TY - JOUR T1 - <strong/><strong>The potential of trastuzumab Fab-DTPA as a vehicle molecule for theranostic application in HER2 receptor expressing cancers </strong> JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 548 LP - 548 VL - 61 IS - supplement 1 AU - Yogesh Rathore AU - Jaya Shukla AU - Bhagwant Mittal AU - Rajender Kumar AU - Gurpreet Singh AU - Ishita Laroiya AU - Amanjit Bal Y1 - 2020/05/01 UR - http://jnm.snmjournals.org/content/61/supplement_1/548.abstract N2 - 548Introduction: The trastuzumab (MW 141kDa) is monoclonal antibody against HER2 receptor protien. We demonstrated the feasibility of Ga-68 Trastuzumab Fab (45kDa) for clinical imaging. Methods: The Fab was generated by immobilized papain beads in phosphate buffer (1M, pH 8) containing L-Cystine followed by purification through amicon ultrafiltration device (cut off 30 kDa). Fab was quanified at 280 nm. The extinction coefficient (71780) was calculated based on the amino acid sequence of Fab with PDB data bank. Fab was characterized by the MALDI-TOF and SDS-PAGE. Further Fab was used for conjugation (of bifunctional chelating agent DTPA. The conjugation was standardized, for different molar ratios of Fab and DTPA, reaction volume, time, pH and temperature. The purification of conjugated Fab-DTPA was done using Micro Bio-Spin P-6 Chromatography Columns. The average number of conjugated bifunctional chelating agent per molecule Fab was calculated by using MALDI-TOF. The affinity of Fab and DTPA conjugated Fab were compared with the trastuzumab antibody by using the biolayer interferometry technique (BLI) using ForteBio Octet RED 96. The radiolabelling reaction conditions were optimized for both Ga-68 and Lu-177. The quality control included radiochemical purity using TLC in sodium citrate mobile phase, pyrogenicity using PTS, and sterility by tryptic soy broth culture media. The SKBR-3 cell (106) were cultured in 96 well plates and incubated with a varied amount of Lu-177-DTPA-Fab. The affinity and percentage of cell death (SKBR-3 HER2 positive cell) with Lu-177-DTPA-Fab was calculated by using flow cytometry assay. After obtaing permission from the IEC and informed consent from patients, F-18 FDG PET and IHC proven HER2 positive patients (n=5 for Ga-68 fab, n=4 for Lu-177 DTPA-Fab) were recruited. Images of Ga-68-DTPA-Fab (2.4-4.1 mCi) PET/CT at 2,3 h and Lu-177 DTPA-Fab (10-15 mCi) SPECT/CT up to 5 days were aquired. Results: MALDI-TOF and SDS-PAGE demonstrated 45kDa mw of Fab. MALDI-TOF confirmed 2DTPA/Fab at 1:10 molar ratio. The Kd value for HER2 receptor with trastuzumab, Fab, and conjugated DTPA-Fab was 0.2 nM, 0.57 nM, and 20 nM respectively. high labelling of Ga-68-DTPA-Fab (≥95%) and Lu-177-DTPA-Fab (≥98%) eliminated the purification step. Also both formulation were sterile and pyrogen free. Lu-177-DTPA-Fab (2.312- 18.5 MBq) showed good apoptosis (1.5-42.5%) in Her2 SKBR-3 cell. Apart from liver, High kidney and bladder activity on Ga-68-DTPA-Fab PET/CT MIP and Lu-177 DTPA-Fab were observed. Uptake in primary lesion (similar to FDG) was observed with both tracers. SUVmax with Ga-68-DTPA-Fab PET/CT was increased from 3.1 at 2h to 5.5 at 3 h. However, lymph nodes were not visualized due to high blood pool activity. Liver uptake and blood pool was reduced on 5 day Lu-177 DTPA-Fab image and lesions, primary and lymph nodes, were better visualized. Conclusions: Fab-DTPA can be used as theranostic molecule for Her2 expressing tumors. ER -