APTAMER TECHNOLOGY – From Postal Codes to GPS: Building Better Drug Conjugates With Aptamers


Orthoclone OKT3 was the first therapeutic monoclonal anti­body brought to market in 1986 for the treatment of kidney trans­plant rejection.1 Following this, the biotherapeutic market has continued to expand, representing the fastest-growing pharma­ceutical market sector. Predicted annual growth of 14% with an­nual sales of over $120 billion in this sector compares favorably to growth of just 6.8% for traditional pharmaceuticals.2,3 Indeed, last year, five of the top-ten best-selling drugs were antibody ther­apeutics, and antibody technology has a proven track record in developing effective therapies with good safety profiles across on­cology, hematology, cardiology, immunology, and autoimmu­nity.4-6 The increased interest in this sector has spurred advances in immunotherapy, gene and cell therapies, and affinity reagent technologies. However, these advances have also revealed limi­tations in antibody technology that prevent these molecules from meeting the developing potential of precision medicine.2,3 Such limitations include the large size of anti­body molecules (Figure 1), which hampers their ability to penetrate tissues in vivo, and their structural complexity, which requires expensive and difficult to optimize mam­malian manufacturing systems.

Size comparison of an antibody (left) versus an aptamer (right). Antibodies are approximately 10 times the size of aptamers. The larger size and molecular weight of antibodies (~150 kDa) can impede tissue penetration compared to the smaller size and molecular weight of aptamers (~5-18 kDa).

Click image to enlarge

A new breed of affinity binders that can be used as an alternative to antibody therapeutics has the potential to reshape the industry, delivering new medicines with improved safety and efficacy profiles and reduced healthcare costs.


Conjugating drug molecules to an affinity ligand produces targeted therapies that can enhance the selectivity and hence the performance of the therapeutic agent while simultaneously reducing off-target interactions, which lead to adverse side ef­fects.7 Antibody-drug conjugates (ADCs) allow the combination of the targeted pre­cision of antibodies with:

  1. the potency of small molecules for tar­geted therapy
  2. oligonucleotide therapeutics (such as siRNA) – for the targeted cellular deliv­ery of gene therapy
  3. an additional affinity reagent as a bis­pecific modality for increased thera­peutic targeting and receptor activation

From the introduction of the first ADC in 2001, these molecules have undergone multiple rounds of refinement, including developing new toxic payloads, increasing the linker stability, and assessing the po­tential for antibody alternatives.8 Though the high target binding affinities of mono­clonal antibodies reduce off-target effects, the large size of these biologics has been shown to restrict their diffusion through tis­sue in vivo and limit the accessibility of ADCs to certain epitopes.9 Their high mo­lecular weight also reduces the rate of renal clearance. While this can be benefi­cial in that it reduces dosing frequency, it can also be detrimental in that long circu­lation times increase the potential for off-target effects and the development of anti-drug antibodies.

Antibodies are composed of multiple polypeptide chains that are variably glyco­sylated and connected through numerous disulfide bonds. This structural complexity poses a significant challenge to the scale-up of their manufacture, requiring expen­sive mammalian expression systems and often leading to batch consistency issues.10 Additionally, for ADC production, the final antibody must be further modified with the therapeutic payload. Site-specific chemistries, such as maleimide linkages on cysteine residues, can still lead to prob­lems in generating a homogenous product due to the number of cysteine residues present in the antibody molecule and their varying accessibility and chemical reactiv­ity. While there has been considerable progress in this area, significant chal­lenges remain in controlling the anti­body:drug ratio and purification of the final product.

Delivery of the final ADC cargo to the cell’s cytoplasm for its effector function is not straightforward with antibodies. Due to the large size of antibodies, cellular internalization can be retarded, requiring highly stable linkers to prevent premature linker cleavage and release of the cargo before it reaches the intended site of ac­tion.11


The use of nucleic acid aptamers as the targeting moiety overcomes many of these pitfalls. Aptamers are small, single-stranded oligonucleotide molecules iso­lated from large libraries, using highly tunable in vitro processes. Based upon their nucleotide sequences, aptamers form a variety of structures and can be selected to specifically bind to their target mole­cules in the same way as antibodies. How­ever, aptamers also offer manufacturing advantages as they are generated via well-defined solid-phase synthetic processes, for batch consistency and sim­ple modification with a range of cargo.12 Due to their small size, aptamers have a short half-life in vivo, offering reduced tox­icity concerns compared to antibodies, which can stay in the patient’s circulation for weeks. To delay renal clearance, strate­gies must be adopted, such as PEGylation, which increase half-life where required.13 Aptamer-drug conjugates (ApDCs) are offering new potential for this therapeutic approach to provide effective treatments for a range of diseases.

The first aptamer therapeutic, pegap­tanib sodium (Macugen) was licensed in 2004 for the treatment of neovascular (wet) age-related macular degeneration.14 A further 10 aptamer therapeutics have successfully entered clinical development for conditions ranging from oncology and inflammation to diabetes and coagulation.

NOXXON Pharma’s unique aptamer-based Spiegelmers® are L-enantiomers and consequently not subject to nuclease activity for increased retention. Ongoing trials for Spiegelmer therapeutics include Phase 1/2 trials for the treatment of pan­creatic cancer and brain cancer in differ­ent combinations and Phase 1 trials for the treatment of solid tumors.15 Iveric Bio have progressed their complement 5 targeting aptamer-based therapeutic for the treat­ment of age-related macular degenera­tion to Phase 3, and for the treatment of autosomal recessive Stargardt disease to Phase 2.16

The advantages that aptamer thera­peutics hold over standard antibodies and antibody-related binders, have led to them being investigated as direct therapeutics and as delivery vehicles for a range of cargo.17 Many examples of ApDCs have been generated with cargo ranging from chemotherapeutics and photosensitizers to siRNA (Table 1).7

Examples of aptamer-drug conjugates used in targeted drug delivery.


