Preliminary evaluation of Lu-177-labeled knottin peptides for integrin receptor-targeted radionuclide therapy

Department of Nuclear Medicine, Shanghai Ruijin Hospital, Shanghai Jiaotong University, 2nd Ruijin Rd, Shanghai, 200025, People's Republic of China.
European Journal of Nuclear Medicine (Impact Factor: 5.38). 12/2010; 38(4):613-22. DOI: 10.1007/s00259-010-1684-x
Source: PubMed


Cystine knot peptides (knottins) 2.5D and 2.5F were recently engineered to bind integrin receptors with high affinity and specificity. These receptors are overexpressed on the surface of a variety of malignant human tumor cells and tumor neovasculature. In this study, 2.5D and 2.5F were labeled with a therapeutic radionuclide, (177)Lu, and the resulting radiopeptides were then evaluated as potential radiotherapeutic agents in a murine model of human glioma xenografts.
Knottins 2.5D and 2.5F were synthesized using solid phase peptide synthesis, folded in vitro, and site-specifically coupled with 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) at their N terminus for (177)Lu radiolabeling. The stability of the radiopeptides (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F was tested in both phosphate-buffered saline (PBS) and mouse serum. Cell uptake assays of the radiolabeled peptides were performed in U87MG integrin-expressing human glioma cells. The biodistribution studies of both (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F were examined in U87MG tumor-bearing athymic nu/nu mice. Radiation absorbed doses for the major tissues of a human adult male were calculated based on the mouse biodistribution results.
DOTA-2.5D and DOTA-2.5F were labeled with (177)Lu at over 55% efficiency. High radiochemical purity for both radiocomplexes (> 95%) could be achieved after high performance liquid chromatography (HPLC) purification. Both radiopeptides were stable in PBS and mouse serum. Compared to (177)Lu-DOTA-2.5D (0.39 and 0.26 %ID/g at 2 and 24 h, respectively), (177)Lu-DOTA-2.5F showed much higher tumor uptake (2.16 and 0.78 %ID/g at 2 and 24 h, respectively). It also displayed higher tumor to blood ratios than that of (177)Lu-DOTA-2.5D (31.8 vs 18.7 at 24 h and 52.6 vs 20.6 at 72 h). Calculation of radiodosimetry for (177)Lu-DOTA-2.5D and (177)Lu-DOTA-2.5F suggested that tumor and kidney were tissues with the highest radiation absorbed doses. Moreover, (177)Lu-DOTA-2.5F had a higher tumor to kidney radiation absorbed dose ratio than that of (177)Lu-DOTA-2.5D.
Cystine knot peptides can be successfully radiolabeled with (177)Lu for potential therapeutic applications. Knottin 2.5F labeled with (177)Lu exhibits favorable distribution in murine U87MG xenograft model; thus, it is a promising agent for radionuclide therapy of integrin-positive tumors.

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    • "The power of theranostics is already established in the clinic where 68 Ga/ 177 Lu-labeled peptides targeting the somatostain receptor are successfully being used for imaging and treatment of neuroendocrine tumors [18]. Several new targets are currently being evaluated in pre-clinical cancer models including integrins [19], HER-2 [20] [21], gastrin-releasing peptide (GRP) [22] and vascular endothelial growth factor (VEGF) [23]. 177 Lu is a low-energy betaemitter (~1.5 mm maximum penetration in soft tissue) capable of inducing cytotoxic effects in tumors but not surrounding tissue by providing both a 'cross-fire'-and 'bystander' effect from direct betaparticles and Auger electrons, respectively [24]. "
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    ABSTRACT: The proposition of uPAR as a potential target in cancer therapy is advanced by its predominant expression at the invasive front of colorectal cancer (CRC) and its value as prognostic biomarker for poor survival in this disease. In this study, we provide the first in vivo proof-of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. A DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with (64)Cu and (177)Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29 cells were inoculated in Nude mice and treated with (177)Lu-DOTA-AE105 once a visible tumor had formed. To evaluate the true effect of the targeted radiotherapy, two controls groups were included in this study, one receiving a (177)Lu-labeled non-binding control peptide and one receiving vehicle. All animals were treated day 0 and 7. A parallel (18)F-FLT PET/CT study was performed on day 0, 1, 3 and 6. Dosimetry calculations were based on a biodistribution study, where organs and tissue of interest were collected 0.5, 1.0, 2.0, 4.0 and 24h post injection of (177)Lu-DOTA-AE105. Toxicity was assessed by recording mouse weight and by H&E staining of kidneys in each treatment group. uPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using (64)Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using (177)Lu-DOTA-AE105, where a significant effect on tumor size and the number of uPAR-positive cells in the tumor was found (p<0.05). Evaluations of biodistribution and dosimetry revealed highest accumulation of radioactivity in kidneys and tumor tissue. (18)F-FLT PET/CT imaging study revealed a significant correlation between (18)F-FLT tumor uptake and efficacy of the radionuclide therapy. A histological examination of the kidneys from one animal in each treatment group did not reveal any gross abnormalities and the general performance of all treated animals also showed no indications of radioactivity-induced toxicity. These findings document for the first time the in vivo efficacy of an uPAR-targeted radionuclide therapeutic intervention on both tumor size and its content of uPAR expressing cells thus setting the stage for future translation into clinical use.
    Full-text · Article · Jun 2012 · Nuclear Medicine and Biology
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    • "But due to the lower tumor uptake of the RGD monomer, multiple-dose administration was necessary to achieve therapeutic efficacy [106]. Recently, 177Lu labeled two knottin peptides (2.5D and 2.5F), and RGD peptides targeting a range of integrins (αvβ3/αvβ5/α5β) were tested for potential radiotherapy in a mouse model of human glioma [107, 108]. 177Lu-DOTA-2.5F "
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    Full-text · Article · May 2012
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    • "Several knottin mutants that bind to integrin receptors (αvβ3/αvβ5 or αvβ3/αvβ5/α5β1) with low nanomolar affinity have been identified 51, 52, and radionuclide and optical dye labeled such knottin peptides have demonstrated favorable in vivo tumor targeting properties 53-56. Recently, two knottin peptides (2.5D and 2.5F: targeting integrin αvβ3/αvβ5 and αvβ3/αvβ5/α5β1, respectively) were radiolabeled with a therapeutic radionuclide 177Lu, and the resulting radiopharmaceuticals were evaluated for potential radiotherapy in a mouse model of human glioma 57. Compared to 177Lu-DOTA-2.5D, "
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