Preclinical Evaluation of 111 In-Labeled PEGylated Maytansine Nimotuzumab Drug Conjugates in EGFR-Positive Cancer Models

J Nucl Med. 2019 Aug;60(8):1103-1110. doi: 10.2967/jnumed.118.220095. Epub 2019 Jan 17.


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. Methods: Here we have used noninvasive small-animal SPECT/CT imaging and ex vivo biodistribution to understand the in vivo behavior 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. The drug-to-antibody of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-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 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 toward 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 that 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. These data provide insights for evaluating the pharmacokinetics and normal -tissue toxicity and in determining dosing rate of PEGylated ADCs.

Keywords: ADCs; EGFR; PEGylated maytansine; microSPECT/CT imaging; nimotuzumab.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal, Humanized / pharmacology*
  • Cell Line, Tumor
  • Chromatography, High Pressure Liquid
  • ErbB Receptors / metabolism
  • Flow Cytometry
  • HT29 Cells
  • Humans
  • Immunoconjugates*
  • Indium Radioisotopes*
  • Interferometry
  • Kinetics
  • Maytansine / pharmacology*
  • Mice
  • Mice, Inbred BALB C
  • Neoplasm Transplantation
  • Neoplasms / metabolism*
  • Polyethylene Glycols / chemistry
  • Single Photon Emission Computed Tomography Computed Tomography
  • Spectrophotometry, Ultraviolet
  • Tissue Distribution
  • Trastuzumab / pharmacology


  • Antibodies, Monoclonal, Humanized
  • Immunoconjugates
  • Indium Radioisotopes
  • Maytansine
  • Polyethylene Glycols
  • nimotuzumab
  • EGFR protein, human
  • ErbB Receptors
  • Trastuzumab