title: Mutagenic Deimmunization of Diphtheria Toxin for Use in Biologic Drug Development.
abstract: BACKGROUND
  Targeted toxins require multiple treatments and therefore must be deimmunized. We report a method of protein deimmunization based on the point mutation of highly hydrophilic R, K, D, E, and Q amino acids on the molecular surface of truncated diphtheria-toxin (DT390).
  
  
  METHODS
  Based on their surface position derived from an X-ray-crystallographic model, residues were chosen for point mutation that were located in prominent positions on the molecular surface and away from the catalytic site. Mice were immunized with a targeted toxin containing either a mutated DT390 containing seven critical point mutations or the non-mutated parental toxin form.
  
  
  RESULTS
  Serum analysis revealed a significant 90% reduction in anti-toxin antibodies in mice immunized with the mutant, but not the parental drug form despite multiple immunizations. The experiment was repeated in a second strain of mice with a different MHC-haplotype to address whether point mutation removed T or B cell epitopes. Findings were identical indicating that B cell epitopes were eliminated from DT. The mutant drug form lost only minimal activity in vitro as well as in vivo.
  
  
  CONCLUSIONS
  These findings indicate that this method may be effective for deimmunizing of other proteins and that discovery of a deimmunized form of DT may lead to the development of more effective targeted toxin.
authors.affiliation: University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN 55423, USA. joerg.schmohl@uni-tuebingen.de.|Department for Hematology and Oncology, Department of Medicine 2, University Hospital of Tuebingen, Tuebingen 72076, Germany. joerg.schmohl@uni-tuebingen.de.
authors.forename: Joerg U
authors.initials: JU
authors.lastname: Schmohl
authors.affiliation: University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN 55423, USA. todhu001@umn.edu.
authors.forename: Deborah
authors.initials: D
authors.lastname: Todhunter
authors.affiliation: University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN 55423, USA. ohxxx021@umn.edu.
authors.forename: Seung
authors.initials: S
authors.lastname: Oh
authors.affiliation: University of Minnesota Masonic Cancer Center, Section of Molecular Cancer Therapeutics, Therapeutic Radiology-Radiation Oncology, University of Minnesota, Minneapolis, MN 55423, USA. valle001@umn.edu.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Vallera
mesh_terms: D000818:Animals; D057134:Antibodies, Neutralizing / Q000097:blood; D000970:Antineoplastic Agents / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D018360:Crystallography, X-Ray; D004167:Diphtheria Toxin / Q000235:genetics* / Q000276:immunology*; D055808:Drug Discovery / Q000379:methods*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D020128:Inhibitory Concentration 50; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008819:Mice, Nude; D010957:Plasmids; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays
chemical_list: D057134:Antibodies, Neutralizing; D000970:Antineoplastic Agents; C525855:DTEGF13 cytotoxin; D004167:Diphtheria Toxin; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins
keywords: biologic drug; cancer; cancer treatment; deimmunization; diphtheria; toxin