Progress 01/01/03 to 06/30/04
Outputs
Introduction: Copper-64-labeled monoclonal antibodies (mAbs) have caused complete tumor responses at very low radiation doses in rodent models. This project addressed three Specific Aims: 1) Optimize the conjugation and labeling chemistry of and internalizing monoclonal antibody (mAb), cBR96, and a non-internalizing mAb, cT84.66, and perform 24 hour pilot biodistributions; 2) Determine comprehensive biodistributions for tumor dosimetry and confirm in vitro internalization and efflux characteristics for both mAbs; 3) Perform a radioimmunotherapy (RIT) trial comparing 64Cu-labeled cBR96 and cT84.66. This model was designed to test the hypothesis that cellular internalization is critical in causing tumor cell death at low radiation doses. Methods: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was conjugated to cBR96 and cT84.66. Conjugates were labeled with 64Cu citrate, pH 5.5, at 43C for 1 hour and purified by spin-column gel filtration chromatography.
Serum stability, specific activity of labeling, radiochemical purity, and immunoreactivity were determined. Biodistributions were performed in female nude mice bearing LS174T colon carcinoma xenografts at time points over 48 hours for both mAbs (n=5 per time point). An RIT trial was performed with control groups (n=10) treated with saline, unlabeled DOTA-cBR96 or DOTA-cT84.66 and experimental groups (n=9) treated with 0.89 mCi 64Cu-DOTA-cBR96 or 0.71 mCi 64Cu-DOTA-cT84.66. Two groups (n=3) treated with therapeutic doses were imaged with co-registered positron emission tomography (PET) and computed tomography (CT) at points over 48 h. Results: Conjugates labeled near-quantitatively to 15mCi/mg with >99% radiochemical purity. Serum stability and immunoreactivity were >99%. The biodistributions of the 64Cu-labeled mAbs was statistically similar. Liver, spleen, kidney, and tumor uptakes were similar at 24 h for both conjugates (p = 0.05). Tumor absorbed radiation doses were estimated to
be 1128 rad/mCi for 64Cu-DOTA-cBR96 and 1409 rad/mCi for 64Cu-DOTA-cT84.66. The therapy study revealed a statistically significant difference (P<0.0001) between experimental and control groups, but no difference between the 64Cu-labeled mAbs. PET imaging confirmed the similarity of the tumor doses between the mAbs. Conclusions: The in vitro and biodistribution data establish the suitability of this two-antibody model of colon cancer to evaluate 64Cu-RIT. The results of the therapy study suggest that internalization is not the only critical step in cytotoxicity of 64Cu radiopharmaceuticals. The PET dosimetry data establishes this novel technology as a potent and valid alternative to traditional biodistribution studies.
Impacts
This research examines the potential role of internalization for the literature evidence suggesting that copper-64 labeled radiopharmaceuticals cause regressions of cancer in experimental models at very low tumor-absorbed radiation doses. The experiments conducted to date establish a two-antibody model for comparison of internalizing and non-internalizing antibodies in a single animal model of cancer. This research is intended to lead to the exploration and development of novel radiopharmaceuticals and radiopharmaceutical targets.
Publications
- Bryan JN, Lewis MR, Henry CJ, Owen NK, Zhang J, Mohsin H, Jia F, Sivaguru G, Anderson CJ, Development of a two-antibody model for the evaluation of copper-64 radioimmunotherapy. Veterinary and Comparative Oncology 2004; 2(2):82-90.
