title: Immunotoxin FN18-CRM9 induces stronger T cell signaling than unconjugated monoclonal antibody FN18.
abstract: BACKGROUND
  The T-cell receptor (TCR)/CD3 complex is the target of therapeutic strategies aimed at prolonging allograft survival. The immunotoxin FN18-CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated diphtheria toxin CRM9, is useful for prolonging allograft survival in preclinical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 complex, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein tyrosine phosphorylation and ligand/receptor internalization in purified monkey peripheral blood T cells.
  
  
  METHODS
  Purified normal rhesus monkey T cells were incubated with unconjugated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization.
  
  
  RESULTS
  Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with similar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues and in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internalization, suggesting that tyrosine phosphorylation is involved in the internalization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-treated cells, there was a gradual decrease in cellular CD3 protein levels within 24 and 48 hr; such a decrease was not observed with the control protein Csk.
  
  
  CONCLUSIONS
  Our findings suggest that the conjugation of the mutated diphtheria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated cross-linking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosphorylation and CD3 internalization. This may in turn contribute to the greater efficacy of the immunotoxin in prolonging allograft survival.
authors.affiliation: Department of Surgery, Division of Transplantation, University of Wisconsin, Clinical Sciences Center H4/749, 600 Highland Avenue, Madison, WI 53792. hamawy@surgery.wisc.edu.
authors.forename: M M
authors.initials: MM
authors.lastname: Hamawy
authors.forename: M
authors.initials: M
authors.lastname: Tsuchida
authors.forename: C S
authors.initials: CS
authors.lastname: Cho
authors.forename: E R
authors.initials: ER
authors.lastname: Manthei
authors.forename: J H
authors.initials: JH
authors.lastname: Fechner
authors.forename: S J
authors.initials: SJ
authors.lastname: Knechtle
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D001692:Biological Transport / Q000187:drug effects; D017252:CD3 Complex / Q000187:drug effects / Q000378:metabolism; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D000922:Immunotoxins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D008253:Macaca mulatta; D008297:Male; D010766:Phosphorylation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015398:Signal Transduction / Q000502:physiology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D004167:Diphtheria Toxin; D004791:Enzyme Inhibitors; C114317:FN18-CRM9; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D019833:Genistein