title: A universal combinatorial design of antibody framework to graft distinct CDR sequences: a bioinformatics approach.
abstract: Antibody (Ab) humanization is crucial to generate clinically relevant biologics from hybridoma-derived monoclonal antibodies (mAbs). In this study, we integrated antibody structural information from the Protein Data Bank with known back-to-mouse mutational data to build a universal consensus of framework positions (10 heavy and 7 light) critical for the preservation of the functional conformation of the Complimentarity Determining Region of antibodies. On the basis of FR consensus, we describe here a universal combinatorial library suitable for humanizing exogenous antibodies by CDR-grafting. The six CDRs of the murine anti-human EGFR Fab M225 were grafted onto a distinct (low FR sequence similarity to M225) human FR sequence that incorporates at the 17 FR consensus positions the permutations of the naturally observed amino acid diversities. Ten clones were selected from the combinatorial library expressing phage-displayed humanized M225 Fabs. Surprisingly, 2 of the 10 clones were found to bind EGFR with stronger affinity than M225. Cell-based assays demonstrated that the 10 selected clones retained epitope specificity by blocking EGFR phosphorylation and thus hindering cellular proliferation. Our results suggest that there is a universal and structurally rigid near-CDR set of FR positions that cooperatively support the binding conformation of CDRs.
authors.affiliation: Department of Antibody Technology, ImClone Systems, a Wholly-Owned Subsidiary of Eli Lilly and Company, Alexandria Center for Life Sciences, New York, New York 10016, USA. jaafar.haidar@imclone.com
authors.forename: Jaafar N
authors.initials: JN
authors.lastname: Haidar
authors.forename: Qing-An
authors.initials: QA
authors.lastname: Yuan
authors.forename: Lin
authors.initials: L
authors.lastname: Zeng
authors.forename: Mark
authors.initials: M
authors.lastname: Snavely
authors.forename: Xenia
authors.initials: X
authors.lastname: Luna
authors.forename: Haifan
authors.initials: H
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.forename: Dale L
authors.initials: DL
authors.lastname: Ludwig
authors.forename: Zhenping
authors.initials: Z
authors.lastname: Zhu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D045744:Cell Line, Tumor; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D019295:Computational Biology / Q000379:methods*; D066246:ErbB Receptors / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D019151:Peptide Library
chemical_list: D000906:Antibodies; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D066246:ErbB Receptors