Modifying the biodistribution of the anti TAG-72 peptide T3-15 peptide by multivalent PEG conjugation

Objectives In a search for peptides with high binding affinities for TAG-72 tumor antigen, we earlier identified T3-15. However, the free ^99mTc-labeled T3-15 was found to clear too rapidly from circulation in mice. We have now investigated a multivalent complex of the peptide using a PEG-tetraglutaryl with 4 NHS groups to increase the circulation time and tumor binding.

Methods The N-terminal amine of the peptide was preferentially conjugated with PEG-tetraglutaryl NHS at pH 6.5 and at a PEG to peptide molar ratio of 1:5. Thereafter, the PEG-T3-72 peptide was conjugated on an available lysine with NHS-MAG3 for radiolabeling with ^99mTc. The effect on membrane integrity of PEG-T3-15, T3-15 and PEG alone on TAG-72 positive LS-174T cells was determined by an LDH membrane integrity assay, the IC50 was determined by cell competition and the biodistribution was compared to T3-15 by necropsy following IV administration in mice with LS-174T tumors.

Results SDS-PAGE showed that an average of 2 peptides per PEG were attached and successful ^99mTc labeling was confirmed by C18 HPLC analysis. No cytotoxic effects were evident in the membrane integrity assay for PEG-T3-15, T3-15 or PEG through 24h. The cell binding assay demonstrated a 2-fold improved IC50 for PEG-T3-15 of 0.15 nM over that for the T3-15 peptide of 0.29 nM. In mice the PEG-T3-15 peptide cleared more slowly than T3-15 alone, with blood levels about 3-fold higher at 30 to 90 min. However, high accumulation in liver and spleen of 18% and 15% respectively at 90 min reduced the tumor accumulation such that the tumor to blood ratios were largely unchanged with and without the PEG.

Conclusions While the multivalent T3-15 peptide-PEG construct improved the IC50 over the free peptide without cytotoxicity and slowed blood clearance, tumor accumulation did not improve despite its multivalence, possibly due to the surprising increased accumulation in liver and spleen