We have constructed a drug delivery vehicle that targets the epidermal growth factor receptor (EGFR) and its mutant isoform EGFRvIII. The monoclonal antibody, cetuximab, previously known as C225, which binds to both EGFR and EGFRvIII, was covalently linked via its Fc region to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. As measured by mass spectrometry and UV/vis spectroscopy, the resulting bioconjugate, designated C225-G5-MTX, contained 12.6 molecules of methotrexate per unit of dendrimer. Specific binding and cytotoxicity of the bioconjugate was evaluated against the EGFR-expressing rat glioma cell line F98_EGFR. Using a competitive binding assay, it was shown that the bioconjugate retained its affinity for F98_EGFR cells, with a 0.8 log unit reduction in its EC₅₀. Only cetuximab completely inhibited binding of the bioconjugate, which was unaffected by methotrexate or dendrimer. Cetuximab alone was not cytotoxic to F98_EGFR cells at the concentration tested, whereas the IC₅₀ of the bioconjugate was 220 nmol/L, which was a 2.7 log unit decrease in toxicity over that of free methotrexate. The biodistribution of C225-G5-MTX in rats bearing i.c. implants of either F98_EGFR or F98_WT gliomas was determined 24 hours following convection enhanced delivery of ¹²⁵I-labeled bioconjugate. At this time, 62.9 ± 14.7% ID/g tumor was localized in rats bearing F98_EGFR gliomas versus 11.3 ± 3.6% ID/g tumor in animals bearing F98_WT gliomas, thereby showing specific molecular targeting of the tumor. The corresponding radioactivity of normal brain from the F98_EGFR tumor-bearing right and non-tumor-bearing left cerebral hemisphere were 5.8 ± 3.4% and 0.8 ± 0.6% ID/g, respectively. Based on these results, therapy studies were initiated in F98_EGFR glioma-bearing rats. Animals that received C225-G5-MTX, cetuximab, or free methotrexate had median survival times of 15, 17, and 19.5 days, respectively, which were not statistically different from each other or untreated control animals. Our results, which are both positive and negative, show that specific molecular targeting is but one of several requirements that must be fulfilled if an antibody-drug bioconjugate will be therapeutically useful. [Mol Cancer Ther 2006;5(1):52–9]