These studies demonstrate the feasibility of targeted therapy for the treatment of disseminated peritoneal disease using (212)Pb-labeled Herceptin as an in vivo generator of (212)Bi. In vitro studies compare the potential of the bismuth radioisotopes, (213)Bi and (212)Bi, to that of (212)Pb. Overall, (212)Pb results in a higher therapeutic index than either bismuth radioisotope, requiring lower radioactivity (microCi) for effective cytotoxic response. A pilot radioimmunotherapy (RIT) experiment treating mice bearing 5 d LS-174T intraperitoneally (i.p.) xenografts determined a maximum tolerated dose (MTD) of 20-40 microCi with i.p. administration. A specific dose response was observed and 10 microCi was selected as the effective operating dose for future experiments. Median survival of tumor-bearing mice receiving 10 microCi increased from 19 to 56 days (p = 0.008). The efficacy of (212)Pb-Herceptin was also assessed in a human pancreatic carcinoma xenograft (Shaw; i.p.) animal model previously reported as unresponsive to 213Bi-Herceptin (p = 0.002). Multiple dosing of (212)Pb-Herceptin was evaluated in both animal models. The median survival of mice bearing 3 d LS-174T i.p. xenografts increased to 110 days, with up to 3 doses of (212)Pb-Herceptin given at approximately monthly intervals; however, there was no evidence of a correlation with the second and third doses (p = 0.98). No improvement in median survival was noted with a similar regimen in the Shaw xenograft model.
First, CED can be used to perfuse the brainstem safely and effectively with macromolecules. Second, a large-molecular-weight imaging tracer can be used successfully to deliver, monitor in vivo, and control the distribution of small- and large-molecular-weight putative therapeutic agents for treatment of intrinsic brainstem processes.
Cetuximab is a recombinant, human/mouse chimeric IgG1, monoclonal antibody (mAb) that binds to the epidermal growth factor receptor (EGFR/HER1). Cetuximab is approved for the treatment of patients with HER1-expressing metastatic colorectal cancer. Limitations in currently reported radiolabeled cetuximab for PET applications prompted the development of 86Y-CHX-A”-DTPA-cetuximab as an alternative for imaging HER1-expressing cancer. 86Y-CHX-A”-DTPA-cetuximab can also serve as a surrogate marker for 90Y therapy.
Methods
Bifunctional chelate, CHX-A”-DTPA was conjugated to cetuximab and radiolabeled with 86Y. In vitro immunoreactivity was assessed in HER1-expressing A431 cells. In vivo biodistribution, PET imaging and non-compartmental pharmacokinetics were performed on mice bearing HER1-expressing human colorectal (LS-174T and HT29), prostate (PC-3 and DU145), ovarian (SKOV3) and pancreatic (SHAW) tumor xenografts. Receptor blockage was demonstrated by co-injection of either 0.1 or 0.2 mg cetuximab.
Results
86Y-CHX-A”-DTPA-cetuximab was routinely prepared with a specific activity of 1.5– 2 GBq/mg and in vitro immunoreactivity ranging from 65–75 %. Biodistribution and PET imaging studies demonstrated high HER1-specific tumor uptake of the radiotracer and clearance from non-specific organs. In LS-174T tumor bearing mice injected with the 86Y-CHX-A”-DTPA-cetuximab alone, 86Y-CHX-A”-DTPA-cetuximab plus 0.1 mg cetuximab or 0.2 mg cetuximab, the tumor uptake values at 3 d were 29.3 ± 4.2, 10.4 ± 0.5 and 6.4 ± 0.3 % ID/g, respectively, demonstrating dose-dependent blockage of the target. Tumors were clearly visualized 1 d after injecting 3.8–4.0 MBq 86Y-CHX-A”-DTPA-cetuximab. Quantitative PET revealed highest tumor uptake in LS-174T (29.55 ± 2.67 % ID/cc) and lowest tumor uptake in PC-3 (15.92 ± 1.55 % ID/cc) xenografts at 3 d after injection. Tumor uptake values quantified by PET were closely correlated (r2= 0.9, n=18) to values determined by biodistribution studies.
