Purpose: The expression of extra domain B (ED-B) fibronectin is always associated with angiogenic processes and can be exclusively observed in tissues undergoing growth and/or extensive remodeling. Due to this selective expression, ED-B fibronectin is an interesting target for radioimmunotherapy of malignant diseases. The aim of this study was to identify the most appropriate ED-B-targeting radioimmunoconjugate for the therapy of solid tumors. Experimental Design: Three ED-B fibronectin-binding human antibody formats of L19 were investigated: dimeric single-chain Fv (f50 kDa), ''small immunoprotein'' (SIP, f80 kDa), and immunoglobulin G1 (IgG1, f150 kDa). These L19 derivatives were either labeled with I-125 or with In-111 (using MX-diethylenetriaminepentaacetic acid, MX-DTPA). Pharmacokinetics and tumor accumulation of the radiolabeled immunoconjugates were investigated in F9 (murine teratocarcinoma) tumor-bearing mice. Subsequently, dosimetry for the corresponding therapeutic isotopes I-13-1andY-90 was done. After testing the myelotoxicity of I-131-L19-SIP and I-131-L19-IgG1in non-tumor-bearing mice, the therapeutic efficacy of these iodinated antibody formats was finally investigated in F9 tumor-bearing mice. Results: The most favorable therapeutic index was found for I-131-L19-SIP followed by I-131-L19-IgG1. The therapeutic index of all In-111-labeled derivatives was significantly inferior. Considering the bone marrow as the dose-limiting organ, it was calculated that activities of 74 MBq I-131-L19-SIP and 25 MBq I-131-L19-IgG1 could be injected per mouse without causing severe myelotoxicity. The best therapeutic efficacy was observed using I-131-L19-SIP, resulting in significant tumor growth delay and prolonged survival after a single injection. Conclusion: Compared with other L19-based radioimmunoconjugates, I-131-L19-SIP is characterized by superior antitumor efficacy and toxicity profile in the F9 teratocarcinoma animal model. These results indicate that ED-B fibronectin-targeted radioimmunotherapy using I-131-L19-SIP has potential to be applied to treatment of solid cancers.

The successful use of peptides as potential radiopharmaceuticals essentially requires the modification of the bioactive peptide hormones to introduce chelators for radiolabeling. In this study, four Y 1/Y 2 receptor-selective NPY analogues with different receptor subtype specificities have been investigated. For in vitro studies, the cold metal surrogate was used. Gallium and indium complexes were introduced by using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid as bifunctional chelator. The peptides were synthesized by solid-phase peptide synthesis (SPPS), the chelator was coupled either at the N-terminus or at the N(epsilon) side chain of Lys(4) of the resin-bound peptide, and the labeling was performed in solution after cleavage. Competitive binding assays showed high binding affinity of the receptor-selective analogues at NPY receptor expressing cells. To test internalization of the novel peptide analogues and the metabolic stability in human blood plasma, the corresponding 5(6)-carboxyfluorescein (CF) analogues were prepared and investigated. One of the most promising analogues, the Y 1-receptor selective [Lys(DOTA)(4), Phe(7), Pro(34)]NPY was labeled with (111)In and injected into nude mice that bear MCF-7 breast cancer xenografts, and biodistribution studies were performed. In vitro and in vivo studies suggest that receptor-selective analogues of NPY have promising characteristics for future applications in nuclear medicine for breast tumor diagnosis and therapy.

