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A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A(165)), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.

Purpose: Neurotensin (NT) and its high affinity receptor (NTR1) are involved in several neoplastic processes. Thus, NT-based radiopharmaceuticals are potential tracers for targeted diagnosis and therapy of NTR-positive tumours. A new analogue based on NT(8-13), NT-XIX, with the three enzymatic cleavage sites stabilised, was synthesised and tested., Methods: The synthesis was performed by Boc strategy. Labelling with (99m)Tc/(188)Re was performed using the tricarbonyl technique. Metabolic stability was tested in vitro and in vivo. NT-XIX was further characterised in vitro in HT-29 cells and in vivo in nude mice with HT-29 xenografts., Results: NT-XIX showed much longer half-lives than non-stabilised analogues. Binding to NTR1 was highly specific, although the affinity was lower than that of natural NT. Bound activity rapidly internalised into HT-29 cells and 50% remained trapped after 24 h. In the time-course biodistribution, the highest uptake was found in the tumour at all p.i. times. In vivo uptake was specific, and accumulation of activity in the kidneys was low. Radioactivity clearance from healthy organs was faster than that from the tumour, resulting in improved tumour-to-tissue ratios and good SPECT/CT imaging. Treatment with (188)Re-NT-XIX (30 MBq, in three or four fractions) decreased tumour growth by 50% after 3 weeks., Conclusion: The high in vivo stability and the favourable in vivo behaviour makes NT-XIX an excellent candidate for the imaging and therapy of NTR1-positive tumours.

Radiolabeled bombesin (BBS) analogues are promising pharmaceuticals for imaging of cancer cells expressing gastrin-releasing peptide receptors (GRPR). However, most of the radiolabeled BBS derivatives show a high accumulation of activity in the liver and a strong hepatobiliary excretion, both unfavorable for imaging and therapy of abdominal lesions. For this reason, we introduced hydrophilic carbohydrated linker moieties into our BBS analogues to reduce the abdominal accumulation and to improve the tumor-to-background ratios. A stabilized BBS(7-14) sequence bearing the (NalphaHis)Ac-chelator was modified with amino acid linkers containing a lysine or propargylglycine residue. The epsilon-amino group of a lysine was either coupled to shikimic acid or reacted with glucose to form the Amadori conjugate. Alternatively, a glucose was attached to the peptide via "click" chemistry with the propargylglycine side chain. The peptides were synthesized on Rink amide resin using solid-phase peptide synthesis and labeled with 99mTc using the tricarbonyl technique. Binding and degradation were tested in Vitro in GRPR-expressing PC-3 cells. Biodistribution and SPECT/CT imaging studies were performed in nude mice bearing PC-3 tumor xenografts. The new peptides showed a log D between -0.2 and -0.5 and kept the high affinity for GRPR with Kd values of <0.5 nM. In Vitro, they were rapidly internalized into the tumor cells and showed an increased cellular retention and stability (t(1/2 )>35 min). In ViVo, all new compounds exhibited higher tumor-to-background ratios compared to the nonglycated reference. Thus, the best results were obtained with the triazole coupled glucose with a 4-fold increased uptake and retention in tumor tissue (3.6 and 2.5%ID/g at 1.5 h and 5 h p.i, respectively) and a significantly reduced accumulation in the liver (0.6 vs 2.4%ID/g, 1.5 h p.i., respectively). Apart from higher tumor-to-liver ratios (17-fold, 1.5 h p.i.), both tumor-to-kidney and tumor-to-blood ratios could be significantly improved by a factor of 1.5 and 2.7, respectively (1.5 h p.i., P<0.05). The imaging studies proved the reduction of abdominal background, and tumor xenografts could clearly be visualized. In conclusion, the introduction of a carbohydrated linker substantially improved the biodistribution properties of BBS analogues labeled with the 99mTc-tricarbonyl core.

Unlabelled: (99m)Tc-technetium ((99m)Tc) and (188)Re-rhenium ((188)Re) represent an interesting pair of radionuclides for diagnosis and therapy. The aim of this study was to synthesize and characterize in vitro/in vivo the first (188)Re-folate derivative [(188)Re(CO)(3)-picolylamine monoacetic acid 188/Re-OANA-folate (2)] for potential targeted radionuclide therapy of FR-positive tumors. The data were compared with those of the isostructural (99m)Tc-analog [(99m)Tc-PAMA folate (1)] reported previously., Methods: In vitro stability of compound 2 was tested in phosphate-buffered saline and human plasma. Cell binding experiments were performed with FR-positive human KB cells. Biodistribution was assessed in female nude mice, bearing KB tumor xenografts., Results: Cell binding experiments showed high and FR-specific uptake. In vivo, compound 2 accumulated specifically in the FR-positive tumors with maximal values 4 h post injection (p.i.) ['2: 1.87+/-0.04 percent injected dose per gram of weight tissue (% ID/g) vs. '1: 2.33+/-0.36% ID/g]. Unfavorably high retention of radioactivity was found in FR-positive kidneys (12.04+/-0.62% ID/g; 4 h p.i.). Tumor-to-blood ratio of radioactivity ('2: 14.5+/-1.32, 4 h p.i.) was lower than for compound '1 (58.0+/-12.2, 4 h p.i.), whereas tumor-to-kidney ratios were in the same range ('2: 0.15+/-0.01 vs. '1: 0.13+/-0.02, 4 h p.i.). Preadministration of the antifolate pemetrexed significantly improved the tumor-to-kidney ratio (2: 1.59+/-0.30, 4 h p.i.)., Conclusions: The isostructural radiofolates 1 and '2 displayed almost identical pharmacokinetic profiles and accumulated both specifically in FR-positive tumors. However, only the coapplication of the antifolate pemetrexed improved the biodistribution of the radiotracers in such ways that a potential therapeutic application of compound '2 can be envisaged in the future.

