(177)Lu/ (90)Y intermediate-affinity monoclonal antibodies targeting EGFR and HER2/c-neu: preparation and preclinical evaluation.

The epidermal growth factor receptor (EGFR) is a rational target of anticancer therapies due to its overexpression in a variety of malignant epithelial tumors. Nevertheless, this antigen is also present in normal tissues. Consequently, monoclonal antibodies which selectively bind to EGFR-overexpressing tumors will be choice drug candidates for development of radioimmunoconjugates (RIC). Nimotuzumab (h-R3) and trastuzumab are monoclonal antibodies (mAbs) which would preferentially target tissues with EGFR and HER2 overexpression, respectively. In this chapter, we describe preparation and evaluation of the targeting properties of RIC formed by (177)Lu/(90)Y and monoclonal antibodies which selectively target EGFR- and HER2/c-neu-overexpressing tissues. mAbs were labeled with n.c.a. (177)Lu/(90)Y using bifunctional chelating agents. RIC binding properties and toxicity were evaluated in vitro using cell lines with varying antigen expression. In vivo tumor targeting properties of RIC were evaluated in mice bearing colorectal (SNU-C2B) and A431 tumor xenografts. RICs were prepared with specific activities up to 2 GBq/mg without significant loss in biological activity. (90)Y-h-R3/trastuzumab increased cell growth inhibition compared with unmodified mAbs or (90)YCl(3) alone in cell lines with overexpression of the target antigen. (177)Lu-h-R3 showed significantly higher uptake in A431 (22.8 ± 3.1% ID/g) than in SNU-C2B (8.8 ± 4.1% ID/g) xenografts at 72 h post injection, indicating strong association between tumor uptake and EGFR expression levels.

Measurement of protein synthesis: in vitro comparison of (68)Ga-DOTA-puromycin, [ (3)H]tyrosine, and 2-fluoro-[ (3)H]tyrosine.

AIM: Puromycin has played an important role in our understanding of the eukaryotic ribosome and protein synthesis. It has been known for more than 40 years that this antibiotic is a universal protein synthesis inhibitor that acts as a structural analog of an aminoacyl-transfer RNA (aa-tRNA) in eukaryotic ribosomes. Due to the role of enzymes and their synthesis in situations of need (DNA damage, e.g., after chemo- or radiation therapy), determination of protein synthesis is important for control of antitumor therapy, to enhance long-term survival of tumor patients, and to minimize side-effects of therapy. Multiple attempts to reach this goal have been made through the last decades, mostly using radiolabeled amino acids, with limited or unsatisfactory success. The aim of this study is to estimate the possibility of determining protein synthesis ratios by using (68)Ga-DOTA-puromycin ((68)Ga-DOTA-Pur), [(3)H]tyrosine, and 2-fluoro-[(3)H]tyrosine and to estimate the possibility of different pathways due to the fluorination of tyrosine. METHODS: DOTA-puromycin was synthesized using a puromycin-tethered controlled-pore glass (CPG) support by the usual protocol for automated DNA and RNA synthesis following our design. (68)Ga was obtained from a (68)Ge/(68)Ga generator as described previously by Zhernosekov et al. (J Nucl Med 48:1741-1748, 2007). The purified eluate was used for labeling of DOTA-puromycin at 95°C for 20 min. [(3)H]Tyrosine and 2-fluoro-[(3)H]tyrosine of the highest purity available were purchased from Moravek (Bera, USA) or Amersham Biosciences (Hammersmith, UK). In vitro uptake and protein incorporation as well as in vitro inhibition experiments using cycloheximide to inhibit protein synthesis were carried out for all three substances in DU145 prostate carcinoma cells (ATCC, USA). (68)Ga-DOTA-Pur was additionally used for µPET imaging of Walker carcinomas and AT1 tumors in rats. Dynamic scans were performed for 45 min after IV application (tail vein) of 20-25 MBq (68)Ga-DOTA-Pur. RESULTS: No significant differences in the behavior of [(3)H]tyrosine and 2-fluoro-[(3)H]tyrosine were observed. Uptake of both tyrosine derivatives was decreased by inhibition of protein synthesis, but only to a level of 45-55% of initial uptake, indicating no direct link between tyrosine uptake and protein synthesis. In contrast, (68)Ga-DOTA-Pur uptake was directly linked to ribosomal activity and, therefore, to protein synthesis. (68)Ga-DOTA-Pur µPET imaging in rats revealed high tumor-to-background ratios and clearly defined regions of interest in the investigated tumors. SUMMARY: Whereas the metabolic pathway of (68)Ga-DOTA-Pur is directly connected with the process of protein synthesis and shows high tumor uptake during µPET imaging, neither [(3)H]tyrosine nor 2-fluoro-[(3)H]tyrosine can be considered useful for determination of protein synthesis.