Despite more than three decades since the discovery of the first aptamers, development of these molecules through the clinic has been limited. This lag period is not unexpected in the uptake of a new technology, and many of the perceived limitations of therapeutic aptamers have been addressed through new advances:

Susceptibility to ubiquitous nucleases within the body – The issue of stability has been largely overcome by modifying the nucleic acid backbone with the substitution of 2’-hydroxyl groups in RNA aptamers with 2’-fluoro, 2’-amino or 2’-O-methyl groups, and through 3’-end capping with inverted thymidine, other blocking mole­cules or the circularisation of aptamers.30

Rapid clearance by renal filtration – Chemical conjugation with high molecular weight polyethylene glycol (PEG), choles­terol or other carrier molecules helps to re­duce the rate of renal clearance and improve aptamer retention in vivo. In some cases this strategy has been shown to extend aptamer half-life beyond 48 hours, thus improving their pharmacoki­netic properties.30 However, for situations such as the development of imaging reagents or “hit-and-run” treatments with highly toxic payloads (where long retention times are detrimental) rapid clearance may be beneficial for ApDCs. The ability to tune aptamer therapeutic half-life in this way is beneficial.


As aptamer technology is increasingly explored for its therapeutic potential, there are a number of additional advantages to these molecules that make them particu­larly efficient for drug delivery.

Biomarker-Free Selection & Screening
The in vitro selection process for cell-targeting aptamers allows the discrimina­tion of cell phenotypes, such as diseased versus healthy. Each cell type can be in­cluded in the aptamer selection strategy as part of multiple positive and negative se­lection rounds, removing the need for a priori knowledge of disease-specific bio­markers. Aptamers that bind to the dis­eased cells and not to the healthy cell population can subsequently be used to pull-down the specific biomarker that they target.31 In this way, aptamer selection can be used for biomarker discovery and vali­dation in one complete step.

Simple, Cost-Effective Manufacturing
Aptamers can be synthesized using well-defined and characterized chemical processes, with no requirement for cell sys­tems. This makes aptamer production more cost-effective and simpler to scale up than comparative protein-based therapeu­tics. For ApDCs that are delivering oligonucleotide therapeutics, such as siRNA or antisense oligonucleotides, there is also the potential to synthesize the ApDC as a single contiguous molecule, removing the need for additional conjugation and purification of the final aptamer with the drug cargo. This greatly reduces the pro­duction costs, increases standardization of the product and improves yields relative to processes requiring post synthesis conju­gation.

Rapid Internalization for Drug Delivery
As aptamers are approximately one-tenth the size of antibodies, they can be readily taken up by cells through a number of mechanisms, most commonly endocy­tosis and micropinocytosis.32 Aptamer up­take is ultimately determined by the specific interaction with their target, mak­ing them very appealing for the delivery of cargo to the cell interior for targeted chemotherapy or gene therapy applica­tions.

Delivery to Challenging Tissues In Vivo
Delivery of therapeutics across the blood-brain barrier has long been chal­lenging, resulting in limited treatment op­tions for many brain and central nervous system diseases. Many targeted antibody therapeutics for neurological conditions have failed to show clinical benefit and pose serious risk of eliciting an immune re­sponse. Aptamers have shown to be a promising class of therapeutic for the po­tential treatment of brain disorders, as they are small, non-immunogenic, and able to permeate the blood-brain barrier with rel­ative ease compared to larger antibody molecules.28,33

A second challenging site for targeted delivery of oligonucleotide therapeutics is the kidney.34 Despite increased approval of oligonucleotide therapeutics, targeting these molecules to extrahepatic tissues re­mains challenging. Due to the small size of oligonucleotides, targeting to the kidney is difficult as it promotes the rapid renal fil­tration of the intended therapeutic, limiting the potential effect. To overcome this prob­lem, siRNA and antisense oligonucleotides can be conjugated to aptamers that are both highly specific for targeted delivery, and with high affinity to prevent the thera­peutic being cleared from the kidney. Ap­tamer Group is currently working in collaboration with AstraZeneca to develop such solutions for the treatment of kidney disease using aptamer technology.35


As the potential of biotherapeutics continues to expand and the limitations of antibody technologies are increasingly being highlighted, a new breed of oligonucleotide-based affinity binders is showing their value as more molecules progress steadily toward clinical applica­tion. Aptamers offer many advantages over antibodies and protein-based binders, including ease of formatting and production, improved cell internalization, and the ability to target new diseases, such as in the CNS. With many of the perceived limitations of aptamers having been suc­cessfully addressed, aptamers are coming of age. These novel therapeutics have the potential to deliver effective treatments that can transform patients’ lives whilst offering essential cost savings for healthcare providers who urgently need alternatives in the face of the high cost of antibody therapeutics.


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Dr. David Bunka is the CTO of Aptamer Group. He has spent over 20 years developing nucleic acid aptamers against a wide variety of targets, including small molecules, disease-associated proteins, several cancer-associated cell-lines, viruses and tissue biopsies. He earned his PhD in Molecular Biology from University of Leeds.

Dr. Emily Robinson leads the therapeutic development team at Aptamer Group to support early drug discovery for partners and collaborators. She has research experience in the evaluation of dysregulated signaling pathways in cancer and the identification of novel drug targets. She earned her PhD in Cancer Immunology from University of Leeds.