Targeted Drug Delivery using Aptamer Drug Conjugates (ApDC)

While many small molecule therapeutics are highly effective, they often have poor specificity. Systemic administration therefore leads to considerable side-effects; as is evident with chemotherapeutics used to treat cancer. In conditions with high mortality rates (such as cancer), patients are often willing to accept side-effects, in order to maximize the effect of treatment on the cancer. However, these off-target effects could be reduced if the drug can be more effectively delivered to the diseased tissues. Examples of this targeting have be achieved through the use of Antibody Drug Conjugates (ADCs).

Antibody drug conjugates are an important class of highly potent targeted therapies for the treatment of a wide variety of conditions (including cancer). Unlike traditional chemotherapies, ADCs are intended to target and kill only the cancer cells and avoid damaging the healthy cells, tissues etc. ADCs are complex molecules composed of a targeting antibody linked to a biologically active cytotoxic payload or drug. By combining the targeting capabilities of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs, ADCs should permit treatment of the condition without the harmful side-effects.

Basic structure of an Antibody Drug Conjugate (ADC) showing the 3 core components.
Cartoon representation of the mode of action of Antibody or Aptamer Drug Conjugates (ADC or ApDC respectively). The drug molecule (red spheres) are attached targeting aptamer / antibody. The targeting moiety binds to a cell surface protein / receptor and is taken up by the cells. The toxic cargo is then released; resulting in cell death in this case.

The broad steps involved in ADC development are:

  1. Identification and production of a suitable cell surface antigen.
  2. Isolation of a polyclonal antibody, then screening to isolate the best performing monoclonal antibodies.
  3. Humanise the antibody to limit recognition of the ADC by the patient’s immune system and reduce clearance from the blood stream.
  4. Extensive ‘reformatting’ to create the final ADC, which involves:
  • Identification of suitable drug conjugates, characterisation of their mode of action, potential off-target effects, etc
  • Identification of a suitable linker to attach the drug – needs to be stable in the circulation (to prevent ‘early release’ of the conjugate), but labile enough to efficiently release the payload once the target is reached. It is also important that the linker does not inactivate the drug
  • Attachment of the drug such that it does not hinder the antibody-antigen interaction. This conjugation typically utilises either cysteine or lysine residues, which limits the number of drug molecules that may be attached and the ADC’s efficacy
  • The antibody:drug ratio (ADR) must be optimised to deliver an appropriate amount of drug without hindering the antibody interaction. Since a protein contains numerous conjugation sites (usually amino acids containing hydroxyl, amine, or thiol groups) ADC drug products are heterogeneous mixtures of differently labelled molecules, with different efficacy.

When considering the scale of R&D, refinement, reengineering, and reformatting required in ADC development, perhaps it is more surprising that four ADCs have made it to the clinic.

Nucleic acid aptamers have the potential to overcome the limitations associated with antibodies.

  1. In vitro selection methodologies allow aptamers to be isolated against a wider variety of targets, including whole cells, tissues, and microorganisms. This avoids steps involved in identification of a cell-surface antigen.
  2. Aptamers can be selected based on the ability to discriminate between closely related cell or tissue types.
  3. Process modifications ensure that the resulting aptamers are taken up by the target cell line
  4. Aptamers are synthetic and have low inherent immunogenicity, avoiding any need to ‘humanise’ them.
  5. ‘Reformatting’ is considerably simpler for aptamers
  • Drug conjugates and appropriate linkers still need to be identified and characterised
  • Drug conjugates can be attached at a specific position on the aptamer during synthesis. This eliminates the problems associated with conjugates disrupting the active site / structure of the aptamer.
  • Site specific labelling of aptamers allows simple control of Aptamer:Drug Ratio (ApDR). Aptamers are readily appended with a ‘tail’ which allows greater ApDRs.

The Advantages of Aptamers:

  • Simplified process – Hypothesis-free, in vitro selection of cell or tissue targeting aptamers, coupled with synthetic production processes, greatly simplify ApDC development.
  • Small Size– the small size of aptamers gives improved tissue penetration and uptake.
  • Routine chemistry – A comparable range of drug conjugates, linker chemistries, etc are available for use with aptamers.
  • Avoid labelling issues – aptamer library can be prepared with the drug and linker conjugate in place, then directly select the aptamer with the drug in situ. This ensures that the resulting ApDC will recognise its target.
  • Simplified conjugation – Unlike antibodies, the site of attachment to the aptamer can be controlled, eliminating the possibility that the conjugate will hinder target binding and giving greater control over Aptamer: Drug Ratio (ApDR). It is also possible to append the aptamer with a nucleic acid ‘tail’ (or other branched linker molecule) to aid drug conjugation.
  • Diverse cargos – Aptamers can be used to deliver a wide variety of cargos, including chemotherapeutic agents, siRNA, synthetic virions, synthetic genes etc.
Nucleic acid aptamers were selected which discriminate between a model cancer cell line and the healthy equivalent cell line in a hypothesis-free selection approach. The resulting aptamer shows clear preferential binding to the cancer cells (upper panels) and no binding to the healthy control (lower panels).
Internalisation time-course shows that FITC-labelled aptamers initially bind to their target on the cell surface (far left panel). The aptamers are gradually internalised over the course of 5 minutes. This demonstrates aptamer mediated delivery of a fluorophore. The fluorophore could be replaced with a therapeutic agent.
Schematic representation of an Aptamer Drug Conjugate (ApDC) showing the 3 core components. Unlike Antibody Drug Conjugates (ADCs), ApDCs require fewer steps to prepare as they do not require the extensive ‘reformatting’ needed for ADCs.
Aptamers can be appended to other types of cargo. In this case, the aptamer (pink) is attached to an Antisense Oligonucleotide (ASO) (red and blue), but this may be an siRNA, a virus, a nanoparticle, synthetic gene or other aptamers.
Aptamer based delivery of a cytotoxic compound shows cell-specific killing by the Aptamer Drug Conjugates (ApDCs) in two different model systems. Flow Cytometry was used to demonstrate ApDC mediated cell-death in a Myeloma model (left panels). No effect was seen for the Healthy cell line control (upper left), but a clear killing effect is seen for the Myeloma line (bottom left), when the ApDC is present. In a separate example, a viability assay was used to demonstrate the effect of an ApDC against a prostate cancer cell model (right panel). The target cells (orange bars) show reduced viability when treated with the ApDC (but not the separate components); the non-target control cells (blue bars) show no response to the ApDC or it’s components.