Conclusion
This study demonstrates the feasibility in preparation of high specific activity 86Y-CHX-A”-DTPA-cetuximab and its application for quantitative non-invasive PET imaging of HER1-expressing tumors. 86Y-CHX-A”-DTPA-cetuximab offers an attractive alternative to previously labeled cetuximab for PET and warrants further investigation for clinical translation.
Although radioimmunotherapy with radiolabeled intact monoclonal antibodies has demonstrated efficacy in the treatment of lymphoma, it provides low tumor-to-normal-tissue radionuclide target ratios and unwanted prolonged radiation exposure to the bone marrow. To overcome these obstacles, the administration of the radionuclide was separated from that of the antibody by using an anti-IL-2 receptor ␣ antibody single chain Fv-streptavidin fusion protein, followed by radiolabeled biotin to treat lymphoma or leukemia xenografted mice. This Pretarget approach provided extremely rapid and effective tumor targeting, permitting the use of short-lived ␣-emitting radionuclides. With the -emitter 90 Y, all of the 10 lymphoma-xenografted mice were cured. With the ␣-emitter 213 Bi, significant efficacy was obtained in treating leukemic mice, and, furthermore, when combined with immunotherapy, 7 of 10 leukemic mice were cured. Thus, Pretarget radioimmunotherapy is very promising and could represent the next generation in the treatment of lymphoma and leukemia.
The studies reported herein demonstrate the efficacy of ␣-particle-targeted radiation therapy of peritoneal disease with Herceptin as the targeting vehicle. Using the CHX-A-DTPA linker, Herceptin was radiolabeled with indium-111 and bismuth-213 with high efficiency without compromising immunoreactivity. A pilot radioimmunotherapy study treating mice bearing 5-day LS-174T (i.p.) xenografts, a low but uniform HER2 expressing, human colon carcinoma, with a single dose of 213 Bi-CHX-A"-Herceptin, proved disappointing. This defined the effect of tumor burden/size on tumor response to radioimmunotherapy with ␣-radiation. A more successful experiment with a lower tumor burden (3 days) in mice followed. A specific dose-response (P ؍ 0.009) was observed, and although a maximum-tolerated dose was not determined, a dose of 500 to 750 Ci was selected as the operating dose for future experiments based on changes in animal weight. Median survival was increased from 20.5 days for the mock-treated mice to 43 and 59 days with 500 and 750 Ci, respectively. The therapeutic effectiveness of 213 Bi-CHX-A"-Herceptin was also evaluated in a second animal model for peritoneal disease with a human pancreatic carcinoma (Shaw). The results of this study were not as dramatic as with the former model, and higher doses were required to obtain an increase in survival of the mice (P ؍ 0.001).
The monoclonal antibody cetuximab binds to EGFR and thus provides an opportunity to create both imaging and therapies that target this receptor. The potential of cetuximab as a radioimmunoconjugate using the acyclic bifunctional chelator, CHX-A”-DTPA was investigated. The pharmacokinetic behavior in the blood was determined in mice with and without tumors. Tumor targeting and scintigraphic imaging were evaluated in mice bearing xenografts of LS-174T (colorectal), SHAW (pancreatic), SKOV3 (ovarian), DU145 (prostate) and HT-29 (colorectal). Excellent tumor targeting was observed in each of the models with peak tumor uptakes of 59.8±18.1, 22.5±4.7, 33.3±5.7, 18.2±7.8 and 41.7±10.8 %ID/g at 48-72 hr, respectively. In contrast, the highest tumor %ID/g obtained in mice bearing melanoma (A375) xenografts was 6.3±1.1 at 72 hr. The biodistribution of 111In-cetuximab was also evaluated in non-tumor bearing mice. The highest %ID/g was observed in the liver (9.3±1.3 at 24 hr) and the salivary glands 8.1±2.8 at 72 hr). Scintigraphy showed excellent tumor targeting at 24 hr. Blood pool was evident as expected but cleared over time. At 168 hr the tumor was clearly discernible with negligible background.
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