Antibody engineering

IMGT Primer-DB

IMGT Sequence databases

IMGT Sequence analysis tools

IMGT Genome database

IMGT Genome analysis tools

IMGT 3D structure database

IMGT Structure analysis tools

IMGT Web resources

IMGT links

Other links

Antibody engineering

Combinatorial libraries & phage displays

Reagents monoclonal antibodies

Antibody glycosylation and effector properties

Antibody humanization

Alemtuzumab CAMPATH-1H (Humanized antibodies)

K20 (Humanized antibodies)

  • Protein display V-DOMAIN:
  • Colliers de Perles:

FR-IMGT and CDR-IMGT

CDR-IMGT delimitation for grafting

Excerpt of:

Lefranc M-P. IMGT® immunoglobulin repertoire analysis and antibody humanization.

In: Alt, F.W, Honjo, T, Radbruch A. and Reth, M. (Eds.), Molecular Biology of B cells, Second edition, Academic Press, Elsevier Ltd, London, UK, Chapter 26, 2014, PP. 481-514. dx.doi.org, ISBN : 978-0-12-397933-9. LIGM: 438
page 507-508 (source above to be quoted)

‘The objective of antibody humanization is to graft at the DNA level the CDR of an antibody V domain, from mouse (or other species) and of a given specificity, onto a human V domain framework, thus preserving the specificity of the original (murine or other species) antibody while decreasing its immunogenicity (95). IMGT/DomainGapAlign (9, 24, 25) is the reference tool for antibody humanization design based on CDR grafting. Indeed, it precisely defines the CDR-IMGT to be grafted and helps selecting the most appropriate human FR-IMGT by providing the alignment of the amino acid sequences between the mouse (or other species) and the closest human V-DOMAIN.

Analyses performed on humanized therapeutic antibodies underline the importance of a correct delimitation of the CDR and FR. As an example, two amino acid changes were required in the first version of the humanized VH of alemtuzumab, in order to restore the specificity and affinity of the original rat antibody. The positions of these amino acid changes (S28>F and S35>F) are now known to be located in the CDR1-IMGT and should have been directly grafted, but at the time of this mAb humanization they were considered as belonging to the FR according to the Kabat numbering (92). In contrast, positions 66-74 were, at the same time, considered as belonging to the CDR according to the Kabat numbering, whereas they clearly belong to the FR2-IMGT and the corresponding sequence should have been ‘human’ instead of being grafted from the ‘rat’ sequence (IMGT® http://www.imgt.org, The IMGT Biotechnology page > Antibody humanization > Alemtuzumab).’

REFERENCES

(9) Ehrenmann, F., Kaas, Q., and Lefranc, M.-P. (2010). IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucl Acids Res. 38, D301-307.

(24) Ehrenmann, F., and Lefranc, M-P. (2011). IMGT/DomainGapAlign: IMGT Standardized Analysis of Amino Acid Sequences of Variable, Constant, and Groove Domains (IG, TR, MH, IgSF, MhSF). Cold Spring Harb Protoc. 6, 737-749. pii: pdb.prot5636. doi: 10.1101/pdb.prot5636.

(25) Ehrenmann, F., and Lefranc, M.-P. (2012). IMGT/DomainGapAlign: the IMGT® tool for the analysis of IG, TR, MHC, IgSF and MhcSF domain amino acid polymorphism. In “Immunogenetics” (F. Christiansen and B. Tait, Eds.), Chap. 33. Humana Press, Springer, New York. Methods Mol Biol. 882, 605-633.

(92) Kabat, E.A., Wu, T.T., Perry, H.M., Gottesman K.S., and Foeller C. (1991). Sequences of proteins of immunological interest, Washington, DC: U.S. Department of Health and Human Services (USDHHS), National Institute of Health NIH Publication, 91-3242.

(95) Riechmann, L., Clark M, Waldmann H, Winter G. (1988). Reshaping human antibodies for therapy. Nature 332, 323-327.

Amino acid interactions between FR-IMGT and CDR-IMGT

Excerpt of:

Lefranc M-P. IMGT® immunoglobulin repertoire analysis and antibody humanization.

In: Alt, F.W, Honjo, T, Radbruch A. and Reth, M. (Eds.), Molecular Biology of B cells, Second edition, Academic Press, Elsevier Ltd, London, UK, Chapter 26, 2014, PP. 481-514. dx.doi.org, ISBN : 978-0-12-397933-9. LIGM: 438
page 508 (source above to be quoted)

‘IMGT Colliers de Perles from crystallized 3D structures in IMGT/3Dstructure-DB (8-10) highlight two conserved hydrogen bonds between FR-IMGT and CDR-IMGT positions: FR2-IMGT 39 with CDR2-IMGT 56 (or 57) and FR2-IMGT 40 with CDR3-IMGT 105. Antibody engineering and humanization should therefore preserve these bondings which stabilize the loops. It is also worthwhile to note that, in VH CDR3, the stem of the CDR3 loop is stabilized by a conserved salt bridge between R106 (arginine contributed by the 3’V-REGION) and D116 (aspartate contributed by the 5’J-REGION of the Homo sapiens IGHJ2, IGHJ3, IGHJ4, IGHJ5 or IGHJ6) (IMGT® http://www.imgt.org, IMGT Repertoire > Proteins and alleles > Alignments of alleles).’

REFERENCES

(8) Kaas, Q., Ruiz, M., and Lefranc, M.-P. (2004). IMGT/3Dstructure-DB and IMGT/StructuralQuery, a database and a tool for immunoglobulin, T cell receptor and MHC structural data. Nucl Acids Res. 32, D208-210.

(9) Ehrenmann, F., Kaas, Q., and Lefranc, M.-P. (2010). IMGT/3Dstructure-DB and IMGT/DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF and MhcSF. Nucl Acids Res. 38, D301-307.

