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Antibody humanization and engineering:
what do we learn from IMGT® standardization
http://www.imgt.org
Marie-Paule LefrancIMGT Founder and Director
Professor University Montpellier 2, CNRS, Montpellier, France
5th Annual European Antibody Congress 2009Geneva, Switzerland, 30 November - 2 December 2009
http://www.imgt.org
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http://www.imgt.orgcreated in 1989
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IMGT standards based on IMGT-ONTOLOGY
http://www.imgt.org
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IMGT-ONTOLOGY seven axioms:
To share, reuse and represent knowledgein Immunogenetics and Life Sciences
IMGT-ONTOLOGY
CLASSIFICATION
NUMEROTATION
DESCRIPTION
ORIENTATION
LOCALIZATION
Giudicelli and Lefranc, Bioinformatics (1999)
IDENTIFICATION OBTENTION
http://www.imgt.org
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CLASSIFICATION axiom
group
subgroup
allele
locus
is a member of an instance of
is a member of an instance of
is a variant of an instance of
is ordered in an instance of
IGLV
IGLV2
IGLV2-11
IGLV2-11*02
human IGL(22q11.2)
is ordered in
is a member of
is a variant of
is a member of
gene
« Concepts » « Instances »
http://www.imgt.org
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Concepts of CLASSIFICATION
1. The IMGT-ONTOLOGY main concepts of classification• include ‘group’, ‘subgroup’, ‘gene’, ‘allele’.• have allowed to set up the nomenclature of the
immunoglobulin (IG) and T cell receptor (TR) genes(V, D, J, C genes).
2. IMGT gene names have been approved by the HUGO Nomenclature Committee (HGNC) in 1999.
3. New alleles are validated by the WHO-IUIS/IMGTnomenclature committee and entered in IMGT/GENE-DB.
4. IMGT/GENE-DB is the international reference database for IG genes (direct links from NCBI Entrez Gene) and alleles.
http://www.imgt.org
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Concepts of CLASSIFICATION
1. The IMGT-ONTOLOGY main concepts of classification• include ‘group’, ‘subgroup’, ‘gene’, ‘allele’.• have allowed to set up the nomenclature of the
immunoglobulin (IG) and T cell receptor (TR) genes(V, D, J, C genes).
2. IMGT gene names have been approved by the HUGO Nomenclature Committee (HGNC) in 1999.
3. New alleles are validated by the WHO-IUIS/IMGTnomenclature committee and entered in IMGT/GENE-DB.
4. IMGT/GENE-DB is the international reference database for IG genes (direct links from NCBI Entrez Gene) and alleles.
http://www.imgt.org
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FR1-IMGT
V-GENEV-EXON
FR2-IMGT FR3-IMGT
L-PART1
V-REGION
CC5 ’UTR 3 ’UTR
CD
R3
-IMG
T
DONOR-SPLICE
W
V-GENE V-EXON
FR3-IMGT CDR3-IMGT
L-PART1 DONOR-SPLICE
V-REGION FR1-IMGT
Label 1 Label 2
V-REGION CDR3-IMGT
Relations entre Labels
DESCRIPTION axiom
PROTOTYPE for a V-GENE http://www.imgt.org
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Concepts of DESCRIPTION
1. The IMGT-ONTOLOGY concepts of description:• comprise the standardized IMGT labels and their relations.• have allowed to describe the IG (or antibody) and TR
sequences and structures, whatever the receptor type,the chain type or the species.
2. IMGT labels are used in all IMGT® databases and tools for the description of:
• nucleotide and amino acid sequences (IMGT/LIGM-DB…)• 2D and 3D structures (IMGT/3Dstructure-DB…).
3. Sequence Ontology (SO) includes IMGT labels.
4. IMGT® databases can be queried using labels (a big ‘plus’compared to generalist databases).
http://www.imgt.org
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Concepts of DESCRIPTION
1. The IMGT-ONTOLOGY concepts of description:• comprise the standardized IMGT labels and their relations.• have allowed to describe the IG (or antibody) and TR
sequences and structures, whatever the receptor type, the chain type or the species.
2. IMGT labels are used in all IMGT® databases and tools for the description of:
• nucleotide and amino acid sequences (IMGT/LIGM-DB…)• 2D and 3D structures (IMGT/3Dstructure-DB…).
3. Sequence Ontology (SO) includes IMGT labels.
4. IMGT® databases can be queried using labels (a big ‘plus’compared to generalist databases).
http://www.imgt.org
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DESCRIPTION
IMGT/LIGM-DB
IMGT-ONTOLOGY:277 IMGT labels for sequences
285 IMGT labels for 3D structures
139 446 sequences from 238 species
http://www.imgt.org
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NUMEROTATION axiom
Lefranc et al. Dev. Comp. Immunol. 27, 55-77 (2003)
CDR-IMGT lengths[8.10.12]
http://www.imgt.org
IMGT Collier de Perles
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NUMEROTATION axiom
Lefranc et al. Dev. Comp. Immunol. 27, 55-77 (2003)
CDR-IMGT lengths[8.10.12]
Based on the IMGT unique numbering
- conserved AA (and codons) are always at the same positions:
23 1st-CYS41 CONSERVED-TRP89 hydrophobic
104 2nd-CYS118 J-PHE, J-TRP
- delimitation of the FR-IMGT and CDR-IMGT is standardized
- CDR-IMGT lengths are crucialinformation
http://www.imgt.org
IMGT Collier de Perles
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Concepts of NUMEROTATION
1. The IMGT-ONTOLOGY concepts of numerotation include:• IMGT unique numbering• IMGT Collier de Perles.
2. The concepts bridge the gap between sequences and 3D structures, at the amino acid (codon) level, for:
• the variable domains (V-DOMAIN) • the constant domains (C-DOMAIN).
