Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies

G. Huls, D. Gestel, J. van der Linden, E.E. Moret, T. Logtenberg

Research output: Contribution to journalArticleAcademicpeer-review

20 Citations (Scopus)


We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.

Original languageEnglish
Pages (from-to)163-171
Number of pages9
JournalCancer Immunology, Immunotherapy
Issue number3
Publication statusPublished - 2001


  • Affinity maturation
  • DNA shuffling
  • Ep-CAM
  • Immunotherapy
  • Molecular modeling
  • Phage display


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