Abstract
1228
Introduction: The monoclonal anti HER2 antibody (145 kDa) is used for the treatment of HER2 positive primary and metastatic disease. Trastuzumab through the antigen detecting site bind at domain IV of HER2 receptor, stop the dimerization of receptor and inhibit the tumor growth signaling. The fragment of trastuzumab bearing antigen binding site, Fab (45 kDa), is explored for imaging HER2 expressing breast cancer. The Fab was generated with enzyme digestion of Trastuzumab using insoluble papain enzyme tagged beads in 1M phosphate buffer (pH 7) at temperature 37°C and 850 rpm for 22 hours. The purification of generated Fab was carried out with Amicon ultrafiltration cutoff 30 kDa tube. The characterization of purified Fab was done by the MALDI and SDS-PAGE, The quantification of Fab after digestion was determined by the UV-VIS spectrophotometer and nanodrop. The Fab was conjugated with NOTA, a bifunctional chelating agent. The molar ratio of NOTA : Fab were optimized by the repetition of experiments for different molar ratios (10:1, 15:1, 20:1, 25:1), reaction volume, incubation time and temperature for maximum yield. The purification of conjugated NOTA-Fab from unconjugated chelating agent was performed with PD-10 (desalting) column. The number of chelating molecules in conjugated fab were calculated by mass difference of conjugated and unconjugated Fab obtained by mass spectra of MALDI. The radiolabeling of NOTA-Fab with freshly eluted Ga-68 from Ge-68/Ga-68 was optimized. Radiolabeled Ga-68 NOTA-Fab was separated using PD-10 column and passed through 0.22 μm filter for sterility. The quality control included-radiochemical purity by paper chromatography using sodium-citrate (pH-5.5) as mobile phase, apyrogenicity with PTS, sterility in culture broth for 7 days. Patients (n=5), histopathological proven for HER expression, were recruited,F-18 FDG PET/CT was done. After obtaining permission from Institute and informed written consent from patients,Ga-68 NOTA-Fab (3-5 mCi) was injected and PET/CT was acquired after 1.5 and 3 hr. Scans were analyzed by two nuclear medicine physicians. Papain split the whole antibody into three fragments, (45 kDa) Fab (2), Fc, and was demonstrated on mass spectra. The integrity of Fab on SDS-PAGE was seen in the form of distinct bands, variable of heavy and light chain, in reduce condition (with β-mercaptoethanol) while trastuzumab have two bands (heavy and light chain), and in non-reduced conditions, Fab and trastuzumab at different molecular weight portions. The NOTA conjugation was standardized at 4°C and 22-24 hours incubation period. The number of NOTA conjugates at 25:1 molar concentration was 1to2. Labeling efficiency of Ga-68 NOTA-Fab was 48%. After purification, the radiochemical purity was >90%. It was sterile and apyrogenic. Ga-68 NOTA-Fab MIP PET/CT shown high kidney and bladder activity, demonstrating renal clearance. at 3h, the blood pool activity of Ga-68 NOTA-Fab was reduced and uptake in lesions was increased (mean SUVmax at 1.5 and 3 hr was 2.4 and 3.5). The liver uptake was noted, however liver mets could be visualized as seen in F-18 FDG PET/CT. To best of our knowledge this is the first human study using Ga-68 NOTA-Fab. The Ga-68 NOTA-Fab has potential for targeting HER2 positive lesions. This can be utilized to evaluate the response of trastuzumab therapy and for selection of patients for trastuzumab radioimmunotherapy.