title: Humanization of the bispecific epidermal growth factor receptor x CD3 diabody and its efficacy as a potential clinical reagent.
abstract: OBJECTIVE
  Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody.
  
  
  METHODS
  We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementarity-determining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice.
  
  
  RESULTS
  The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma.
  
  
  CONCLUSIONS
  The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.
authors.affiliation: Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Sendai, Japan.
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Asano
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sone
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.forename: Kouhei
authors.initials: K
authors.lastname: Tsumoto
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hayashi
authors.forename: Yu
authors.initials: Y
authors.lastname: Katayose
authors.forename: Michiaki
authors.initials: M
authors.lastname: Unno
authors.forename: Toshio
authors.initials: T
authors.lastname: Kudo
authors.forename: Izumi
authors.initials: I
authors.lastname: Kumagai
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D000937:Antigen-Antibody Reactions; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D017252:CD3 Complex / Q000276:immunology; D016466:CHO Cells; D002277:Carcinoma / Q000188:drug therapy* / Q000276:immunology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor; D066246:ErbB Receptors / Q000276:immunology; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D011485:Protein Binding; D014183:Transplantation, Heterologous; D023041:Xenograft Model Antitumor Assays
chemical_list: D018033:Antibodies, Bispecific; D000970:Antineoplastic Agents; D017252:CD3 Complex; D066246:ErbB Receptors