Radiosynoviorthesis of knees by means of 166Ho-holmium-boro-macroaggregates.

The aim of this study was to evaluate adverse and therapeutic effects of applicated holmium-boro-macroaggregates (HBMAs) in the radiosynoviorthesis (RSO) of knees in patients suffering from chronic synovitis. We started RSO of the knees by means of a new radiopharmaceutical (RF) HBMA in patients with gonarthrosis, rheumatoid arthritis, chronic synovitis, psoriatic arthritis, and gout arthropathy. Seventeen (17) intra-articular injections were performed in 15 patients who were receiving a mean activity of 972 MBq (range, 904-1057) of 166Ho-HBMA. Patient inclusion to the study followed a series of inclusion and exclusion criterions. The patients were hospitalized for 3 days. Side-effects were evaluated during their hospital stay and again after 6-8 weeks. Static scintigraphy of knee joints and measurements of blood radioactivity were performed. Therapeutic effects were evaluated after 6-8 weeks and at 6 months. In 2 hours and 2 days following the application, we proved, by means of knee and inguinal scintigraphy, only insignificant radiopharmaceutical leakage from the joint cavity to the inguinal lymph nodes in 4 patients. In the treated patients, no serious adverse effects occurred. Nine (9) patients were without complaints, 4 patients had slight knee exudation, and 2 patients had great exudation. Therapeutic effects were as follows: 2 patients were without pain, 9 were with lower pain, 3 were with the same pain, and 1 patient was with increased pain. Joint motion was improved in 7 patients, remained the same in 7 patients, and was impaired in 1 patient. Analgesics consumption was lower in 5 patients, the same in 9 patients, and greater in 1 patient. Knee exudation was absent in 2 patients, lower in 4 patients, the same in 6 patients, and greater in 3 patients. In 3 patients it was necessary to do surgical RSO. This RF can extend the range of clinically used radiopharmaceuticals for RSO and to supplement space between 90Y with high energy and 186Re with 169Er with lower beta energy. The energy of 166Ho is suitable for great and medium joints (i.e., knees, hips, shoulders, elbows, wrists, and ankles).

Preparation of Holmium-166 Labelled Macroaggregates for radionuclide synovectomy.

BACKGROUND: Radionuclide synovectomy (radiation synovectomy) is an alternative method that cures patients with rheumatoid arthritis diseases without surgery. During treatment, the suspension of the (166)Ho-macroaggregates radioactive particles ((166)Ho-MA) is administrated via intra-articular injection into the target joint to destroy the inflamed synovium. MATERIAL AND METHODS: The isotope of (166)Ho (E(beta) max = 1.84 MeV, T((1/2)) = 26.8 hr) was prepared by the (165)Ho(n, gamma)(166)Ho reaction in the LWR-15 nuclear reactor (8-10 MW) using approximate neutron flux 10(14) cm-2s-1. RESULTS: The particles of Ho-Macroaggregates with suitable dimension 1-20 microm and the mean diameter of 8.5 microm were prepared. High in-vitro stability was obtained after incubation of neutron-irradiated Ho-Macroaggregates in saline solution (0.9% NaCl). The in-vivo stability on rats was verified as well. CONCLUSIONS: High in-vivo and in-vitro stability as well as supporting gamma radiation of Ho-166 make the Macroaggregates a prospective agent for radionuclide synovectomy. The method of preparation is relatively easy and allows for the production of particles of a suitable dimension with a sufficient amount of radioactivity of Ho-166 for the treatment of the rheumatoid arthritis.

Preparation and preclinical evaluation of 177Lu-nimotuzumab targeting epidermal growth factor receptor overexpressing tumors.

