Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation

J Biol Chem. 2013 Mar 15;288(11):7688-7696. doi: 10.1074/jbc.M112.445502. Epub 2013 Jan 25.


A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.

MeSH terms

  • Animals
  • Antibodies / chemistry*
  • Antibodies, Monoclonal / chemistry
  • Antigens / chemistry
  • Base Sequence
  • Binding, Competitive
  • Cell Separation
  • Codon
  • Complementarity Determining Regions / metabolism*
  • Cytokines / metabolism
  • Flow Cytometry
  • HEK293 Cells
  • Humans
  • In Vitro Techniques
  • Mice
  • Models, Genetic
  • Molecular Sequence Data
  • Mutation*
  • Protein Engineering / methods
  • Signal Transduction


  • Antibodies
  • Antibodies, Monoclonal
  • Antigens
  • Codon
  • Complementarity Determining Regions
  • Cytokines