Pre-clinical experience with Re-188-RC-160, a radiolabeled somatostatin analog for use in peptide-targeted radiotherapy.

The clinical potential of radiolabeled peptides such as octreotide and VIP has been widely established for tumor localization. Radiotherapy based on the tumor binding potential of the peptides and the radiotoxic effects of beta- or a-emitting radionuclides is an extension of such applications. Rhenium-188 (T1/2 16.9 hr, beta-max 2.1 MeV) coupled to the analogue RC-160 has been used to establish the feasibility of treating tumors with radiolabeled peptides, and our experience with this approach is summarized. In three different experimental tumor models (human prostate, mammary gland, and small cell lung carcinomas) in nude mice, treatment resulted in significant reduction or elimination of tumor burden. Two routes of administration were used: intra-lesional injection (prostate carcinoma) and intra-cavity injection (mammary and SCLC). Re-188-labeled negative control peptides bound to tumor cells to a low extent and did not exhibit therapeutic benefit. RC-160 by itself did not result in therapeutic benefit. Tumors which did not bind Re-188-RC-160 did not evidence a therapeutic benefit. Uncoupled Re-188 (control) was rapidly excreted via the urinary bladder and did not accumulate in either tumors or normal tissues even following direct injection. Instant radiolabeling kits containing 200 micrograms of RC-160 were labeled with < 3000 MBq of Re-188 in 30 minutes with no need for subsequent purification. These studies establish the conceptual feasibility of targeted radiotherapy based on the local or regional administration of radiolabeled peptides.

Radiolabeling of epidermal growth factor with 99mTc and in vivo localization following intracerebral injection into normal and glioma-bearing rats.

High grade gliomas may have amplified expression of the epidermal growth factor receptor (EGFR) gene c-erb-B, which often is associated with increased expression of transmembrane EGFR. The purpose of the present study was to develop a method for labeling EGF with 99mTc and to determine whether the resulting radioligand would localize, following intracerebral injection, in rats bearing EGFR-positive gliomas. EGF has a relatively low molecular mass (approximately 6 kDa) compared to monoclonal antibodies, and this has allowed smaller bioconjugates, which should diffuse more rapidly within the brain and more effectively target disseminated glioma cells, to be constructed. In the present study, EGF has been labeled with either 131I or 99mTc, and in vitro uptake of the resulting radioligand has been investigated using C6EGFR rat glioma cells, which had been transfected with the EGFR gene. Cellular uptake of 131I radioactivity peaked after approximately 30 min of incubation with [131I]EGF, following which time it declined, while 99mTc radioactivity continued to increase over a 6 h incubation with [99mTc]-EGF. To determine if radiolabeled EGF had in vivo tumor-localizing properties, C6EGFR glioma cells were implanted stereotactically into the brains of Fischer rats. Four weeks later, either 99mTc- or 131I-labeled EGF was injected intracerebrally into normal or glioma-bearing animals using the same stereotactic coordinates. External gamma scintigraphy revealed that 131I radioactivity disappeared rapidly from the brain regions of tumor-bearing animals compared to 99mTc, approximately 50% of which remained in the tumor for up to 12 h. In contrast, only approximately 20% remained in the brains of non-tumor-bearing animals after 6 h. These studies are the first to describe a method for radiolabeling EGF with 99mTc and to detect it by external scintigraphy in the brains of tumor-bearing animals.

Preparation of 188Re-RC-160 somatostatin analog: a peptide for local/regional radiotherapy.

Conditions are described for the preparation of 188Re-RC-160, a radiolabeled synthetic peptide derived from an analogue of somatostatin, using a lyophilized labeling kit containing RC-160 (200 micrograms) and stannous tartrate to which is added carrier-free 188Re (beta, Emax, 2.12 MeV; gamma, 155 keV, 10%; T1/2 = 16.7 h). The kits have been radiolabeled with > 2960 MBq (80 mCi) with a high labeling efficiency and no need for subsequent purification. Radiolabeling results in one major peak when analyzed by reverse-phase (RP) HPLC. Presumptive radiolysis was detected at 2.5 h post-labeling; however, a convenient method is described for stabilizing the kits against radiolysis by the post-labeling addition of ascorbic acid.

Re-188 labelled antibodies.

Monoclonal antibodies can be directly labelled with 188Re using a simple one-step radiolabelling kit. Using B72.3 as a model antibody, the formulation was optimized and kits were made and tested and compared to data previously reported for the same antibody labelled with other radioisotopes. Labelling with Re-188 was carried out with the eluate of a W-188/Re-188 generator from Oak Ridge National Laboratory. Fresh generator eluate was added to the pre-reduced lyophilized antibody and the mixture allowed to incubate overnight at room temperature. The radiochemical purity, immunoreactive fraction, and biodistribution in normal and LS174T tumor bearing nude mice was determined. The radiochemical purity was 88 +/- 7%, the immunoreactive fraction was 68.46 +/- 3.8%. The immunoreactive fraction was higher than any previously reported for this antibody when labelled with other radioisotopes. At 48 h, 7.9 +/- 2.4% of the injected dose per gram was found in the tumor. The biodistribution and tumor uptake of Re-188 labelled B72.3 was similar to that previously reported for Re-186 and In-111 labelled B72.3.

Quantitation of radiolabeled antibody binding to cells by thin-layer chromatography.

Thin layer chromatography (TLC) was used to monitor binding of radiolabeled antibodies to cells. Labeled antibodies were reacted with cells and aliquots chromatographed on serum-blocked, ITLC strips. The cell-antibody complexes remain at the origin and unbound antibody migrates with the solvent front. The antibody binding was estimated from the ratio of radioactivity at the origin compared to the total applied. Separations are completed in about 10 min. This method does not use centrifugation or wash steps, and provides an inexpensive and self-contained system to evaluate radioligand binding. Cell binding assay results using this method are approximately the same as those obtained using bead- or cell-type assays.

Affinity thin-layer chromatography test of the immunoreactive fraction of radiolabeled antibodies.

A simple, rapid and self-contained system for assaying the immunoreactive fraction of radiolabeled antibodies was developed using affinity thin-layer chromatography (ATLC). ATLC combines use of solid-phase-bound antigen and conventional TLC. The technique is an improvement over existing means of measuring immunoreactive fraction (bead-type or cell-type assays) in that it has neither wash steps nor centrifugation steps, yet provides results essentially identical to those obtained with the more time-consuming assays. ATLC is accomplished using chromatography strips that are coated with antigen material in a discrete region near the origin. The antigen-coated strips are then blocked in serum, air-dried and stored. For use, radiolabeled antibody is spotted at the origin, and the strip is developed using a buffered solvent. Immunoreactive antibody binds to the antigen at or near the origin, while radioactivity not associated with immunoreactive antibody migrates with the solvent front. Antigen-negative strips (serum-blocked only) are used to measure "nonspecific" binding. The ATLC development time is about 16 min, and the results can be obtained in about 30 min. The assay described in this report uses antigens from colon tumor and is suitable for use with B72.3 and other colon cancer-reactive antibodies.

Similarity of copper and technetium binding sites in human IgG.

Kits for direct labeling of IgG with 99mTc were used without modification for the preparation of [67Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind 67Cu. The direct labeling of reduced IgG with 67Cu was highly efficient, resulting in approx. 95% 67Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of 99mTc bound to it. The results demonstrate a direct relationship between copper- and 99mTc-binding sites in IgG.