OBJECTIVES: Nimotuzumab (h-R3) is a humanized monoclonal antibody (mAb) which recognizes the external domain of the epidermal growth factor receptor (EGFR) with high specificity. It was demonstrated that h-R3 has a unique clinical profile for immunotherapy of adult gliomas and pediatric pontine gliomas. The aim of this work was to evaluate the conjugate (177)Lu-h-R3 as a potential radioimmunoconjugate for radioimmunotherapy (RIT) of tumors overexpressing EGFR. METHODS: h-R3 was modified with the macrocylcic ligand S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA) and the acyclic ligand S-2-(4-Isothiocyanatobenzyl)-diethylenetriamine pentaacetic acid (p-SCN-Bn-DTPA); the immunoconjugates were labeled with no-carried added (177)Lu. Specificity and affinity were tested using radioimmunoassays in a cell line overexpressing EGFR. Biodistribution in mice, healthy or bearing A431 epithelial carcinoma xenografts, was performed for 11 days. Tumor uptake, the influence of the nature of the chelate and the way of administration were studied. Absorbed dose in tumor and selected organs was calculated using the OLINDA/EXM software; the data from the animals was extrapolated to humans. RESULTS: (177)Lu-h-R3 conjugates were obtained with specific activity up to 915 MBq/mg without significant loss of immunoreactivity. The binding of (177)Lu-h-R3 conjugates to A431 cells showed to be EGFR specific, and the affinity was similar to native h-R3. Tumor uptake reached a maximum value of 22.4±3.1 %ID/g at 72 h and remained ~20% ID/g over 1 week. Locoregional application showed better tumor/nontumor ratios than intravenous application. CONCLUSIONS: (177)Lu-h-R3 should be considered for further evaluations as a potential radiopharmaceutical for RIT of tumors overexpressing EGFR.

Preclinical evaluation of (177)lu-nimotuzumab: a potential tool for radioimmunotherapy of epidermal growth factor receptor-overexpressing tumors.

BACKGROUND: The humanized monoclonal antibody Nimotuzumab (h-R3) has demonstrated an exceptional and better clinical profile than other monoclonal antibodies for immunotherapy of epidermal growth factor receptor-overexpressing tumors. This work deals with the preparation and radiolabeling optimization of (177)Lu-Nimotuzumab and their preclinical evaluation. METHODS: Nimotuzumab was conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (p-SCN-Bn-DOTA), testing different molar ratios. The immunoconjugates were characterized. The radiolabeling with (177)Lu was optimized. Radioimmunoconjugates stability was tested in 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA) excess and human serum. In vitro studies were performed in tumor model cell lines. Receptor-specific binding was tested by competitive inhibition. (177)Lu-Nimotuzumab in vivo studies were conducted in healthy and xenograft animals. RESULTS: Nimotuzumab conjugates were obtained with high purity. Radiolabeling yield and specific activities ranged from 63.6% to 94.5% and from 748 to 1142 MBq/mg, respectively. The stability in DTPA excess and human serum was 95.9% and 93.2% after 10 days, respectively. The radioimmunoconjugate showed specific receptor binding in tumor cell lines. Biodistribution in healthy animals showed the typical behavior of the immunoconjugates based on monoclonal antibodies. The study in xenografts mice demonstrated uptake of (177)Lu-Nimotuzumab in the tumor and reticuloendothelial organs. CONCLUSIONS: (177)Lu-Nimotuzumab was obtained with high purity and specific activities under optimal conditions without significant loss in immunoreactivity and might be a potential radioimmunoconjugate for radioimmunotherapy of tumors with epidermal growth factor receptor overexpression.

Complexation and biodistribution study of 111In and 90Y complexes of bifunctional phosphinic acid analogs of H4dota.

In this study, the complexation rates of two new phosphinate H(4)dota (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) analogs, H(5)do3ap(PrA) and H(4)do3ap(ABn), and H(4)dota itself were compared under identical conditions (H(5)do3ap(PrA)=10-{[(2-carboxyethyl)hydroxyphosphoryl]methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid; H(4)do3ap(ABn)=10-{[(4-aminophenyl)hydroxyphosphoryl]methyl}-1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid). The biodistribution of their (111)In and (90)Y complexes in healthy rats was also studied. Unlike the observation obtained under "chemical" conditions where differences between the ligands were observed no such differences in complexation rates were found under radiochemical conditions. The ligands bind the radiometals similarly to H(4)dota. So, "chemical" formation kinetic data should be transferred into radiochemical conditions only with high care and "radiochemical" complexation experiments should be run as part of standard in vitro studies with new ligands considered as potential radiopharmaceuticals. Pharmacokinetic studies in rats showed similar distribution characteristics for both phosphinate H(4)dota analogs radiolabelled with (111)In and (90)Y when compared with that of the (111)In-H(4)dota complex. No specific uptake in any organ and tissue of rats was determined. The phosphinate complexes are not accumulated in calcified tissues.