Preclinical Evaluation of In-Labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-Positive Cancer Models.

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 behavior of these ADCs is needed to move these compounds to the clinic. Here we have used noninvasive small-animal SPECT/CT imaging and ex vivo biodistribution to understand the in vivo behavior of PEG-DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG-DM1. We generated immunoconjugates with low (nimotuzumab-PEG-DM1-Low) and high (nimotuzumab-PEG-DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG-DM1-Low and nimotuzumab-PEG-DM1-High was 3.5 and 7.3, respectively. Quality control was performed using ultraviolet spectrophotometry, size-exclusion high-performance liquid chromatography, bioanalyzer, biolayer interferometry, and flow cytometry in EGFR-positive DLD-1 cells. These immunoconjugates were conjugated with DOTA and radiolabeled with In. The in vitro binding and internalization rates of In-nimotuzumab, In-nimotuzumab-PEG-DM1-Low, and In-nimotuzumab-PEG-DM1-High were characterized. Furthermore, the pharmacokinetics, biodistribution, and imaging characteristics were evaluated in normal and DLD-1 tumor-bearing mice. Flow cytometry and biolayer interferometry showed a trend toward decreasing EGFR affinity with increasing number of PEG-DM1 on the antibody. Despite the lower overall cellular binding of the PEG-DM1 radioimmunoconjugates, internalization was higher for PEG-DM1 ADCs than for the non-PEGylated ADC in the following order: In-nimotuzumab-PEG-DM1-High > In-nimotuzumab-PEG-DM1-Low > In-nimotuzumab. Nuclear uptake of In-nimotuzumab-PEG-DM1-High was 4.4-fold higher than In-nimotuzumab. Pharmacokinetics and biodistribution showed that In-nimotuzumab-PEG-DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of In-nimotuzumab was similar to In-nimotuzumab-PEG-DM1-Low but was significantly higher than for In-nimotuzumab-PEG-DM1-High. Tumor-to-background ratios for In-nimotuzumab and In-nimotuzumab-PEG-DM1-Low were higher than for In-nimotuzumab-PEG-DM1-High. The results show that conjugation of multiple PEG-DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG-DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of In-nimotuzumab-PEG-DM1-High, with a slow overall whole-body clearance rate. These data provide insights for evaluating the pharmacokinetics and normal -tissue toxicity and in determining dosing rate of PEGylated ADCs.