title: Construction of an immunotoxin, D2C7-(scdsFv)-PE38KDEL, targeting EGFRwt and EGFRvIII for brain tumor therapy.
abstract: OBJECTIVE
  The EGF receptor gene (EGFR) is most frequently amplified and overexpressed, along with its deletion mutant, EGFRvIII, in glioblastoma. We tested the preclinical efficacy of the recombinant immunotoxin, D2C7-(scdsFv)-PE38KDEL, which is reactive with a 55-amino acid (AA) region present in the extracellular domain of both EGFRwt (583-637 AAs) and EGFRvIII (292-346 AAs) proteins.
  
  
  METHODS
  The binding affinity and specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII were measured by surface-plasmon resonance and flow cytometry. In vitro cytotoxicity of D2C7-(scdsFv)-PE38KDEL was measured by inhibition of protein synthesis in human EGFRwt-transfected NR6 (NR6W), human EGFRvIII-transfected NR6 (NR6M), EGFRwt-overexpressing A431-epidermoid-carcinoma, and glioblastoma xenograft cells (43, D08-0493MG, D2159MG, and D270MG). In vivo antitumor efficacy of D2C7-(scdsFv)-PE38KDEL was evaluated using 43, NR6M, and D270MG orthotopic tumor models.
  
  
  RESULTS
  The KD of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII was 1.6×10(-9) mol/L and 1.3×10(-9) mol/L, respectively. Flow cytometry with NR6W and NR6M cells confirmed the specificity of D2C7-(scdsFv)-PE38KDEL for EGFRwt and EGFRvIII. The D2C7-(scdsFv)-PE38KDEL IC50 was 0.18 to 2.5 ng/mL on cells expressing EGFRwt (NR6W, A431, 43, and D08-0493MG). The D2C7-(scdsFv)-PE38KDEL IC50 was approximately 0.25 ng/mL on EGFRvIII-expressing cells (NR6M) and on EGFRwt- and EGFRvIII-expressing glioblastoma xenograft cells (D2159MG and D270MG). Significantly, in intracranial tumor models of 43, NR6M, and D270MG, treatment with D2C7-(scdsFv)-PE38KDEL by convection-enhanced delivery prolonged survival by 310% (P=0.006), 28% (P=0.002), and 166% (P=0.001), respectively.
  
  
  CONCLUSIONS
  In preclinical studies, the D2C7-(scdsFv)-PE38KDEL immunotoxin exhibited significant potential for treating brain tumors expressing EGFRwt, EGFRvIII, or both.
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: Vidyalakshmi
authors.initials: V
authors.lastname: Chandramohan
authors.forename: Xuhui
authors.initials: X
authors.lastname: Bao
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Keir
authors.forename: Charles N
authors.initials: CN
authors.lastname: Pegram
authors.forename: Scott E
authors.initials: SE
authors.lastname: Szafranski
authors.forename: Hailan
authors.initials: H
authors.lastname: Piao
authors.forename: Carol J
authors.initials: CJ
authors.lastname: Wikstrand
authors.forename: Roger E
authors.initials: RE
authors.lastname: McLendon
authors.forename: Chien-Tsun
authors.initials: CT
authors.lastname: Kuan
authors.forename: Ira H
authors.initials: IH
authors.lastname: Pastan
authors.forename: Darell D
authors.initials: DD
authors.lastname: Bigner
mesh_terms: D000951:Antigens, Neoplasm / Q000276:immunology; D001932:Brain Neoplasms / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D000939:Epitopes / Q000302:isolation & purification; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D005909:Glioblastoma / Q000276:immunology* / Q000473:pathology / Q000628:therapy; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology; D000922:Immunotoxins / Q000008:administration & dosage* / Q000235:genetics; D020349:Surface Plasmon Resonance; D023041:Xenograft Model Antitumor Assays
chemical_list: D000951:Antigens, Neoplasm; D000939:Epitopes; D007074:Immunoglobulin G; D000922:Immunotoxins; C512478:EGFR protein, human; D066246:ErbB Receptors