Effects of humanization and gene shuffling on immunogenicity and antigen binding of anti-TAG-72 single-chain Fvs

Gabriela Pavlinkova, David Colcher, Barbara J.M. Booth, Apollina Goel, Uwe A. Wittel, Surinder K. Batra

Research output: Contribution to journalArticlepeer-review

28 Scopus citations


One major constraint in the clinical application of murine monoclonal antibodies (MAbs) is the development of a human antimurine antibody response. The immunogenicity of MAbs can be minimized by replacing nonhuman regions with corresponding human sequences. The studies reported in our article were undertaken to analyze the immunoreactivity and the immunogenicity of the CC49 single-chain antibody fragments (scFvs): (i) an scFv construct comprised of mouse CC49 VL and VH (m/m scFv), (ii) a light chain shuffled scFv with human VL (Hum4 VL) and mouse CC49 VH (h/m scFv), and (iii) a humanized scFv assembled from Hum4 VL and CC49 VH complementary determining regions (CDRs) grafted onto a VH framework of MAb 21/28′ CL (h/CDR scFv). The CC49 scFvs competed for an antigen binding site with CC49 IgG in a similar fashion in a competition radioimmunoassay and were able to inhibit the binding of CC49 IgG to the antigen completely. The immunogenicity of CC49 scFvs was tested using sera with antiidiotypic antibodies to MAb CC49 obtained from patients treated by CC49 IgG in clinical trials. All tested sera exhibited the highest reactivity to the m/m scFv. However, the sera demonstrated differential reactivities to h/CDR scFv and h/m scFv. Replacement of the mouse chain in h/m scFv and h/CDR scFv decreased or completely averted serum reactivity. Our studies compared for the first time the antigen binding and immunogenicity of different scFv constructs containing the mouse, CDR grafted or human variable chains. These results indicate that the humanized CC49 scFv is potentially an important agent for imaging and therapeutic applications with TAG-72-positive tumors.

Original languageEnglish (US)
Pages (from-to)717-726
Number of pages10
JournalInternational Journal of Cancer
Issue number5
StatePublished - Dec 1 2001


  • Antibody engineering
  • Antigen binding
  • Antiidiotypic antibodies
  • Monoclonal antibodies
  • Single-chain antibodies

ASJC Scopus subject areas

  • Oncology
  • Cancer Research


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