Purpose: For the assessment of folate-based radiopharmaceuticals, human nasopharyngeal KB carcinoma cells are traditionally used although nasopharyngeal cancer is rare. On the other hand, the folate receptor (FR) is frequently overexpressed on diverse cancer types, the highest frequency (>90%) being on ovarian carcinomas. The goal of our study was the in vitro and in vivo assessment of different FR-positive human carcinoma cells. In addition, a murine sarcoma cell line was assessed as a pre-clinical alternative to human xenograft models., Methods: FR-positive human nasopharyngeal, cervical, ovarian and colorectal cancer cell lines and the transgenic mouse sarcoma (24JK-FBP) cell line were targeted with a novel 99mTc-tricarbonyl folate derivative 2. Comparative in vitro cell binding studies were carried out under standardised folate-deficient conditions. In vivo studies were performed in nude mice and C6 black mice., Results: The in vitro cell experiments revealed only FR-specific binding (unspecific <0.02%), ranging from 3.5% to 52% of complex 2 owing to variable levels of FR expression of the cell lines. In vivo tumour uptake of radiotracer 2 varied less than in vitro. It ranged from 0.66+/-0.17% ID/g (LoVo) through 1.16+/-0.64% ID/g (IGROV-1) and 1.55+/-0.43% ID/g (24JK-FBP) to 2.33+/-0.36% ID/g (KB) 4 h p.i., Conclusion: These pre-clinical studies indicate that in vitro data obtained in FR-positive cancer cells do not necessarily correspond with or predict in vivo radiofolate uptake in corresponding (xeno)grafts. In addition, the murine 24JK-FBP cell line proved to be a valuable pre-clinical alternative to human tumour models.

Two neuropeptides, bombesin (BBS) and neurotensin (NT) and their radiolabeled analogues, have great potential for tumour targeting, either for diagnosis (e.g., with 99mTc) or therapy (e.g., with 90Y or 188Re). In this study, we investigated NT(8-13) and BBS(7-14) analogues with Nalpha-histidinyl acetate linked to the N-terminus of the peptide. This His-derivative forms a stable and inert tridentate complex with the 99mTc(CO)3 and the 188Re(CO)3 moieties. The stability of 99mTc-labeled neurotensin and bombesin analogues was tested in human plasma samples and in tumour cell cultures in the presence and absence of specific enzyme inhibitors. The inhibitor of ACE (angiotensin converting enzyme) was the most effective in inhibiting the peptide cleavage of both NT(8-13) and BBS(7-14). In agreement with this finding, the replacement of Ile12 by tert-leucine (NT) and Leu13 by cyclohexylalanin (BBS) brought about a better stability. With NT(8-13) analogues, higher tumour to nontarget (t/nt) ratios and the same affinity to the receptor was observed, but with BBS(7-14) derivatives the affinity was lower and the t/nt ratio was not significantly improved. Toxicity tests showed no effect in mice of up to a five-hundred-fold higher dose than planned for patient application, which started successfully with NT(8-13) analogues.

The application of the beta particle-emitting nuclide 67Cu in radioimmunotherapy is reviewed. The production of the nuclide is outlined, and different production modes are discussed with an emphasis on cyclotron production. A short survey of copper chelators currently used for antibody labelling and their impact on the pharmacokinetics of 67Cu-labelled immunoconjugates is provided. Protocols for antibody labelling with 67Cu as well as quality control procedures for 67Cu-labelled antibodies are described. Preclinical data on the biological properties of 67Cu-labelled immunoconjugates are reported and discussed. 67Cu-labelled antibodies show higher and more persistent tumour uptake than their radioiodinated counterparts due to accumulation of labelled metabolites in tumour cells. Biodistribution of 67Cu-labelled antibody fragments has been improved by selection of negatively charged chelators and peptide linkers. Pharmacokinetic analysis of the accumulated dose in tumour and critical organs such as the kidney and liver indicates that, despite this improvement, intact 67Cu-labelled antibodies achieve higher tumour uptake and better therapeutic ratios than 67Cu-labelled antibody fragments and that they are at present the logical choice for clinical studies. Clinical studies using 67Cu-labelled antibodies in lymphoma, colon carcinoma and bladder cancer patients are reviewed. Some of the advantages over radioiodinated antibodies found in the preclinical work, such as higher tumour uptake and better tumour/blood ratios, have also been found with systemic application in lymphoma and colon carcinoma. However, in both lymphoma and colon carcinoma patients, the radiation dose to the liver has been found to be higher from 67Cu- than from 131I-labelled antibodies. The intravesical application of 67Cu-labelled antibody has been shown to be a promising approach for targetting cytotoxic radiation to superficial bladder tumours, without detectable systemic absorption. Given the favourable properties of 67Cu-labelled antibodies, it is the reliable availability of the 67Cu nuclide which is the limiting factor for their more widespread evaluation in radioimmunotherapy trials.