Introduction

Use of aptamers as a therapeutic targeting moiety holds the promise of building on the success of antibody drug conjugates. Aptamers have advantages over monoclonal antibodies as a targeting moiety including: (i) a smaller size that enables better tissue/tumor penetration; (ii) chemical synthesis which allows rapid and affordable scale-up; (iii) a superior safety profile from lack of immunogenicity and short half-life which reduces off-target adverse events; and (iv) room temperature stability for longer half-lives. Therefore, the use of aptamers in conjunction with drugs holds promise for tumor management.  

We have identified a novel therapeutic target for certain cancers (COF-01 is the proprietary target code name) using analysis of proteomics data and immunohistochemistry from patient-derived tumors. Here, we present a new therapeutic approach that targets COF-01-positive cells using next generation aptamers (Raptamers). Raptamers that bind tumor antigens such as COF-01, with high affinity and specificity are well-suited for development of Raptamer-drug conjugates (RapDCs). 

Methods

During the Raptamer selection process, library beads and a tagged target were incubated, pulled down with magnetic particles to recover sequences that were then re-introduced to the target in split fractions under varying conditions. After a second magnetic pull-down, putative Raptamers were released into solution and ready for amplification and next-generation sequencing. After analyses of sequencing data, Raptamer candidates were selected for synthesis. The binding affinity of the Raptamer candidates was then tested using biolayer interferometry. RapDCs were developed by conjugating the Raptamers to monomethyl auristatin E via a cleavable linker. The final RapDC product was purified by HPLC to obtain >90% purity. In vitro cytotoxicity was done using the Cell Titer Glo® (Promega) luminescent assay. IC50 values will be calculated for each RapDC in each human cell line.

Results

We have generated novel next-generation base-modified ssDNA aptamers (Raptamers) against COF-01 using a cell- and protein-based selection approach that utilizes our proprietary bead-based oligonucleotide combinatorial libraries. The binding affinities of these COF-01 Raptamer candidates were determined by concentration curves using biolayer interferometry (Octet®). The dissociation constants for the COF-01 Raptamers ranged from 80 nM – 200 nM. Anti-COF-01 Raptamer-drug conjugates (Rap-DCs) were then developed by conjugating the Raptamers to monomethyl auristatin F via a cleavable linker. RapDC in vitro efficacy was tested in COF-01-positive human skin cancer cells, gastric cancer cells, pancreatic cancer cells and COF-01-negative lung cancer cells. Our in vitro efficacy data for the COF-01 Rap-DCs has shown that the COF-01 Rap-DCs can selectively induce cytotoxicity in COF-01-positive human cells. There was a significant and specific decrease in cell viability of COF-01-positive cells when compared to COF-01-negative control cells.

Conclusion

We have generated high affinity Raptamers against a novel therapeutic target, COF-01. RapDCs were developed with Raptamers attached to a single MMAE with a valine-citrulline linker. We have examined the in vitro efficacy of COF -01 RapDCs. There was a significant and specific decrease in cell viability of COF-01-positive human cancer cells when compared to COF-01-negative control cells. Our next studies include examining the in vivo efficacy of RapDCs in cell-derived xenograft models of various types of cancers. This sudy provides proof-of concept for the use of high-affinity Raptamers against novel molecular targets, such as COF-01. This platform can be used to develop Raptamers against emergent neo-antigens on cancer cells that have escaped chemotherapy or other targeted therapies. We can rapidly generate high-affinity and specific Raptamers against a range of targets.