RT Journal Article SR Electronic T1 Preclinical Evaluation of 111In-labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-positive Cancer Models JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP jnumed.118.220095 DO 10.2967/jnumed.118.220095 A1 Siddesh V Hartimath A1 Elahe Alizadeh A1 Viswas Raja Solomon A1 Rufael Chekol A1 Wendy Bernhard A1 Wayne Hill A1 Angel Parada Casaco A1 Kris Barreto A1 Clarence Ronald Geyer A1 Humphrey Fonge YR 2019 UL http://jnm.snmjournals.org/content/early/2019/01/17/jnumed.118.220095.abstract AB Background: Epidermal growth factor receptor I (EGFR) is overexpressed in most cancers of epithelial origin. Antibody drug conjugates (ADCs) with PEGylated-maytansine (PEG-DM1) show promise in vitro and in vivo. However, in vivo biodistribution data for ADCs with PEG-DM1 have not been reported. Development of methods to understand the real-time in vivo behaviour of these ADCs is needed to move these compounds to the clinic. Methods: Here we have used non-invasive µSPECT/CT imaging and ex vivo biodistribution to understand the in vivo behaviour of PEG6-DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG6-DM1. We generated immunoconjugates with low (nimotuzumab-PEG6-DM1-Low) and high (nimotuzumab-PEG6-DM1-High) drug to antibody ratios (DAR). The DAR of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was 3.5 and 7.3, respectively. Quality control was performed using UV spectrophotometry, size exclusion HPLC, bioanalyzer, biolayer interferometry, and flow cytometry in EGFR-positive DLD-1 cells. These immunoconjugates were conjugated with DOTA and radiolabeled with 111In. The in vitro binding and internalization rates of 111In-nimotuzumab, 111In-nimotuzumab-PEG6-DM1-Low and 111In-nimotuzumab-PEG6-DM1-High were characterized. Furthermore, the pharmacokinetics, biodistribution and imaging characteristics were evaluated in normal and DLD-1 tumor bearing mice. Results: Flow cytometry and biolayer interferometry showed a trend towards decreasing EGFR affinity with increasing number of PEG6-DM1 on the antibody. Despite the lower overall cellular binding of the PEG6-DM1 radioimmunoconjugates, internalization was higher for PEG6-DM1 ADCs than for the non-PEGylated ADC in the following order: 111In-nimotuzumab-PEG6-DM1-High > 111In-nimotuzumab-PEG6-DM1-Low > 111In-nimotuzumab. Nuclear uptake of 111In-nimotuzumab-PEG6-DM1-High was 4.4-fold higher than 111In-nimotuzumab. Pharmacokinetics and biodistribution showed 111In-nimotuzumab-PEG6-DM1-High had the slowest blood and whole body clearance rate. Uptake in DLD-1 tumors of 111In-nimotuzumab was similar to 111In-nimotuzumab-PEG6-DM1-Low but was significantly higher than for 111In-nimotuzumab-PEG6-DM1-High. Tumor-to-background ratios for 111In-nimotuzumab and 111In-nimotuzumab-PEG6-DM1-Low were higher than for 111In-nimotuzumab-PEG6-DM1-High. Conclusion: The results show that conjugation of multiple PEG6-DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG6-DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111In-nimotuzumab-PEG6-DM1-High with a slow overall whole body clearance rate. This data provides insights for evaluating the pharmacokinetics, normal tissue toxicity and in determining dosing rate of PEGylated ADCs.