Targeted transfer of a functionally active sodium iodide symporter (NIS) into tumour cells may be used for radioiodine therapy of cancer. Therefore, we investigated radioiodine uptake in a hepatoma cell line in vitro and in vivo after transfer of the sodium iodide symporter ( hNIS) gene under the control of a tumour-specific regulatory element, the promoter of the glucose transporter 1 gene (GTI-1.3). Employing a self-inactivating bicistronic retroviral vector for the transfer of the hNIS and the hygromycin resistance genes, rat Morris hepatoma (MH3924A) cells were infected with retroviral particles and hNIS-expressing cell lines were generated by hygromycin selection. (125)I(-) uptake and efflux were determined in genetically modified and wild type hepatoma cells. In addition, the iodide distribution in rats bearing wild type and genetically modified hepatomas was monitored. hNIS-expressing MH3924A cell lines accumulated up to 30 times more iodide than wild type hepatoma cells, with a maximal iodide uptake after 30 min incubation time. Competition experiments in the presence of sodium perchlorate revealed a decrease in the iodide uptake (80-84% decrease). Moreover, ouabain led to a loss of accumulated I(-) (81% decrease) whereas 4,4'-diisothiocyano-2,2'-disulphonic acid stilbene (DIDS) increased the I(-) uptake into cells (87% increase). However, a rapid efflux of the radioactivity (70%) was observed 20 min after (125)I(-)-containing medium had been replaced by non-radioactive medium. Lithium had no significant effect on iodide efflux. In rats, the hNIS-expressing tumours accumulated 22 times more iodide than the contralateral wild type tumour. In accordance with the in vitro data, we also observed a rapid efflux of the radioactivity out of the tumour in vivo. Dosimetric calculations resulted in an absorbed dose of 85 mGy in the wild type tumour and 830 mGy in the hNIS-expressing tumour after administration of 18.5 MBq (131)I. In conclusion, transduction of the hNIS gene under the control of the GLUT1 promoter element induces iodide transport in Morris hepatoma cells in vitro and in vivo. However, for therapeutic application additional conditions need to be defined which inhibit the iodide efflux out of the tumour cells.

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Suicide gene therapy of malignant melanoma essentially requires efficient gene transfer and highly selective therapeutic gene expression. To achieve this, recombinant adeno-associated virus (rAAV) particles were constructed containing the tissue-specific promoter of the human melanoma inhibitory activity (hMIA) gene combined with four copies of the enhancer element of the murine tyrosinase gene. Three melanoma and one cervix carcinoma cell line were infected with rAAV particles carrying a reporter gene under control of the enhancer/hMIA promoter in order to determine transcriptional activity and specificity of this system. Viral particles containing the enhancer/hMIA promoter mediated reporter gene activity only in melanoma cells, whereas infection with a cytomegalovirus (CMV)-based promoter construct induced unspecific gene expression. Correspondingly, transient transduction with viral particles bearing the HSVtk gene under the control of the enhancer/MIA promoter elements followed by treatment with ganciclovir (GCV) resulted in growth inhibition only in melanoma cells, whereas the CMV promoter-based construct induced unspecific cytotoxicity. In vivo experiments in nude mice demonstrated that tumors originating from human melanoma cells disappeared after stable, but not transient transduction with vectors bearing the HSVtk gene under the control of the enhancer/hMIA promoter in response to GCV application. In face of higher transduction efficiency, these rAAV particles might therefore be a useful tool for suicide gene therapy of malignant melanoma.

In order to achieve tumor-specific targeting of adeno-associated virus (AAV)-mediated gene expression, the promoter of the glucose transporter isoform 1 (GLUT1) gene was cloned upstream of the enhanced green fluorescence protein (EGFP) and the herpes simplex virus thymidine kinase (HSVtk) gene. FACS analysis performed at 48 h after transient infection with rAAV/cytomegalovirus (CMV)egfp viral particles revealed an increase of fluorescence in all the cell lines tested. However, EGFP expression under control of the GLUT1 promoter element (rAAV/GTI-1.3egfp) was limited to the tumor cells and oncogene-transformed cells. Evidence for phosphorylation of the HSVtk substrates ganciclovir (GCV) and 125 I-deoxycytidine was found in all transfected tumor cell lines compared to noninfected controls (HCT116: 111%; MH3924A: 130%; HaCaT-RT3: 257% increase), but not in HaCaT and HUVEC cells. Furthermore, tumor cells and the oncogene-transformed (ras) cell line HaCaT-RT3 showed a GCV-induced reduction in cell number (HCT116: À71%; MH3924A: À43% and HaCaT-RT3: À31%). No statistically relevant cytotoxic effect was observed in HaCaT (6% decrease) and HUVEC cells (2% decrease). Furthermore, a reduction of 3 H-thymidine incorporation into the DNA was seen after treatment with GCV (HCT116: 38%; MH3924A: 33% and HaCaT-RT3: 37% decrease). In a therapy study of HSVtkexpressing tumors with GCV, we achieved total tumor remission.