Preparation and preclinical characterization of 111In-DTPA-Anti-MUC1 as a radioimmunoconjugate for diagnosis of breast cancer by single-photon emission computed tomography.

Context: Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer. Aims: The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS. Settings and Design: In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported. Subjects and Methods: 111In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals. Statistical Analysis Used: Statistical analyses were performed using SPSS 10.0. Results: 111In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site. Conclusions: 111In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors.

Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors.

In this study, [111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [111 In]In-DOTA-hIgG and [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology.

La imagen nuclear con anticuerpos monoclonales radiomarcados: viejas aplicaciones con nuevos enfoques / Nuclear imaging with radiolabeled monoclonal antibodies: old applications with new aproaches

Resumen Los anticuerpos monoclonales marcados con radionucleidos fueron en sus inicios ampliamente empleados para el estudio de diversas enfermedades, fundamentalmente oncológicas mediante la inmunogammagrafía. Estos fueron poco a poco sustituidos por moléculas con mejores prestaciones como los péptidos y la 18F-fluordesoxiglucosa (18F-FDG). No obstante, en el presente siglo, la amplia introducción de la inmunoterapia produjo un cambio de paradigma en cuanto al empleo de los anticuerpos monoclonales radiomarcados para la adecuada selección y seguimiento de los pacientes a ser tratados con inmunoterapia, resurgiendo la inmunotomografía por emisión de fótón único (inmuno-SPECT), la inmunotomografía por emisión de positrones (inmuno-PET) y la imagen corregistrada con la tomografía axial computarizada (TAC), como modalidades de gran valor en el manejo del cáncer. El objetivo del presente trabajo fue brindar una panorámica acerca de la evolución de la imagen nuclear con anticuerpos monoclonales radiomarcados y sus principales aplicaciones en el tiempo, fundamentalmente en el estudio de los pacientes con cáncer.

Abstract In the beginning, radionuclide-labeled monoclonal antibodies were widely employed for the study of various diseases, mainly oncological, by immunoscintigraphy. They were gradually replaced by molecules with better performance such as peptides and 18F-FDG. However, in the present century, the wide introduction of immunotherapy produced a paradigm shift in the use of radiolabeled monoclonal antibodies for the proper selection and follow-up of patients to be treated with immunotherapy, re-emerging of the immune-single photon emission tomography (immuno-SPECT), the immune-positron emission tomography (immuno-PET) and the co-registered image with computed tomography (CT) as imaging modalities of great value in the management of cancer. The aim of the present work was to provide an overview of the evolution of nuclear imaging with radiolabeled monoclonal antibodies and their main applications over the time, mainly in the study of patients with cancer.

Preclinical characterization and validation of a dual-labeled trastuzumab-based imaging agent for diagnosing breast cancer.

OBJECTIVE: The combination of both nuclear and fluorescent reporters provides unique opportunities for noninvasive nuclear imaging with subsequent fluorescence image-guided resection and pathology. Our objective was to synthesize and optimize a dual-labeled trastuzumab-based imaging agent that can be used to validate an optical imaging agent with potential use in identifying tumor metastases in human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. METHODS: [(111)In]-DTPA-trastuzumab-IRDye 800 was synthesized by a three-step procedure. Purity, stability, immunoreactivity, internalization and biodistribution were explored in HER2+ SKBR-3 cells. Biodistribution of [(111)In]-DTPA-trastuzumab-IRDye 800 was performed in a SKBR-3 xenograft model. RESULTS: [(111)In]-DTPA-trastuzumab-IRDye 800 demonstrated high purity by both chemical and fluorometric determinations. Both flow cytometry and the Lindmo assay demonstrated a high binding affinity of [(111)In]-DTPA-trastuzumab-IRDye 800 to HER2-overexpressing cells. The dual-labeled conjugate was stable in PBS, but not in serum after 24 h at 37 °C. Larger molecules (>150 kD) were seen after a 24 h-incubation in human serum. Biodistribution studies revealed tumor-specific accumulation of [(111)In]-DTPA-trastuzumab-IRDye 800 in SKBR-3 tumors, and tumor uptakes at 24 and 48 h were (12.42±1.72)% and (9.96±1.05)%, respectively, following intravenous administration. The tumor-to-muscle ratio was 9.13±1.68 at 24 h, and increased to 12.79±2.13 at 48 h. Liver and kidney showed marked uptake of the dual-labeled imaging agent. CONCLUSIONS: [(111)In]-DTPA-trastuzumab-IRDye 800 is an effective diagnostic biomarker that can be used to validate dual-labeled, molecularly targeted imaging agents and can allow these agents to be translated into clinical practice for identifying HER2+ lesions.

99mTc carbonyl DTPA-rituximab: preparation and preliminary bioevaluation.

