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. 2013 Mar 19;6(3):340-57.
doi: 10.3390/ph6030340.

A review of therapeutic aptamer conjugates with emphasis on new approaches

John G Bruno  1
Affiliations

A review of therapeutic aptamer conjugates with emphasis on new approaches

John G Bruno. Pharmaceuticals (Basel). .

Abstract

The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies.

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Figures

Figure 1
Figure 1
The author's patented [13] chemical scheme for attachment of proteins such as Fc fragments for opsonization [10] and C1qrs to invoke complement-mediated lysis of Gram negative bacteria [11], cancer cells [12,15], or parasites by direct coupling to the unpaired 3' adenine overhang on double-stranded (ds) PCR products imposed by Taq DNA polymerase. Although a sulfo-EGS bifunctional linker (ethylene glycol bis(sulfosuccinimidylsuccinate)) is shown, low levels of glutaraldehyde [43] or other bifunctional linkers could be used as well.
Figure 2
Figure 2
Schematic of the putative DNA aptamer-C1qrs conjugate-mediated triggering of the classical complement system to kill Gram negative bacteria and other thin-walled (cancer and some parasite) target cells by complement-mediated lysis.
Figure 3
Figure 3
Spread plates from an anti-E. coli O111 lipopolysaccharide (LPS) aptamer-C1q killing experiment [11]. The “antibiotic” effect due to aptamer-C1q triggering of the complement system is especially visible in the lower panel where the full test Group 1 shows few, if any, colonies while control Group 2 shows a noteworthy number of colonies across the same two dilutions. The compositions of each group were as follows: Group 1-full test group with aptamer-C1q conjugate and all other complement components. Group 2-control group for the alternative pathway which contained no aptamer-C1q and simply assessed bacterial colony counts in the presence of human serum. Group 3- was a positive growth control group with no serum or aptamer-C1q added. Group 4- contained bacteria treated only with the aptamer-C1q conjugate, but may have triggered some bacterial killing due to contaminating traces of other complement or serum proteins. Ten-fold serial dilutions are shown across the top of the top panel. The bottom panel shows only the 10−6 and 10−7 dilutions for Groups 1 and 2 from a second experiment.
Figure 4
Figure 4
Further examples of aptamer-biotin-streptavidin-C1q killing of Gram negative bacterial species. Top panel shows results for E. coli strain K12 with clear cytopathic effect at 10−3 dilution and beyond. Bottom panel shows killing of Campylobacter jejuni at the same dilutions. “Oligoteins” indicate where aptamer-biotin-streptavidin-C1q conjugates were used.
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