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内容記述 |
Objective: Antibody N-linked glycans play an important role in pharmacodynamics and pharmacokinetics [1]. In particular, it has been reported that the presence or absence of glycans in IgG1 antibody significantly affects binding to active Fc receptors [2, 3], and may also alter IgG2 antibody pharmacokinetics. In this study, to verify the pharmacokinetics of IgG2 antibody with and without sugar chains, we synthesized IgG2 antibody-labeled compounds with partially hydrolyzed sugar chains and investigated their pharmacokinetics by PET imaging.Method: The EGFR-targeting antibody Vectibix (panitumumab) was chosen as a model of IgG2 because it is robust, well characterized, and easily available. 89Zr was used as the radionuclide because of its residualizing properties and its physical half-life of approximately 3.3 d, which ideally complements the biological half-life of IgG2.. The glycan hydrolysis of panitumumab was prepared with the use of EndoS2 (which hydrolyzes the chitobiose core of the asparagine-linked glycans). The reaction between DFO and panitumumab was achieved via the random reaction of p-SCN-Bn-DFO with the lysine moieties in the antibody. PET imaging was performed in a mouse model seeded with DLD-1 derived from human colon cancer.Results: To the panitumumab (25.0 mg/1.25 mL), 5.9 μL bed (17.7 μg of enzyme) of immobilized Halo-EndoS2 wild (3 μg/μL bed) was added, and the mixture was shaken rotary for 5 hours at room temperature (24 °C). After filtration of the resin, panitumumab with hydrolyzed glycosyl moiety was obtained. This immunoconjugate was radiolabeled with 89Zr via a standard protocol [4]. The radiochemical yields of 89Zr panitumumab (1) and 89Zr panitumumab with hydrolyzed glycosyl moiety (2) were 72.9 ± 2.3 % and 62.1 ± 9.9 %, respectively. The stability of the radioimmunoconjugates was tested in mouse serum at 37℃, and 2 demonstrated more than 95% stability after 7 days. We conducted initial in vivo evaluation of these radioimmunoconjugates in Balb c nude mice because they are the most commonly used immunodeficient strain used for xenograft models of human cancer. PET images were collected at 7 d after the injection of the radioimmunoconjugates (2.96 MBq). At this time point, PET images revealed that both radioimmunoconjugates accumulated in the EGFR-positive tumors. PET imaging of DLD-1-bearing mice showed the radioactivity accumulation of 8.6 ± 1.0 % ID/g for 1 and 12.5 ± 1.1 % ID/g for 2 in the tumor. The accumulation capacities of 1 and 2 in the liver were 5.5±0.7 %ID/g and 6.3±0.8 %ID/g, respectively. Taken together, these data clearly suggest that 2 showed a significant increase in accumulation in the tumor, compared to 1.Summary: Hydrolysis of panitumumab's N-linked oligosaccharide moiety altered pharmacokinetics and improved the antibody's ability to accumulate in tumor cell.References: [1] Vivier D, et al (2019) J. Nucl. Med., 60, 1174-1182. [2] Kristensen LK, et al (2019) Theranostics, 15, 4409-4420. [3] Vivier D, et al (2020) Theranostics, 60, 1746-1757. [4] Chang AJ, et al (2013) Mol Imaging, 12, 17-27.Acknowledgements: This work was supported by JSPS KAKENHI Grant Number 21K07714. This work was supported by AMED under Grant Number 21zf0127003h001. |