The anti CD20 antibody Rituximab was conjugated with para isothiocyanato benzyl diethylene triamine penta acetic acid (p-NCS-Bz-DTPA) and subsequent radiolabeling with (99m)Tc was carried out via the (99m)Tc carbonyl synthon. The (99m)Tc labeled antibody conjugate exhibited >95% radiochemical purity after purification and retained good in vitro stability when studied up to 24h at room temperature. In vitro cell binding studies carried out in Raji cells expressing CD20 antigen validated the biological efficacy of the preparation.

4to Seminario Internacional y 4to Taller Nacional "Uso y desarrollo de productos de la industria isotópica para la salud" / 4th International Seminar and 4th National Workshop on "Use and Development of Health-Related Industrial Isotope Products". Scientific report

El trabajo presenta una reseña científica de los principales temas abordados en el 4to Seminario Internacional y 4to Taller Nacional "Uso y Desarrollo de Productos de la Industria Isotópica para la Salud" celebrado los días 7, 8 y 9 de diciembre de 2010 en conmemoración al XV Aniversario del Centro de Isótopos.

The paper presents an analysis of the main topics discussed at the 4th International Seminar and 4th National Workshop on "Use and Development of Health-Related Industrial Isotope Products" held on December 7th, 8th and 9th 2010 to commemorate the Fifteenth Anniversary of the Isotope Centre.

Nuclear Medicine in Oncology

Nuclear oncolgy is important in the diagnosis, staging, and long-term surveillance of a number of cancers. Over the past 10 years there has been an explosion of new radioisotopic tracers aimed at detecting, staging and eventually treating tumors. Clinicians and oncologists can now use specific radiolabeled metabolic tracers, monoclonal antibodies, and molecular probes based on the sequencing of the human genome. The current applications of positron emission tomography (PET) in oncology have included characterizing tumor lesions, differentiating recurrent disease from treatment effects, staging tumors, evaluating the extent of disease, and monitoring therapy. The future developments in medicine may use the unique capabilities of PET not only in diagnostic imaging but also in molecular medicine and genetics. Radioimmunoscintigraphy is a technique which uses radiolabeled antibodies to visualize tumors, taking advantage of antigens preferentially expressed by malignant tissue. However, the implementation of radiolabeled antibodies as "magic bullets" for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine monoclonal antibodies. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recently, noninvasive molecular imaging has been developed. Most current molecular imaging strategies are "indirect" and involve the coupling of a "reporter gene" with a complementary "reporter probe". Imaging the level of probe accumulation provides indirect information related to the level of reporter gene expression. In this article, the author discuss the current status of PET, radioimmunoscintigraphy, gene imaging and receptor imaging with a brief review on nuclear oncology.

Radioimmunoscintigraphy Using (99m)Tc-anti-CEA F(ab')(2) Fragment in Rectal Cancer and a Pilot Study for Radioimmunoguided Surgery / 대한핵의학회잡지

PURPOSE: This prospective study was performed to evaluate the usefulness of preoperative radioimmu-noscintigraphy and intraoperative scintimetric examination (radioimmunoguided surgery: RIGS) using (99m)Tc-anti-CEA F(ab')(2), fragment. MATERIALS AND METHODS: Nineteen patients with rectal cancer underwent preoperative whole body planar scintigraphy at 4 hours after injection of (99m)Tc-anti-CEA F(ab')(2), fragment and SPECT imaging at 18 hours. Surgical operation was performed at 24 hours after injection. During laparotomy, radioactivities from intraabdominal viscera were measured by gamma probe. The radioac-tivities from excised tumor and lymph nodes were also measured and compared with pathology. RESULTS: All nineteen patients were confirmed to have adenocarcinomas in the rectum. Twenty-seven of 97 excised lymph node groups had metastasis and 2 patients had liver metastasis in pathology. Preoperative radioim- munoscintigraphy detected primary tumors in 11 patients (sensitivity 55%) and it couId not detect any lymph nodes or liver metastasis. All patients showed high radioactivity in the kidneys, liver, spleen, and major vessels in intraoperative measurement by gamma probe, and tumor activity was not discriminated from background activity. However, ra4ioactivity from excised tumor was higher than normal rectum (T/B ratio; 3.47+/-2.25). When excised lymph node activity/background activity ratio > 1,5 was considered as positive criteria of metastasis, sensitivity, specificity, positive and negative predictive values were 78.6%, 73.9%, 55.0% and 89.5%, respectively. CONCLUSION: Radioimmunoscintigraphy using (99m)Tc-anti-CEA F(ab')(2). has no additional value for preoperative staging and use of early RIGS using (99m)Tc-anti-CEA F(ab')(2)is inappropriate. For early RIGS using (99m)Tc labeled antibodies in rectal cancer patients, further development of more specific antibodies and methods to reduce background activity are needed.