title: Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors.
abstract: Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG and D08-0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody for podoplanin peptide was 2.1 × 10(-8) M, 8.0 × 10(-8) M and 3.9 × 10(-10) M, respectively. In a protein stability assay, NZ-1-(scdsFv)-PE38KDEL retained 33-98% of its activity, whereas that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG and D08-0493MG, and in the medulloblastoma D283MED, D425MED and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in the range of 1.6-29 ng/ml. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor growth delay, averaging 24 days (p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (p ≤ 0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors.
authors.affiliation: Preston Robert Tisch Brain Tumor Center at Duke and Department of Pathology, Duke University Medical Center, Durham, NC.
authors.forename: Vidyalakshmi
authors.initials: V
authors.lastname: Chandramohan
authors.forename: Xuhui
authors.initials: X
authors.lastname: Bao
authors.forename: Mika
authors.initials: M
authors.lastname: Kato Kaneko
authors.forename: Yukinari
authors.initials: Y
authors.lastname: Kato
authors.forename: Stephen T
authors.initials: ST
authors.lastname: Keir
authors.forename: Scott E
authors.initials: SE
authors.lastname: Szafranski
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: D036002:ADP Ribose Transferases / Q000276:immunology* / Q000627:therapeutic use; D001427:Bacterial Toxins / Q000276:immunology* / Q000627:therapeutic use; D001932:Brain Neoplasms / Q000188:drug therapy* / Q000276:immunology*; D045744:Cell Line, Tumor; D005098:Exotoxins / Q000276:immunology* / Q000627:therapeutic use; D005260:Female; D005909:Glioblastoma / Q000188:drug therapy* / Q000276:immunology*; D006801:Humans; D008297:Male; D008527:Medulloblastoma / Q000188:drug therapy* / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000276:immunology*; D037521:Virulence Factors / Q000276:immunology* / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays; D000097668:Pseudomonas aeruginosa Exotoxin A; D000098853:Podoplanin
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C117468:PDPN protein, human; D000098853:Podoplanin