Affinity maturation, humanization, and co-crystallization of a rabbit anti-human ROR2 monoclonal antibody for therapeutic applications

J Biol Chem. 2020 May 1;295(18):5995-6006. doi: 10.1074/jbc.RA120.012791. Epub 2020 Mar 19.


Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell-engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Keywords: X-ray crystallography; affinity maturation; antibody engineering; antigen; cancer therapy; epitope mapping; kringle domain; receptor tyrosine kinase (RTK) ROR2.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / chemistry*
  • Antibodies, Monoclonal, Humanized / immunology*
  • Antibodies, Monoclonal, Humanized / therapeutic use
  • Antibody Affinity*
  • Antibody Specificity
  • CD3 Complex / immunology
  • Cell Line, Tumor
  • Crystallization
  • Humans
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Protein Domains
  • Rabbits
  • Receptor Tyrosine Kinase-like Orphan Receptors / immunology*


  • Antibodies, Monoclonal, Humanized
  • CD3 Complex
  • ROR2 protein, human
  • Receptor Tyrosine Kinase-like Orphan Receptors