Conjugation with In-111-DTPA-poly(Ethylene glycol) improves imaging of Anti-EGF receptor antibody C225

Significant liver uptake often limits the clinical application of radiolabe led antibodies in radioimmunodetection. The purpose of this study was to ev aluate the gamma -imaging properties of an antiepidermal growth factor rece ptor (EGFR) antibody, C225, conjugated with heterofunctional poly(ethylene glycol) (PEG) with 1 terminus of the polymer attached to a radiometal chela tor, diethylenetriaminepentaacetic acid (DTPA). Methods: Two preparations o f PEG-modified C225, one with 20% and the other with 60% amine substitution , were labeled with In-111. The conjugates, (111)in-DTPA-PEG-C225, were inj ected intravenously into nude mice with EGFR-positive A431 tumors. For comp arison, C225 directly labeled with In-111 was also injected. In a competiti ve study, mice with A431 tumors were pretreated intravenously with 100-fold excess of native C225, followed by an injection of In-111-DTPA-PEG-C225 30 min or 20 h later. In addition, In-111-DTIDA-PEG-C225 was injected into mi ce with EGFR-positive MDA-MB-468 tumors and EGFR-negative MDA-MB-435 tumors . Images were acquired at 5 min and at 2, 6, 24, and 48 h after injection o f the radiotracers. Regions of interest (ROIs) were drawn on the computer i mages around the whole body, liver, muscle, and tumor. The counts per pixel in the tumor and normal tissues were calculated. At 48 h, the mice were ki lled and dissected. Blood, liver, muscle, and tumor samples were removed an d the radioactivity of each sample was measured. Results: In A431 tumor xen ografts, the tumor uptake of C225 modified with PEG was not significantly d ifferent than the uptake of unmodified In-111-DTPA-C225. Uptake in the live r, however, was reduced by 38%-45%, and the reduction increased with increa sing degree of PEG substitution. Tumors of A431 and MDA-MB-468 xenografts w ere clearly visualized with In-111-DTPA-PEG-C225, whereas tumors of the MDA -MB-435 xenograft, which expresses low levels of EGFR, were not as readily visible. The tumor-to-blood ratios of In-111-DTPA-PEG-C225 in A431 and MDA- MB-468 xenografts were about 3 fold higher than in MDA-MB-435 xenografts. B locking EGFR by pretreatment with native C225 significantly reduced the upt ake of In-111-DTPA-PEG-C225 in the liver. The tumor-to-blood ratios in mice with A431 tumors were decreased 2.5-2.7 fold after pretreatment with a lar ge excess of C225. Similar results were obtained with MDA-MB-468 tumor xeno grafts. In contrast, the tumor-to-blood ratios in mice with MDA-MB-435 tumo r xenografts were not significantly different in C225-pretreated mice than in nonpretreated mice. Conclusion: These findings indicate that In-111-DTPA -PEG-C225 selectively localized to the tumors expressing high levels of EGF R. PEG-modification of C225 significantly reduced its liver uptake, resulti ng in improved visualization of EGFR-positlve tumors. Using PEG as a linker between the monoclonal antibody and metal chelator is a useful strategy to optimize the imaging characteristics of antibody-based scintigraphic agent s.