Publikationsrepositorium - Helmholtz-Zentrum Dresden-Rossendorf

Objectives:

Complementary L-oligonucleotides (L-ONs) are characterized by high metabolic stability and low immunogenicity, in combination with the absence of natural hybridization targets and therefore represent a potential effector and conjugated with the targeting antibody as binding pairs in pretargeting technologies. PEG modification of the L-DNA will allow the pharmacokinetic tailoring of the effector molecules. Therefore we studied the effect of various sizes of PEG conjugates on the hybridisation of the L-DNA molecules with the complementary L-DNA-Cetuximab conjugates.
Methods:
Cetuximab as targeting probe for the endothelial growth factor receptor was a) modified with 1,4,7 triazacyclononane-1,4,7-triacetic acid (NOTA), b) modified with maleimide moieties by conjugation of the bifunctional cosslinker 4 maleimidobutyric acid N-hydroxysuccinimide ester (GMBS), c) conjugated with the complementary thiol-bearing single strand 17mer-c-L-DNA-SH. The degree of bound NOTA, maleimide and c-L DNA were determined by MALDI-TOF or UV/VIS spectroscopy. HS-PEG-L-DNAs with PEG sizes of 2, 5 and 10 kDa were conjugated with NOTA-maleimide and labelled with ⁶⁴Cu⁺⁺. The hybridisation was carried out in vitro with the radiolabeled L-DNA in different stoichiometrical ratios to NOTA-c-L-DNA-Cetuximab and analysed by agarose gel electrophoresis or UV-spectroscopy.
Results:
Four different L-DNA-Cetuximab-conjugates bearing 2.2±0.8, 2.9±1.1, 4.9±1.4 and 9±2 L-DNA chains (mean±SEM, n=3) were synthesized. PEGs with molecular masses of 2, 5 and 10 kDa did not clearly influence the hybridization of the ⁶⁴Cu-NOTA-PEG-L-DNA-conjugates with the complementary DNA-Cetuximab in vitro. Maximal hybridisation was reached in ratios equal and larger than 1:1 calculated from the DNA amount. The number of bound c-L-DNA on Cetuximab in the radioactive titration experiments were 2, 2.5, 5 and 9.5, which well agreed with 2.2, 2.9, 4.9 and 9, respectively, determined by direct UV/VIS spectroscopy of the c-L-DNA-NOTA-Cetuximab-conjugates.
Conclusion:
The effector ⁶⁴Cu-NOTA-PEG-L-DNA and the targeting probe NOTA-c-L-DNA-Cetuximab were synthesised, characterized and tested. The number of c-L-DNA on the Cetuximab was sufficient for in vitro hybridisation. The size of the PEG spacer did not clearly influence the in vitro hybridisation of the c-L-DNA-NOTA-Cetuximab-conjugates. This characteristic of the studied L-ON derivatives seems to be suitable for in vivo application. The potential of this L-ON approach in the pretargeting technology is currently under in vitro and in vivo investigation.