Conjugates with specific binding to the epidermal growth factor receptor, EGFR, of interest for radionuclide based imaging and therapy were prepared using mouse epidermal growth factor, mEGF, and dextran. In one type of conjugate, mEGF was coupled to dextran by reductive amination in which the free amino group on the mEGF N-terminal reacted with the aldehyde group on the reductive end of dextran. The end-end coupled conjugate could be further activated by the cyanopyridinium agent CDAP, thereby introducing tyrosines to the dextran part. In the other type of conjugate, the cyanylating procedure using CDAP was applied, first to activate dextran and then allowing for the amino terminus of mEGF to randomly attach to the dextran. In the latter case, radionuclide-labelled tyrosines or glycines could be added in the same conjugation step. All types of mEGF-dextran conjugates had EGFR-specific binding since the binding could be displaced by an excess of non-radioactive mEGF. The conjugates were to a large extent internalized in the test cells and the associated radioactivity was retained intracellularly for different times depending on both the type of cells and conjugate applied. Different intracellular 'traffic routes' for the radionuclides are discussed as well as applications for both imaging and therapy.

The effects of dextranation on the biodistribution of mouse epidermal growth factor (mEGF, 6 kDa) were assessed. By reductive amination, mEGF was coupled to 13 and 46 kDa dextran. The two dextranated conjugates and free mEGF were labeled with the positron-emitting nuclide (76)Br (T(1/2) = 16 h). After intravenous administration to Sprague Dawley rats, the radioactivity biodistribution was evaluated by positron emission tomography (PET) and by measurements of dissected tissues. The dextranation prolonged the retention time in blood, especially when the dextran chain was long. [(76)Br]mEGF-dextran conjugates were shown to have significantly, more than 5 times, lower kidney accumulation than the nonconjugated [(76)Br]mEGF. In conclusion, dextranation affects the biodistribution of mEGF in vivo giving a prolonged circulation time, a decreased uptake in kidney, and an increased spleen accumulation.

A conjugate with specific binding to the epidermal growth factor receptor, EGFR, and of interest for clinical tests was prepared using mouse epidermal growth factor, mEGF, and dextran. The mEGF was first coupled to dextran by reductive amination in which the free amino group on the N-terminal of mEGF was reacted with the aldehyde group on the reductive end of the dextran chain. The end-end coupled intermediate was further activated by the cyanopyridinium agent CDAP and tyrosines introduced to the dextran part of the conjugate. The mEGF-dextran-tyrosine conjugate was, with high efficiency, iodinated with the chloramine-T method. Approximately 25-35% of the radioactivity could be removed from the conjugate after exposure to protease K while 65-75% of the radioactivity could be removed after exposure to dextranase. Thus, the largest amount of the iodine was on the dextran part of the conjugate. The iodinated mEGF-dextran-tyrosine had EGFR specific binding since the binding to an EGFR rich human glioma cell line could be displaced by an excess of non-radioactive mEGF. The conjugate was to a large extent internalized in these cells and the administrated radioactivity was thereby retained inside the cells for at least up to 50 h.

The cellular binding properties of a new conjugate, I-125-mEGF-dextran, in which the amino terminus on mEGF was covalently coupled by reductive amination to the reducing end of dextran D14 were analysed. The coupling molar ratio was 1:1 since dextran only contains one aldehyde group and mEGF only has one free amino group available; the amino terminus. The conjugates were I-125-labelled and tested for their receptor binding properties using cultured human glioma, U-343MGaC12:6, cells. The conjugate reached maximal binding around 1.5 or 2 h when incubated at 37 degrees C or 4 degrees C, respectively. The binding was receptor specific since it could be displaced by free mEGF. Dissociation constants were determined at 4 degrees C by using mEGF to displace I-125-mEGF and non-radioactive mEGF-dextran to displace I-125-mEGF-dextran and were 6.6 x 10(-10) and 7.1 x 10(-9) M respectively. Cellular internalisation was studied at 37 degrees C for both I-125-mEGF-dextran and I-125-mEGF and most of the radioactivity was internalized in both cases. However, there was a difference regarding the retention time pattern. It took less than 1 h for the internalised radioactivity delivered with mEGF to decrease to 50% of the initial level while it took about 2.5 h for the conjugate. The studies on this new form of EGF-containing conjugate serve as a model for the design of future dextran containing conjugates employing, for example, antibody fragments or small ligands with tumour specificity.