Development of [64Cu]-DOTA-PR81 radioimmunoconjugate for MUC-1 positive PET imaging.

OBJECTIVE: Breast cancer radioimmunoscintigraphy targeting MUC1 expression is a growing field of work in nuclear medicine research. PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast tumors. In this study, we report production, quality control and preclinical qualifications of a copper-64 labeled PR81 for PET imaging of breast cancer. METHODS: PR81 was conjugated with DOTA-NHS-ester and purified by molecular filtration followed by chelate:mAb ratio determination by spectrophotometric method. DOTA-PR81 was labeled with (64)Cu followed by radiochemical purity, in vitro stability, in vitro internalization and immunoreactivity determination. The tissue biodistribution of the (64)Cu-DOTA-PR81 and (64)Cu-DOTA-hIgG was evaluated in BALB/c mice with breast carcinoma tumors using tissue counting and imaging. RESULTS: The radiochemical purity of radioimmunoconjugate was >95±1.9% (ITLC) (specific activity; 4.6 µCi/µg). The average number of chelators per antibody was 3.4±0.3:1. The (64)Cu-DOTA-PR81 showed immunoreactivity towards MUC1 antigen and MCF7 cell line with significant in vitro stability (>89% in PBS and 78±0.5% in human serum) over 48 h. Maximum internalized activity of radiolabeled PR81 in 4-8 h was 81.5%. The biodistribution and scintigraphy studies showed the accumulation of the complex at the site of tumors with high sensitivity and specificity compared to control probes. CONCLUSION: The results showed that (64)Cu-DOTA-PR81 may be considered as a potential PET tracer for diagnosis and follow-up of MUC1 expression in oncology.

Preclinical evaluation of [¹¹¹ In]-DOTA-trastuzumab for clinical trials.

CONTEXT: Herceptin and its fragments have been radiolabeled and used in the imaging of human epidermal growth factor receptor 2 (HER2)/neu-positive tumors and development of diagnostic kits is of great importance in radiopharmacy. AIMS: In this study, ¹¹¹ In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-trastuzumab (¹¹¹ In-DOTA-trastuzumab) was successively prepared and evaluated for ultimate use in the HER2 antigen imaging in oncology. SETTINGS AND DESIGN: The conjugate was prepared, labeled and evaluated using in vitro (radioimmunoassay [RIA], enzyme-linked immunosorbent assay (ELISA), stability, binding, internalization)/in vivo (bio-distribution, single-photon emission computed tomography [SPECT]) experiments. MATERIALS AND METHODS: ¹¹¹ In-DOTA-trastuzumab was prepared followed by determination of radiochemical purity (RCP), integrity of protein, immunoreactivity of radiolabeled antibody with HER2/neu antigen (by SkBr3 cell line binding and RIA methods) were determined followed by stability tests, internalization studies and the tissue bio-distribution determination in wild-type rats as well as SPECT imaging in SkBr3-bearing mice. STATISTICAL ANALYSIS USED: All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05. RESULTS: ¹¹¹ In-DOTA-trastuzumab was prepared (RCP >95 ± 0.5%, S.A. 5.3 µCi/µg) with the average number of chelators per antibody of 6:1 showing significant immune-reactivity retention using ELISA. In vitro stability was >90% in phosphate buffered saline and 80 ± 0.5% in serum over 48 h. Cell binding was significant (>0.79). In vitro internalization reached up to %12-13 in 10 h. Significant tumor uptake was observed. CONCLUSIONS: In vitro and in vivo/SPECT imaging in SkBr3-bearing mice demonstrated that ¹¹¹ In-DOTA-trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and follow-up of HER2 expression in oncology.

Production and Quality Control of [(67)Ga]-DOTA-trastuzumab for Radioimmunoscintigraphy.

Breast cancer radioimmunoscintigraphy targeting HER2/neu expression is a growing field of work in nuclear medicine research. In this study, trastuzumab was successively labeled with [(67)Ga] GaCl3 after conjugation with DOTA-NHS-ester. The conjugates were purified by molecular filtration, the average number of DOTA conjugated per mAb was calculated and total concentration was determined by spectrophotometric method. DOTA-Trastuzumab was labeled with (67)Ga. Radiochemical purity, integrity of protein after radiolabeling and stability of (67)Ga-DOTA-Trastuzumab were determined followed by biodistribution studies in wild-type rats (30 ± 5.5 µCi, 2, 4 and 24 h p.i.). The radioimmunoconjugate was prepared with a radiochemical purity of higher than 95% (RTLC). The average chelate to antibody ratio (c/a) for the conjugate used in this study was 5.8:1. The final compound was stable in presence of PBS at 37ºC and room temperature. The sample was showed to have similar patterns of migration in the gel electrophoresis similar to the native protein. The accumulation of the radiolabeled antibody in liver, spleen, kidney, heart and other tissues demonstrates. (67)Ga-DOTA-Trastuzumab was prepared as a surrogate for important clinically applicable radionuclides used in SPECT and PET including In-111 and Cu-64 as a model of radiolabeling. It is also a potential compound for molecular imaging of SPECT for diagnosis and treatment studies and follow-up of HER2 expression in oncology.

Development of a (68)Ga-Fluorinated Porphyrin Complex as a Possible PET Imaging Agent.

AIM: Due to the interesting pharmacologic properties of porphyrins, the idea of developing a possible tumor imaging agent using PET by incorporating (68)Ga into a suitable porphyrin ligand was investigated. METHODS: (68)Ga-labeled 5,10,15,20-tetrakis(pentafluoro-13 phenyl) porphyrin ((68)Ga-TFPP) was prepared using freshly eluted [(68)Ga]GaCl3 obtained from a 68Ge/68Ga generator developed in-house and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (H2TFPP) for 60 min at 100°C. RESULTS: The complex was prepared with high radiochemical purity (>99% ITLC, >99% HPLC, specific activity: 13-14 GBq/mmol). Stability of the complex was checked in the final formulation and in human serum for 5 h. The partition coefficient was calculated for the compound (log P = 0.62). The biodistribution of the labeled compound in vital organs of Swiss mice bearing fibrosarcoma tumors was studied using scarification studies and SPECT imaging up to 1 h. The complex was mostly washed out from the circulation through kidneys and liver. The tumor-to-muscle ratio 1 h post injection was 5.13. CONCLUSION: The radiolabeled porphyrin complex demonstrated potential for further imaging studies in other tumor models.