(10) Ehrenmann, F., and Lefranc, M-P. (2011). IMGT/3Dstructure-DB: Querying the IMGT Database for 3D Structures in Immunology and Immunoinformatics (IG or Antibodies, TR, MH, RPI, and FPIA). Cold Spring Harb Protoc. 6, 750-761. pii: pdb.prot5637. doi: 10.1101/pdb.prot5637.

IMGT® tools and databases for antibody humanization

  • Identifying the closest Homo sapiens V gene to be used for the humanization
    • IMGT/DomainGapAlign: This tool analyses the amino acid sequences of the V domains (V-(D)-J-REGION) of immunoglobulins (IG) or antibodies, from Homo sapiens or Mus musculus (or from other species provided that their sequences are in the IMGT reference directory).

    • It allows to identify the closest Homo sapiens V gene at the amino acid level.

  • Taking into account the genomic information of the selected Homo sapiens V gene
    • IMGT/GENE-DB: This database allows to retrieve the genomic germline sequence of the Homo sapiens V gene which has been selected for the humanization.
    • IMGT/GeneFrequency: This tool allows to check if the Homo sapiens V gene selected for the humanization is well represented in IMGT/LIGM-DB.

  • Taking into account the structural information of the selected Homo sapiens V gene
    • IMGT/3Dstructure-DB: This database provides :
        for IG/Ag complexes, the IMGT Contact analysis.
      The IMGT Contact analysis of 3D structures of IG/Ag complexes demonstrates that the CDR-IMGT correspond to the antigen binding sites.
        for each V-DOMAIN, the IMGT Colliers de Perles on 2 layers.
      The IMGT Colliers de Perles on 2 layers show the delimitations of the CDR-IMGT as structural loops and the hydrogen bonds.

    • Only two framework positions (FR2-IMGT C-STRAND 39 and 40) have hydrogen bonds with the CDR-IMGT:
        FR2-IMGT 39 with CDR2-IMGT 56 for VH or 57 for VL,
        FR2-IMGT 40 with CDR3-IMGT 105.
      This means that in the humanization by grafting, positions 39 and 40 may remain ‘mouse’ if required to preserve these hydrogen bonds between the framework and the CDR. A query of the IMGT/3Dstructure-DB database for humanized antibodies with the name of the selected Homo sapiens gene provides the 3D structures in which the Homo sapiens V gene selected for humanization may have already been used.

  • Humanizing the V domain by grafting of the CDR-IMGT onto the Homo sapiens FR-IMGT
    • IMGT/V-QUEST: This tool analyses the nucleotide sequences of the V domains (V-(D)-J-REGION) of immunoglobulins (IG) or antibodies, from Homo sapiens or Mus musculus (or from other species provided that their sequences are annotated - and therefore gapped - in the IMGT reference directory.

    • Running IMGT/V-QUEST for the Mus musculus (or other species) V domain sequence and for the Homo sapiens germline V gene, allows to easily construct the nucleotide sequences of the humanized antibody by copy-paste between the two results of 7. V-REGION translation. With the FR1-IMGT, FR2-IMGT, FR3-IMGT coming from the Homo sapiens germline V gene and between them, the CDR1-IMGT, CDR2-IMGT and CDR3-IMGT from the Mus musculus V domain The FR4-IMGT (from position 118 to the ‘g’ of the DONOR-SPLICE included) can be retrieved from the Mus musculus (or other species) sequence and mutated if required to make it identical to the closest Homo sapiens germline J gene.

  • Visualing the humanized V domain
      IMGT/DomainGapAlign and IMGT/Collier-de-Perles tool allow to visualize the humanized V domain amino acid sequence.

Antibody camelization

Bispecific antibodies

Antibody drug conjugates (ADC)

Transgenic animals for human antibody production

  • Transgenic animals created for the production of human antibodies include :
    • Medarex® UltiMAb Human Antibody Development System® transgenic mice [1]
    • Minilocus transgenic mice [2]
    • Abgenix's XenoMouse® transgenic mouse [3]
    • Double trans-chromosomic mice [4]
    • TransChromo Mouse [5]
    • VelocImmune® mice technology [6]
They are reported, if known, in IMGT/mAb-DB, in Development technology. For a review and future developments, see [7].

Monoclonal antibodies with clinical indications

Fusion proteins for immune applications (FPIA)

External links and resources

References:
[1] Lonberg N et al. Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 368(6474):856-859 (1994).PMID: 8159246
[2] Fishwild DM et al. High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat Biotechnol. 14(7):845-851 (1996). PMID: 9631008 PMID: 9631008
[3] Mendez MJ et al. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nat Genet. 15(2):146-156 (1997). Erratum in: Nat Genet. 16(4):410 (1997). PMID: 9020839
[4] Tomizuka K et al. Double trans-chromosomic mice: maintenance of two individual human chromosome fragments containing Ig heavy and kappa loci and expression of fully human antibodies. Proc Natl Acad Sci U S A. 97(2):722-727 (2000). PMID: 10639146
[5] Ishida I et al. Production of human monoclonal and polyclonal antibodies in TransChromo animals. Cloning Stem Cells 4(1):91-102 (2002). PMID: 12006160
[6] Murphy AJ et al. Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice. Proc Natl Acad Sci U S A. 111(14):5153-5158 (2014). doi: 10.1073/pnas.1324022111. Epub 2014 Mar 25. PMID: 24706856
[7] Brüggemann M et al. Human antibody production in transgenic animals. Arch. Immunol. Ther. Exp. (Warsz) 63(2):101-108 (2015). doi: 10.1007/s00005-014-0322-x. Epub 2014 Dec 3. Review. PMID: 25467949