A novel pretargeting system based on complementary L-oligonucleotides

Objectives: High metabolic stability, low immunogenicity and negligible specificity for naturally binding partners are predominant characteristics of L-configured oligonucleotides. These advantages predestine this substance class
for its use in pretargeted radioimmunotherapy as in vivo recognition system between a tumor-specific antibody and a radiolabeled chelate. We evaluated this new pretargeting system consisting of ⁶⁴Cu labeled NOTA-L-DNA-10kDa-PEG and c-L-DNA modified Cetuximab (C225) in vitro and in vivo.
Methods: C225 was functionalized with NOTA, maleimide moieties and thiol-bearing c-L-DNA. Competition studies were carried out against ⁶⁴Cu labeled standard NOTA₃-C225 in FaDu and A431-cell lysates. In vitro pretargeting studies were done in intact FaDu and A431 cells. PET and biodistribution studies were performed both in FaDu and A431 tumor bearing mice by intravenous injection of 4 nmol NOTA₃-C225-(c-L-DNA)_1,5 and 1 nmol [⁶⁴Cu]Cu-NOTA-L-DNA-10kDa-PEG 24 h later.
Results: We synthesized two Cetuximab derivatives with 1.5 and respective 5 c-L-DNA molecules per antibody. Competition assays showed that affinities are not affected as a result of conjugation with NOTA and c-L-DNA.
PET studies injecting only [⁶⁴Cu]Cu-NOTA₃-C225-(c-L-DNA)_1.5 revealed that a pretargeting interval of 24 h is the best compromise between tumor accumulation, blood background as well as liver uptake. Biodistribution in pretargeted A431 tumor mice is characterized by decreased tumor uptake (see figure). Internalization of antibody within waiting period is the obvious reason and could be confirmed by cellular uptake studies. After 24 h over 2/3 of surface bound antibody was internalized.
Conclusions: The present pretargeting concept shows high potential for further preclinical studies. Use of a noninternalizing antibody is necessary to enhance both tumor uptake and tumor to background ratios.