Poly(ethylene glycol)-conjugated anti-EGF receptor antibody C225 with radiometal chelator attached to the termini of polymer chains

Several biological barriers, including significant liver uptake, limit the clinical application of radiolabeled antibodies in radioimmunoscintigraphy. Here, a general approach is described for radiolabeling of monoclonal antibodies conjugated with poly(ethylene glycol) (PEG). This strategy is demonstrated with C225, a monoclonal antibody directed against epidermal growth factor (EGF) receptor. We synthesized a heterofunctional PEG with one end attached to a radiometal chelator, diethylenetriaminepentaacetic acid (DTPA), and the other end to a protected thiol group, S-acetylthioacetate. After a deprotection step, the resulting DTPA-PEG-SH was conjugated to maleimide-activated C225 to yield DTPA-PEG-C225 conjugate. Characterization of DTPA-PEG-C225 with immunoprecipitation and Western blot analysis revealed that the conjugate was biologically active in binding to the EGF receptor in A431 cells. Competitive EGF receptor binding assay in MDA-MB-468 cells showed that DTPA-PEG-C225, with up to 60% of the amino groups in C225 substituted, retained 66% of C225's binding affinity. Moreover, DTPA-PEG-C225 with increasing degrees of NH₂ substitution from 20% to 70% retained the activity of C225 to induce apoptosis in DiFi cells. More importantly, DTPA-PEG-C225 demonstrated less nonspecific interaction than DTPA-C225. Pharmacokinetic analysis using ¹¹¹In-labeled compounds revealed narrower steady-state distribution of ¹¹¹In-DTPA-PEG-C225 than ¹¹¹In-DTPA-C225, probably due to reduced nonspecific binding of PEG-modified antibody to tissues. The terminal half-life (t_1/2,γ) of ¹¹¹In-DTPA-PEG-C225, 21.1 h, was shorter than that of ¹¹¹In-DTPA-C225, 52.9 h. These data suggest that ¹¹¹In-DTPA-PEG-C225 may provide better imaging characteristics than ¹¹¹In-DTPA-C225, and that using PEG as a linker between the monoclonal antibody and DTPA may be a promising strategy in optimizing the imaging characteristics of immunoscintigraphic agents.