Antibodies… What Are The Alternatives?
The use of antibodies in basic research is very well established with an estimated research reagent market size of $1.4 billion. The value of the therapeutic antibody market is considerably larger, with antibody-based drugs in clinical use to treat conditions such as cancer, rheumatoid arthritis and Crohn’s disease. The recent growth of the antibody therapeutics market has led to a great deal of investment in this field with many novel antibody based drugs currently in development. Monitoring of monoclonal antibody drugs is possible with the use of commercially available anti-biotherapeutic antibodies, which specifically target anti-drug antibodies and can be used to measure free or bound drug in patient samples.
New technology has led to improvements in the generation and manufacture of antibodies. Antibodies can now be produced by recombinant methods and engineered to meet specific requirements. The discovery that camelids (camels, llamas and alpacas) produce fully functional antibodies consisting of only heavy chains has further increased the potential of monoclonal antibodies. These stable, smaller sized ‘nanobodies’ have a number of advantages over their larger immunoglobulin counterparts.
However, over the last 20 years a number of alternatives to antibodies, displaying similar properties but with functional advantages, have been developed. In 1990 a paper by Tuerk and Gold (1) from the University of Colorado, described a method of generating a single stranded RNA molecule that was capable of binding with high affinity and specificity to bacteriophage T4 DNA polymerase. This molecule was termed an aptamer. Aptamers can be single stranded RNA or DNA molecules at around 10-30 kDa in size. Generation of these nucleic acids, capable of mimicking the action of antibodies, has evolved over the years, but is based upon a method known as the systematic evolution of ligands by exponential enrichment, or SELEX. Briefly, a nucleic acid pool is incubated with the target and successful binders are amplified by PCR. This selection cycle is repeated 8-15 times. The resulting nucleic acid aptamers are cloned and sequenced. Aptamers have a number of advantages over antibodies. They are smaller and more stable, while quicker and cheaper to generate than antibodies produced by hybridoma technology. They are of a known sequence and suitable for a wider range of targets, such as smaller and non-immunogenic antigens. Disadvantages have also been noted, especially with regard to therapeutic uses where retention in the bloodstream and control of the duration of action are important. These issues are being addressed, the first FDA approved aptamer for therapeutic use made over $200 million in 2005 (2). Macugen (Pegaptanib) binds vascular endothelial growth factor (VEGF) and prevents loss of vision from abnormal blood vessel growth. Aptamers have also been shown to be particularly useful in tumour imaging and delivery of cytotoxic agents (3).
A number of other antibody alternatives have been developed, these include peptide based aptamers, an example of which was described by Colas et al in 1996 (4). Peptide aptamers were further developed and specific formats commercialised as affimers. Affimers are a proprietary technology by Avacta in Cambridge, UK. Based on two different protein scaffolds, the human protease inhibitor Stefin A, and the plant protein Cystatin A. They are designed to bind with high affinity and specificity with potential uses in research, diagnostics and therapeutics. Their smaller size, 12-14 kDa, and stability has additional benefits over aptamers and antibodies, such as the opportunity for topical application of therapeutics. The ease of generation by phage display screening, and low cost production in bacterial expression systems, could lead to a reduction in costs for development of research tools, diagnostic tests and drug discovery. Avacta currently have a number of affimers at the pre-clinical stage in their therapeutics pipeline.
With technological advances in the generation and manufacture of monoclonal antibodies, and the commercialisation of antibody mimetics, including aptamers and affimers; a broader range of research tools are now available to scientists. Furthermore, both the diagnostic and therapeutic fields could be set to benefit, with many new biopharmaceutical drug candidates in development.
- Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. 249 (4968): 505-510.
- Lakhin, A. V. et al (2013) Aptamers: Problems, Solutions and Prospects. Acta Naturae. 5 (4): 34–43.
- Hicke, B. J. et al (2006) Tumor targeting by an aptamer. J Nucl Med. 47 (4): 668-678.
- Colas, P. et al (1996) Genetic selection of peptide aptamers that recognize and inhibit cyclin-dependent kinase 2. Nature. 380 (6574): 548-550.
Written by Sharon Craggs, Technical Analyst