4. The concepts are used for:• Mutations, polymorphisms• CDR-IMGT lengths• contact analysis, paratope definition.
5. WHO-INN programme requires the CDR-IMGT lengths for antibody.
http://www.imgt.org
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Concepts of NUMEROTATION
1. The IMGT-ONTOLOGY concepts of numerotation include:• IMGT unique numbering• IMGT Collier de Perles.
2. The concepts bridge the gap between sequences and 3D structures, at the amino acid (codon) level, for:
• the variable domains (V-DOMAIN) • the constant domains (C-DOMAIN).
4. The concepts are used for:• Mutations, polymorphisms• CDR-IMGT lengths• contact analysis, paratope definition.
5. WHO-INN programme requires the CDR-IMGT lengths for antibody.
http://www.imgt.org
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Outline
• Standardized analysis of V-DOMAIN- CDR-IMGT and FR-IMGT delimitations- IMGT Collier de Perles- CDR3-IMGT (V-J and V-D-J junctions) - IMGT/JunctionAnalysis
• Towards «Potential immunogenicity evaluation»- The 11 IMGT physicochemical classes- IMGT/DomainGapAlign
• Standardized analysis of antibody/antigen contacts- IMGT/3Dstructure-DB
• Bridging the gap between sequences and 3D structures and vice versa
- IMGT/2Dstructure-DB cards (INN)
http://www.imgt.org
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View from above the CDR-IMGT Side view of the V-DOMAIN
V-J junctionV-D-J junction
V-DOMAIN: VH and V-KAPPA
CDR3-IMGT= Complementarity determining region (105-117)V-D-J junction (104-118), V-J junction (104-118)
V-KAPPAVH V-KAPPAVHhttp://www.imgt.org
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CAMPATH-1H
Alemtuzumab
humanrat
IMGT Biotechnology page (http://www.imgt.org)
VH domain[8.10.12]
2 mutations:S31>T,S28>F
T
http://www.imgt.org
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IMGT/JunctionAnalysis: analysis of the IG and TR junctions
Yousfi Monod et al. Bioinformatics 20, i379-i385 (2004)Pommié et al. J. Mol Recognit. 17, 17-32 (2004)
http://www.imgt.org
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The 11 IMGT physicochemical AA classes
Pommié et al. J. Mol Recognit. 17, 17-32, 2004
http://www.imgt.org
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Magdelaine-Beuzelin C. et al. Crit. Rev. Oncol. Hemat. 64, 210-225 (2007)
Towards «Potential immunogenicity evaluation»
• Comparison with the closest human germline genes • Number of different AA in FR-IMGT
VH alemtuzumab 73 % 14 /91
bevacizumab 72.40 % 23
trastuzumab 81.63 % 9
V-KAPPA alemtuzumab 86.32 % 2 /89
bevacizumab 87.40 % 7
trastuzumab 86.32 % 6
FR-IMGTAA
differences
V-REGIONidentitypercent
http://www.imgt.org
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V-REGIONidentity percent
IMGT/DomainGapAlign
http://www.imgt.org
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Towards «Potential immunogenicity evaluation»
14/91 different AAin FR-IMGT
V-REGIONidentitypercent
• Characteristics of the AA class changes http://www.imgt.org
Hydropathy Volume Physicochemical+ : conserved classes- : different classes
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IMGT/3Dstructure-DB
Hydrogen bonds
http://www.imgt.org
Kaas Q. et al. Nucl. Acids Res. (2004)
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Kaas Q. et al.2004
Contacts VH-(Ligand), V-KAPPA-(Ligand)http://www.imgt.org
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Kaas Q. et al.Nucl. Acids Res. (2004)
Contacts V-KAPPA-(Ligand)http://www.imgt.org
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Kaas Q. et al. Nucl. Acids Res. (2004)
Contacts VH-(Ligand)http://www.imgt.org
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IMGT/2Dstructure-DB
International Nonproprietary Name (INN)
Ehrenmann et al. Nucl. Acids Res. (2010)
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Ehrenmann et al. Nucl. Acids Res. 2010
IMGT/2Dstructure-DB
http://www.imgt.org
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Ehrenmann et al. Nucl. Acids Res. 2010
IMGT/2Dstructure-DB
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Ehrenmann et al. Nucl. Acids Res. 2010
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Ehrenmann et al. Nucl. Acids Res. 2010
IMGT/mAb-DB
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Ehrenmann et al. Nucl. Acids Res. (2010)
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IMGTIMGT®®: 6 databases and 15 online tools: 6 databases and 15 online tools
• Immunoglobulins (IG)(or antibodies)
• T cell receptors (TR) • MHC• IgSF and MhcSF
• Sequences• Genes• Structures
http://www.imgt.org
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Many thanks to the IMGT® team at Montpellier, France
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