PEGylation: an effective therapeutic radiopharmaceutical development strategy

Introduction: Polyethylene glycol (PEG) is a widely used drug modification polymer with low toxicity and low immunogenicity. PEGylation is an effective strategy to alter pharmacokinetic properties, including an enhanced solubility, an improved stability, a reduced renal clearance, and a prolonged circulation halflife in biological fluids, which dramatically improves pharmaceutical value.

Many factors contribute to the effect of PEG on peptide molecules, including the size, the number and the topological structure of attached PEG molecules, and the conjugation position of PEG moieties. To explore how topology and polyvalency, of PEGylation, influence the in vivo fate of peptides, we labeled PD-L1 targeting TPP-1 peptide with PEG molecules with different topological structures. By leveraging with ⁶⁴Cu-PET, we revealed the "topological effects" of PEG molecules in modulating the in vivo pharmacokinetics and their theranostics potential in mouse models.

Methods: Based on the PD-L1-targeted TPP-1 peptide, a monomeric precursor, NOTA-TPP, was generated by conjugation of bifunctional chelator NOTA. Then, we designed and synthesized a series of PEGylated peptide multimers, , NOTA-TPP-(4)PEG, and NOTA-TPP-(8)PEG. Radiolabeled with ⁶⁴Cu, they were evaluated in in vitro and in vivo studies to compare differences in radiochemical and therapeutic abilities. The radiochemical yield (RCY) and molar activity were analyzed by radio HPLC. The stability in mouse serum was evaluated. Therapeutic study was carried out in MC38 tumor-bearing mice models (n=5) (Figure A). After intravenous injection of approximately 37 MBq radioconjugate per mouse, tumor volume, body weight and survival of mouse were recorded on days 6, 9, 12, 15, 19, 21, 23 and 25 post treatment.

Results: Monitored by radio-HPLC, the RCY of [⁶⁴Cu]NOTA-TPP and [⁶⁴Cu]NOTA-TPP-(1)PEG were >98%, while that of [⁶⁴Cu]NOTA-TPP-(4)PEG and [⁶⁴Cu]NOTA-TPP-(8)PEG was >78.2%. The molar activity of [⁶⁴Cu]NOTA-TPP-(1)PEG, [⁶⁴Cu]NOTA-TPP-(4)PEG and [⁶⁴Cu]NOTA-TPP-(8)PEG were more than 37 GBq/μmol, while the molar activity of was more than 74 GBq/μmol. The half-life also changed among different arm number of PEG. The half-life of [⁶⁴Cu]NOTA-TPP-(1)PEG, [⁶⁴Cu]NOTA-TPP-(4)PEG and [⁶⁴Cu]NOTA-TPP-(8)PEG were more than 12 hours, while the half-life of [⁶⁴Cu]NOTA-TPP was only about 80 min.

In the therapeutic study, mice in [⁶⁴Cu]NOTA-TPP group had the most rapid tumor growth and more than 50% tumor-bearing mice were euthanized by day 23 because of excessive tumor volume. In [⁶⁴Cu]NOTA-TPP-(1)PEG and [⁶⁴Cu]NOTA-TPP-(4)PEG group, tumor volume reached 786.28±504.72 mm³ and 753.58±552.22 mm³ at day 21 post injection, respectively, and less than 50% mice were euthanized. In [⁶⁴Cu]NOTA-TPP-(8)PEG group, tumor volume increased insignificantly and kept at a small level of 198.27±237.59 mm³ at day 21 post injection, and all mice survived until the end of the experiment. The normalized average body weight in all therapeutic groups increased slightly.

Conclusions: Through this proof-of-principle study, PEGylation proves to be an effective strategy to improve the stability of peptide precursors. Multimerization via PEGylation significantly improved tumor affinity and retention in blood, and expanded its application in tumor therapy. Moreover, the radiolabeled multimer [⁶⁴Cu]NOTA-TPP-(8)PEG is found an promising radiotheranostic agent for PD-L1 PET imaging and imaging-guided treatment.

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