RationaleEpidermal growth factor receptor (EGFR) upregulation is associated with enhanced proliferation and drug resistance in a number of cancers. Nimotuzumab is a humanized monoclonal antibody with high affinity for EGFR. The objective of this study was to determine if 89Zr-DFO-nimotuzumab could be suitable for human use as a PET probe for quantifying EGFR in vivo.MethodsTo evaluate the pharmacokinetics, biodistribution, microPET imaging, radiation dosimetry, and normal tissue toxicity in tumor and non-tumor bearing mice of 89Zr-desferoxamine-nimotuzumab (89Zr-DFO-nimotuzumab) of a product prepared under GMP conditions. Nimotuzumab was conjugated to DFO and radiolabeled with 89Zr. 89Zr-DFO-nimotuzumab was characterized by in vitro gel-electrophoresis, biolayer interferometry (BLI) and flow cytometry. 89Zr-DFO-nimotuzumab was evaluated in vivo by microPET and ex vivo by biodistribution in healthy and EGFR-positive tumor bearing mice.ResultsFlow cytometry with A431 cells showed no significant difference in the dissociation constant of nimotuzumab (13 ± 2 nM) compared with DFO-nimotuzumab (17 ± 4 nM). PET imaging in mice xenografts showed persistently high tumor uptake with the highest uptake obtained in DLD-1 xenograft (18.3 %IA/cc) at 168 hp.i. The projected human effective dose was low and was 0.184 mSv/MBq (0.679 rem/mCi) in females and 0.205 mSv/MBq (0.757 rem/mCi) in males. There was no apparent normal tissue toxicity as shown by cell blood counts and blood biochemistry analyses at 168-fold and 25-fold excess of the projected human radioactive and mass dose of the agent.Conclusion89Zr-DFO-nimotuzumab had low organ absorbed dose and effective dose that makes it suitable for potential human use.
Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) monoclonal antibody that is approved in many countries for the treatment of EGFR-positive cancers. Near infrared (NIR) fluorescent dye-labeled antibodies represent an attractive class of image-guided surgical probes because of their high specificity, tumor uptake, and low dissociation from tumor cells that express the antigen. In this study, we developed a NIR fluorescent dye-labeled nimotuzumab immunoconjugate, IRDye800CW-nimotuzumab, and evaluated in vitro binding with EGFR-positive cells, in vivo tumor uptake by NIR fluorescent imaging, and ex vivo biodistribution. There was no difference in binding between nimotuzumab and IRDye800CW-nimotuzumab to EGFR-positive cells. In mice bearing EGFR-positive xenografts, IRDye800CW-nimotuzumab uptake peaked at 4 days post injection and slowly decreased thereafter with high levels of accumulation still observed at 28 days post injection. In EGFR-positive xenografts, IRDye800CW-nimotuzumab showed more than 2-fold higher uptake in tumors compared to IRDye800CW-cetuximab. In addition, liver uptake of IRDye800CW-nimotuzumab was two-fold lower than cetuximab. The lower liver uptake of IRDye800CW-nimotuzumab could have implications on the selected dose for clinical trials of the immunoconjugate. In summary, this study shows that nimotuzumab is a good candidate for NIR fluorescent imaging and image-guided surgery.
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 PEG 6 -DM1 ADCs. We developed nimotuzumab ADCs conjugated to PEG 6 -DM1. We generated immunoconjugates with low (nimotuzumab-PEG 6 -DM1-Low) and high (nimotuzumab-PEG 6 -DM1-High) drug-to-antibody ratios. The drug-to-antibody of nimotuzumab-PEG 6 -DM1-Low and nimotuzumab-PEG 6 -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 111 In. The in vitro binding and internalization rates of 111 111 In-nimotuzumab-PEG 6 -DM1-Low, and 111 In-nimotuzumab-PEG 6 -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 PEG 6 -DM1 on the antibody. Despite the lower overall cellular binding of the PEG 6 -DM1 radioimmunoconjugates, internalization was higher for PEG 6 -DM1 ADCs than for the non-PEGylated ADC in the following order: 111 In-nimotuzumab-PEG 6 -DM1-High . 111 Innimotuzumab-PEG 6 -DM1-Low . 111 In-nimotuzumab. Nuclear uptake of 111 In-nimotuzumab-PEG 6 -DM1-High was 4.4-fold higher than 111 In-nimotuzumab. Pharmacokinetics and biodistribution showed that 111 In-nimotuzumab-PEG 6 -DM1-High had the slowest blood and whole-body clearance rate. Uptake in DLD-1 tumors of 111 In-nimotuzumab was similar to 111 In-nimotuzumab-PEG 6 -DM1-Low but was significantly higher than for 111 In-nimotuzumab-PEG 6 -DM1-High. Tumor-to-background ratios for 111 In-nimotuzumab and 111 Innimotuzumab-PEG 6 -DM1-Low were higher than for 111 In-nimotuzumab-PEG 6 -DM1-High. Conclusion: The results show that conjugation of multiple PEG 6 -DM1 reduces the affinity for EGFR in vitro. However, the reduced affinity is counteracted by the high internalization rate of constructs with PEG 6 -DM1 ADCs in vitro. The decreased affinity resulted in low tumor uptake of 111 In-nimotuzumab-PEG 6 -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. Conjugates in EGFR-Positive Cancer ModelsIn-Labeled PEGylated Maytansine Nimotuzumab Drug 111 Preclinical Evaluation of http://jnm.snmj...
Nimotuzumab is a humanized anti-epidermal growth factor receptor I (EGFR) monoclonal antibody. We have developed antibody drug conjugates (ADCs) with nimotuzumab conjugated to PEGylated-maytansine (PEG6-DM1). We generated conjugates with low (nimotuzumab-PEG6-DM1-Low: DAR = 3.5) and high (nimotuzumab-PEG6-DM1-High: DAR = 7.3) drug to antibody ratios (DAR). Quality control was performed using UV spectrophotometry, size exclusion HPLC, bioanalyzer, biolayer interferometry (BLI), and flow cytometry in EGFR-positive DLD-1, MDA-MB-468 (high density EGFR), and HT-29 (very low EGFR density) cells. Control antibody drug conjugates were developed using a human anti-maltose binding protein (MBP) antibody. BLI showed that the binding of nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High was slightly but significantly affected by conjugation of the drug (nimotuzumab KD 0.89 ± 0.02 nM < nimotuzumab-PEG6-DM1-Low KD 1.94 ± 0.02 nM < nimotuzumab-PEG6-DM1-High KD 3.75 ± 0.03 nM). In vitro cytotoxicity was determined following incubation of cells with the immunoconjugates and IC50 values were determined. Nimotuzumab-PEG6-DM1-Low and nimotuzumab-PEG6-DM1-High were used to treat EGFR positive KRAS mutant DLD-1 colorectal cancer xenograft. DLD-1 cells were transduced with a red fluorescent protein (iRFP702) to allow the use of near infrared imaging (NIR) for tumor response monitoring. In vitro potency correlated with the number of drugs on antibody, with nimotuzumab-PEG6-DM1-High showing higher activity than nimotuzumab-PEG6-DM1-Low. Three doses (15 mg/kg) of the ADCs prolonged the survival of DLD-1-iRFP-702 tumor bearing mice as monitored by NIR. Nimotuzumab-PEG6-DM1-Low resulted in 4/6 complete cure while nimotuzumab-PEG6-DM1-High resulted in 2/5 complete cure. The novel ADCs were very effective in a colorectal cancer model in vivo.
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