal of immunology, 181: 6230–6235. Kunik et al (2012). PLoS Comp Biol. 23;8(2). Ab-Ag complexes Structural Alignment ...CRSS----K-S-LLH-S-N-GIT-YLYWYLQ... ...CKAS----Q-D-IN--------S-FLTWFLQ... ...CRAS----Q-D-IS--------N-YLNWYQQ... ...CRAS----S.S.VS----------YIHWFQQ... ...CRAS----E-N-IY--------S-YLAWYQQ...

Structurally aligned positions that systematically bind the antigen Paratome – An automatic structure based Antigen Binding Regions identification method ...CRSS----K-S-LLH-S-N-GIT-YLYWYLQ... ...CKAS----Q-D-IN--------S-FLTWFLQ... ...CRAS----Q-D-IS--------N-YLNWYQQ... ...CRAS----S.S.VS----------YIHWFQQ... ...CRAS----E-N-IY--------S-YLAWYQQ... Structurally aligned positions that systematically bind the antigen ...CRSS----K-S-LLH-S-N-GIT-YLYWYLQ... ...CRAS----S.S.VS----------YIHWFQQ... ...CKAS----Q-D-IN--------S-FLTWFLQ... ...CRAS----E-N-IY--------S-YLAWYQQ... ...CRAS----Q-D-IS--------N-YLNWYQQ... Antigen Binding Regions (ABRs)

How well do the structural consensus regions capture the antigen binding interface?

Where do the differences between CDRs and ABRs stem from? Antigen binding residues coverage Energetic contribution to antigen binding

Comparing ABRs and CDRs Paratome ...ISCRASESVDSYGKSFMHWYQQ...PPKVLIYIASNLESGVP...YYCQQNNEDPPTFG... ...ISCRASESVDSYGKSFMHWYQQ...PPKVLIYIASNLESGVP...YYCQQNNEDPPTFG... Kabat Paratome∩Kabat ΔParatome ΔKabat Kabat: Paratome∩Chothia ΔParatome ΔChothia Chothia: Paratome∩IMGT ΔParatome ΔIMGT IMGT:

Binding sites coverage # of residues in contact with Ag Antigen binding residues coverage Binding sites coverage # of residues in contact with Ag # of residues Residues set 83.5% 1664 3476 Paratome ∩ Kabat 10.77% 212 1018 ΔParatome 1.78% 35 695 ΔKabat 77.08% 1517 3202 Paratome ∩ Chothia 17.23% 339 1292 0.86% 17 348 ΔChothia 79.47% 1564 3077 Paratome ∩ IMGT 14.84% 292 1417 0.76% 15 218 ΔIMGT Structural consensus regions contain the vast majority of antigen binding residues

Energetic contribution to antigen binding ABRs∩CDRs Paratome unique CDRs unique In-silico alanine scan mutagenesis ΔΔG = ΔGmutated_structure - ΔGWT_structure stabilizing ΔΔG < - 0.25 neutral -0.25 ≤ ΔΔG ≤ 0.25 destabilizing ΔΔG > 0.25 important for binding!

not important for binding Energetic contribution to antigen binding - results not important for binding important for binding % Paratome-unique residues are important for binding

www.ofranlab.org/paratome Kunik et al. (2012) Paratome: an online tool for systematic identification of antigen-binding regions in antibodies based on sequence or structure. Nucleic Acids Research. 40(Web Server issue):W521-4.

Summary Identifying the antigen binding interface is an important task Paratome ABRs covers the vast majority of the antigen binding interface Residues identified only by Paratome are energetically important for antigen binding Paratome is available online and is capable of identifying the Antigen Binding Regions from both sequence and structure

Ofran lab, Bar-ilan university, Israel Acknowledgments Yanay Ofran, Bar-ilan University, Israel Bjoren Peters, La Jolla Institute for Allergy and Immunology, USA Shaul Ashkenazi – Lab programmer Ofran lab, Bar-ilan university, Israel

Thank You! poster z10