- Bryan JN, Jia F, Mohsin H, Sivaguru G, Miller WH, Anderson CJ, Henry CJ, and Lewis MR, Comparative uptakes and biodistributions of internalizing versus non-internalizing copper-64 radioimmunoconjugates in cell and animal models of colon cancer. Nuclear Medicine and Biology. Submitted 2005
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Progress 01/01/03 to 03/11/03
Outputs
Objectives: The radionuclide 64Cu [T1/2 = 12.7 h; Beta+ 0.655 MeV (17.4%); Beta- 0.573 MeV (39.0%)] has nuclear properties suitable for both positron emission tomography (PET) imaging and radioimmunotherapy (RIT) of cancer. In laboratory animal models of cancer, internalizing 64Cu-labeled antibodies have effected complete and durable regressions at tumor absorbed radiation doses far below those generally considered to efficacious on the basis of radiation damage alone (Connett et al., Proc. Natl. Acad. Sci. USA 93: 6814; 1996). The purpose of the present studies was to develop a two-antibody model to determine whether tumor cell internalization is critical for enhanced efficacy of 64Cu RIT. Methods: DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) was conjugated to an internalizing antibody, cBR96, and a non-internalizing antibody, cT84.66, respectively. Biodistributions of the 64Cu-labeled conjugates in nude mice bearing LS174T human colorectal
carcinoma xenografts were obtained at time points ranging from 15 min to 48 h. Mouse tumor xenograft dosimetry was calculated using a general Monte Carlo N-Particle Transport Code system (Miller et al., Trans. Am. Nucl. Soc. 89: 689; 2003). Results: The 64Cu-DOTA-cBR96 conjugate demonstrated rapid tumor uptake, reaching 20.2% injected dose per gram of tissue (% ID/g) at 3 h post-injection and peaking at 35.4% ID/g by 24 h. Tumor accumulation of 64Cu-DOTA-cT84.66 was more gradual, 8.19% ID/g at 3 h (p = 0.002), and reached 43.8% ID/g by 24 h, but maximum uptake was not statistically different from 64Cu-DOTA-cBR96 (p = 0.05). Liver, spleen, and kidney uptakes were statistically similar at 24 h for both conjugates (p = 0.05). The LS174T tumor absorbed doses were estimated to be 1128 rad/mCi (304.9 mGy/MBq) for 64Cu-DOTA-cBR96 and 1409 rad/mCi (380.8 mGy/MBq) for 64Cu-DOTA-cT84.66. Conclusion: The biodistributions and tumor dosimetry of these two radiolabeled antibodies were sufficiently
similar for direct comparison of the therapeutic efficacies of internalizing versus non-internalizing 64Cu RIT agents in the same animal model of cancer. RIT studies using this two-antibody model are currently in progress to test the hypothesis that internalization of 64Cu is necessary for enhanced cytotoxic properties at low tumor absorbed doses. These studies will also address the greater hypothesis that internalizing 64Cu radiopharmaceuticals kill tumor cells by mechanisms in addition to classical radiation damage.
Impacts
This research examines the potential role of internalization for the literature evidence suggesting that copper-64 labeled radiopharmaceuticals cause regressions of cancer in experimental models at very low tumor-absorbed radiation doses. The experiments conducted to date establish a two-antibody model for comparison of internalizing and non-internalizing antibodies in a single animal model of cancer. This research is intended to lead to the exploration and development of novel radiopharmaceuticals and radiopharmaceutical targets.
Publications
- Bryan, J.N., Lewis, M.R., Henry, C.J., Owen, N.K., Zhang, J., Mohsin, H., Jia, F., Sivaguru, G., Siegall, C.B., and Anderson, C.J., 2004, Development of a Two-Antibody Model for Evaluation of Copper-64 Radioimmunotherapy. Vet. Comp. Oncol., submitted
- Bryan, J.N., Mohsin, H., Jia, F., Sivaguru, G., Miller, W.H., Siegall, C.B., Anderson, C.J., Henry, C.J., and Lewis, M.R., 2005, Internalizing Versus Non-Internalizing Radiocopper Immunoconjugates: Comparative Tumor Cell Uptake, Biodistributions, and Dosimetry of 64Cu-DOTA-cBR96 and 64Cu-DOTA-cT84.66 in in Vitro and in Vivo Models of LS174T Human Colorectal Carcinoma, for submission to Nucl. Med. Biol
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