Bispecific antisense oligonucleotides with multiple binding sites for the treatment of prostate tumors and their applicability to combination therapy

Antisense oligonucleotides (oligos) against transforming growth factor-alpha (TGF-alpha; MR1) and its binding site, the epidermal growth factor receptor (EGFR; MR2) have proven efficacious against PC-3 and LNCaP prostate tumors when evaluated in both in vitro and in vivo models. To enhance their activity, and also to introduce a significantly different type of multifunctional agent into this field, "bispecific" oligos were constructed containing truncated sequences (derived from MR1 and MR2) recognizing both TGF-alpha and EGFR mRNA internal binding sites, located about their respective AUG initiation codons. Two bispecifics were constructed, each having complementary sequences for TGF-alpha and EGFR mRNA, but differing in their 5' to 3' tandem orientation (TGF-alpha/EGFR [MR12] and EGFR/TGF-alpha [MR21] sequences). These bispecifics were tested in an in vitro system against PC-3 and LNCaP prostate tumor cells, with comparisons made to the original monospecific oligos from which they were derived. Efficacy was also compared when administered either alone or in combination with conventional chemotherupeutic agents. The purpose of this study was: 1) to validate the concept that these newly developed bispecific oligos have antitumor activity; 2) to enhance their efficacy through combination therapy; 3) to identify differences in effectiveness dependent upon binding site orientation; 4) identification of a dominant binding site that can be used to design other bispecifics that target additional tumor regulatory pathways. When fully evaluated against PC-3 cells in a series of experiments, newly developed bispecific oligos are at least as effective as their monospecific counterparts from which they were derived, and the bispecific with the MR21 orientation is notably more effective than the MR1 monospecific by 64% (p = 0.014 by Student t-test and p = 0.068 by the more stringent Mann-Whitney U test). Bispecifics were more effective when administered with chemotherapeutics (producing inhibition of 52.1% and 61.2% for MR12 and MR21, respectively, with Cytoxan (cyclophosphamide) inhibition of 59.0% and 65.1% for MR12 and MR21, respectively, with Taxol (paclitaxel) and 63.0% and 69.4% for MR12 and MR21, respectively, with DES [diethylstilbestrol]). Increasing the oligo concentration above 6.25 mu M with cyclophosphamide had no additional effect. The sequence directed against EGFR was dominant and contributed most to bispecific activity, particularly when inserted 5' to the TGF-alpha binding sequence (MR21 orientation). Bispecific oligos are a significant advance in the design of antisense compounds and could play a role in treating prostate cancer, particularly when they are administered with traditional chemotherapeutics. The truncated portion of the MR2 oligo used here should be included when constructing second-generation bispecifics that target proteins associated with other regulatory pathways, such as apoptosis. (c) 2006 Prous Science. All rights reserved.