Synthesis of 111In-p-SCN-Bn-DTPA-nimotuzumab and its preclinical evaluation in EGFR positive NSCLC animal model

Abstract

Abstract The aim of this study was to investigate labeling of nimotuzumab (h-R3) with 111In using p-SCN-Bn-DTPA as bifunctional chelate, evaluate its targeting potential against SK-LU-1, H226, H650, H661, and HCC4006 non-small cell lung carcinoma (NSCLC) cell lines and correlate epidermal growth factor receptor (EGFR) expression level with internalization kinetics, biodistribution and imaging accuracy using Balb/c mice and New Zealand White rabbit (NZWR) animal model. The amount of p-SCN-Bn-DTPA attached to h-R3 was assessed by measuring relative absorbance at 652 nm with ultraviolet (UV) spectrophotometer. High-performance liquid chromatography (HPLC) was used to determine percent radiochemical purity (%RCP) and in vitro stability using excess amount of diethylenetriamine pentaacetate (DTPA). The in vitro stability in rat serum was estimated using iTLC-SG. EGFR expression level in each tumor was assessed by chemiluminescence. In vivo uptake in different organs of Balb/c mice and non-invasive imaging potential using NZWR bearing HCC4006 tumor, was evaluated with gamma camera. UV spectroscopy has confirmed the attachment of five p-SCN-Bn-DTPA (chelate) with one antibody. The HPLC indicated 98.85 ± 0.14% (n = 3) %RCP with high yield (>96%), specific activity 3.5 ± 0.0.25 mCi per mg and 94.25 ± 0.34% in vitro stability at 37 °C in mice serum. In excess DTPA no considerable transchelation was experiential from the 111In labeled p-SCN-Bn-DTPA-h-R3 to the challenger. The EGFR expression in HCC4006 was higher amongst all with band density of 23.53 relative to 1.00 of H226. Initially internalization was lower which went up 1.05 × 104 molecules per HCC4006 cell in 48 h. The optimal concentration of h-R3 for maximum uptake was 15 μg per animal. Higher uptake in target organ was observed in animal infected with HCC4006 cells. However, in excess pure h-R3 the uptake was significantly reduced indicating tumor specificity. HCC4006 target site was undistinguishable relative to background activity in the initial phase of imaging due to poor uptake. However, within 60 h the HCC4006 tumor was quite apparent. This experiment suggests that at optimal dosage of 111In labeled h-R3 can be used for localization and identification of EGFR positive NSCLC using gamma camera.