1. Why CDRs are not what you think they areor
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

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

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

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

5. 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.

6. 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

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

8. 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...

9. 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)

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

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

12. 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:

13. 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

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

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

16.
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.

17. 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

18. 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

19. Thank You!
poster z10