Aptamerdrug Conjugates For Targeted Cancer Therapy

The clinical utility of radioimmunoconjugates and chemoimmunoconjugates in cancer treatment is well documented with several examples of each in clinical practice or under development at this time.123-125 More recently aptamer conjugates for cancer therapeutic and diagnostic appli-cations,14,23,108,116,118-120,126 biosensors,127-129 flow cytometry,130 ELISAs,131,132 and capillary electrophoresis for separation technology have been discribed.40,133 As noted in Section 16.2, aptamers have distinct advantages over antibodies, and, for the purpose of targeted delivery of toxins, their greatest advantage lies in their relatively smaller size and remarkable stability. The smaller size of aptamers, as compared to antibodies, allows for a more efficient tumor penetration of aptamers, translating to a more efficient delivery of a cytotoxic payload to tumor cells.119 Our research group is interested in the use of aptamer-toxin conjugates for therapeutic applications,126 and we believe that aptamer-toxin conjugates may result in future novel therapeutic applications in oncology and other areas of medicine.

For the preparation of the aptamer-drug conjugates, conventional conjugation strategies that have been used for the preparation of immunoconjugates may also be employed with some modifi-cations.125 For targeted radiotherapy, beta-emitting radioisotopes, such as 131I, 90Y, or 67Cu, or alpha-emitting radioisotopes such as 213Bi or 211At, may be used to develop radioaptamerconju-gates. These radioisotopes may be linked directly to aptamers or immobilized through metal chelators attached to aptamers. For targeted chemotherapy, drug molecules (i.e., doxorubicin, calicheamicin, or auristatin) may be covalently coupled to one or more sites on the aptamer using simple chemistry (i.e., formation of amide, disulfide, or hydrazone bond).134,135 The development of aptamer conjugates is facilitated by site-selective functionalization of aptamers at their 3'-and/or 5'-terminus, making it possible to reproducibly attach an exact number of drugs in a site-

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specific manner. , , Aptamers may also be functionalized within the oligonucleotide chain using modified nucleotides with desired functional groups; however, this latter approach may negatively impact the aptamer conformation and function. In the case of conjugation of toxins to antibodies, the reaction is most commonly not site-directed, and this nonspecific conjugation approach may negatively impact the binding characteristics of antibodies.136 Furthermore, unlike antibodies, the desired linkers or functional groups on aptamers for conjugation on other applications can be easily introduced during the chemical synthesis of these molecules. We have schematically demonstrated several proposed conjugation strategies between aptamers and drugs (Figure 16.4). Typical functional groups modifiable at the 5'- or 3'-ends of aptamers are mostly nucleophiles, including amine (-NH2), sulfhydryl (-SH), andhydrazide (-C(O)NHNH2). Appropriate functional groups on the drugs for immobilization on aptamers may be carboxylic acid (for amide bond formation with amine-modified aptamers), maleimide or unsaturated carbonyl (for thioether formation with sulfhydryl groups on aptamers), activated disulfide (for example, S-pyridyl disulfide) for disulfide

aptamer linker


Site-specific conjugation

FIGURE 16.4 Available conjugation strategies between end-functionalized aptamers and drug molecules. Panels (a) through (d) demonstrate a series of single- and double-conjugation strategies.

formation with sulfhydryl groups on aptamers, and ketone for hydrazone formation with hydrazide on aptamers.136

The covalent bonds formed through the aforementioned strategies are all cleavable in vivo, allowing for the active drug to be released for therapeutic efficacy. The choice of conjugation strategy may allow for a spatial control of this release and improved clinical efficacy. In addition, by altering the size of aptamer-drug complex, the pharmacokinetics properties of the conjugate may be controlled, allowing for temporal control of the drug circulation and clearance. For example, a longer circulation time and reduced renal clearance may be achieved by linking the aptamer-drug conjugates to PEG polymers to increase the size of the complex above the threshold for renal clearance.137 This conjugate form may result in improved drug delivery to cancers as a result of increased systemic circulation time. Intracellular versus extracellular drug release also have unique sets of design considerations.138 In the case of the disulfide linkage approach, the aptamer-drug conjugate is expected to be stable in plasma during circulation with subsequent intracellular cleavage of disulfide bond and drug release due to high intracellular concentration of glutathione (GSH).139 Hydrazone linkage also has favorable properties for conjugation of drugs to aptamers, allowing for a pH sensitive release of the drug in a relatively more acidic environment of the tumor tissues.134,140,141 Therefore, disulfide and hydrazone linkages may be considered when developing aptamer conjugates for cancer therapeutics.

We have recently developed a novel strategy for generating aptamer-drug conjugates by intercalating drugs between nucleic acid bases of the aptamer (Figure 16.5).126 Using doxorubicin (dox) as a model drug and the A10 PSMA aptamer as model aptamer, we have developed a stable aptamer-dox conjugate through physical interactions. The resulting physical conjugate demonstrated the following characteristics: (1) high discrimination between cancer cells expressing target antigen and cells lacking target antigen; and (2) efficient delivery of anti-cancer drug to target cancer cells.126 Specifically, we developed aptamer-dox conjugates and demonstrated the stability of this system in vitro. Using LNCaP prostate cancer cell lines that express the PSMA antigen and PC3 prostate cancer cell lines that do not express the PSMA protein, we demonstrated a differential uptake of the aptamer-dox conjugate by LNCaP cells, translating to a more efficient cellular toxicity and anti-cancer efficacy in vitro (Figure 16.6). These results suggest that physical conjugate strategies such as intercalation may serve as a novel approach for developing aptamer-drug therapeutics.

PSMA aptamer intercalation


Physical conjugate

FIGURE 16.5 A schematic diagram outlining the formation of physical conjugate between aptamers and drugs capable of intercalating into base pairs of aptamers. Doxorubicin, an anti-cancer drug, can be intercalated into a PSMA aptamer to form a 1:1 complex.

FIGURE 16.6 The confocal laser scanning microscopy images of LNCaP (a) and PC3 cells (b) after treatments of the aptamer-doxorubicin physical conjugate (1.5 mM for 2 h.

In addition to small molecule and radioisotopes, bio therapeutics such as small interfering RNAs (siRNA)142-144 and protein-based toxins may also be conjugated to aptamers. For example, siRNAs can be coupled to aptamers through disulfide bond, allowing the release of siRNAs in the active form after cytosolic uptake by the targeted cells. A conjugate of aptamer-siRNA may represent a novel class of therapeutics with widespread application in medicine.116

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