Monoclonal antibodies (mAbs) have naturally evolved as suitable, high affinity and specificity targeting molecules. However, the large size of full-length mAbs yields poor pharmacokinetic properties. A solution to this issue is the use of a multistep administration approach, in which the slower clearing mAb is administered first and allowed to reach the target site selectively, followed by administration of a rapidly clearing small molecule carrier of the cytotoxic or imaging ligand, which bears a cognate receptor for the mAb. Here, we introduce a novel pretargetable RNA based system comprised of locked nucleic acids (LNA) and 2¢O-Methyloligoribonucleotides (2¢OMe-RNA). The duplex shows fast hybridization, high melting temperatures, excellent affinity, and high nuclease stability in plasma. Using a prototype model system with rituximab conjugated to 2¢OMe-RNA (oligo), we demonstrate that LNA-based complementary strand (c-oligo) effectively hybridizes with rituximab-oligo, which is slowly circulating in vivo, despite the high clearance rates of c-oligo.

In prokaryotes, the removal of an N-terminal formyl group by the peptide deformylase is required for post-translational protein processing. Our identification of human mitochondrial PDF (HsPDF) and deformylated peptides in the mitochondria shows that a similar post-translational processing of the 13 human mitochondrial DNA-encoded, and formylated, proteins can occur. Inhibition of HsPDF reduced proliferation of many human cancer lines. We developed a monoclonal antibody to hsPDF; profiling of cancer and normal cell lines by three immunoassays, flow cytometry, immunoprecipitation and in-cell western, showed over-expression of HsPDF in cancer cell lines. This data was confirmed at the mRNA level by RT-PCR. As the c-myc oncogene regulates mitochondria and metabolism in cancer, we investigated its role in regulating HsPDF. In a tet-off c-myc cell line, the expression of HsPDF was regulated by c-myc; inhibiting this protein resulted in the selective inhibition of mitochondrial protein translation, and eventual death of myc-positive, but not myc-negative cells. Furthmore, knocking down c-myc in a Burkitt's Lymphoma cell line significantly reduced the expression of both the mRNA and the protein levels of HsPDF relative to beta-Actin. The mechanism of death by actinonin was apoptosis in two c-myc over-expressing cell lines. Death was initiated with mitochondrial membrane depolarization, followed by inhibition of mitochondrial translation, and eventually caspase-3 activation and apoptosis. To confirm the specificity of PDF in cell death, we used two other hydroxamic acid peptidomimetics that do not target HsPDF. These compounds did not result in apoptotic death. Furthermore, inhibition of translation of the mitochondrial DNA by chloramphenicol and tetracycline, two structurally different inhibitors of the mitochondrial ribosome, which is upstream of deformylation, followed by treatment with actinonin resulted in a delay and marked reduction in apoptosis by actinonin, but not by staurosporine and the HDAC inhibitors SAHA and Trichostatin A. Thus, death was not a result of ATP depletion, but a unique PDF-mediated mechanism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1128. doi:1538-7445.AM2012-1128