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J Nucl Med. 2019 Jan 17. pii: jnumed.118.220095. doi: 10.2967/jnumed.118.220095. [Epub ahead of print]

Preclinical Evaluation of 111In-labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-positive Cancer Models.

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University of Saskatchewan, Canada.
Center for Molecular Immunology, Canada.


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.


ADCs; Animal Imaging; Epidermal Growth Factor Receptor (EGFR); MicroSPECT/CT; Molecular Imaging; Monoclonal Antibodies; Nimotuzumab; Radiochemistry; Radiopharmaceuticals; SPECT; pegylated maytansine


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