Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors
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. What's new? Podoplanin is a glycoprotein that is overexpressed in several types of malignant tumor, including glioblastoma, medulloblastoma, mesothelioma, and germ-cell tumors. In this study, the authors constructed a novel immunotoxin to target podoplanin, by fusing a monoclonal antibody fragment to exotoxin A from Pseudomonas. In preclinical studies, this immunotoxin, called NZ-1-(scdsFv)-PE38KDEL, increased survival by 41%. Its specificity and high binding affinity allow for specific targeting of tumor cells while avoiding adjacent normal tissue, thereby having the potential to improve survival of patients with brain tumors. Copyright © 2012 UICC.
Chandramohan, V; Bao, X; Kaneko, MK; Kato, Y; Keir, ST; Szafranski, SE; Kuan, C-T; Pastan, IH; Bigner, DD
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