Monoclonal antibodies play a vital role in research, diagnostics, and therapeutics. Many advancements have been made to improve the performance of monoclonal antibodies, specifically in the therapeutic area. Though antibodies are an ideal choice for many applications, there are some instances where an aptamer may be a better alternative. Some of the key differences between antibodies and aptamers are highlighted below and outlined in the Aptamers vs. Antibodies table.
Targets for Antibody / Aptamer Development
Traditional antibody production requires an antigen, a molecule that is identified as foreign and triggers a response by the host’s immune system. The process typically begins with multiple injections of a purified protein.1 Small molecules will not elicit an immune response, so they are not viable targets for traditional antibody production. Because antibodies are initially raised in a live animal, it is also difficult to generate antibodies to a compound that is highly toxic. Aptamers are produced in vitro, so they can be selected to a wider range of targets.
Aptamers can be selected to detect non-immunogenic small molecules and metabolites as well as compounds that are toxic to cells. Aptamers can also be selected for binding to live cells, without knowledge of a specific cell surface target.
Antibody / Aptamer Development Processes
Traditional monoclonal antibody development involves several immunizations of a host animal, isolation of antibody-producing cells, and fusion with fast-replicating myeloma cells. This is followed by hybridoma selection and antibody production. Each step is time-intensive, with the entire process realistically taking four to six months. Because antibodies are raised in animals, they are designed to function under physiological conditions.
Traditional aptamer selection is an in vitro process that is typically completed in two to three months, with variations on the selection process being completed even more quickly. Aptamers can be selected to perform in unique buffer conditions or in the presence of specific proteins or chemical compounds; yielding aptamers suited for specific biological, environmental, or industrial applications. (Read more about aptamer selection.)
Access to Tissues and Cells
A typical IgG antibody is ~150 – 170 kDa, versus ~12 – 30 kDa for a 30 to 80 nucleotide aptamer. Large antibody size limits membrane permeability and can create difficulties when targeting dense tissue.13,18 Unique bi-specific antibodies have been engineered to cross the blood-brain barrier, but traditional antibodies cannot.16 Small antibodies (~90 kDa) have been isolated from llamas, sharks, and camels. Fragments of these small antibodies, termed nanobodies (~ 15 kDa), are showing promise as affinity agents but are not widely accessible.8
Aptamers have demonstrated enhanced access to tissues in both in vivo and in vitro imaging.19 Some aptamers and aptamer complexes have been shown to cross the blood-brain barrier9 and even enter cells. Base Pair has selected aptamers for the ability to enter a particular type of cell.
Specificity / Selectivity
A key feature of monoclonal antibodies and aptamers is binding to a specific epitope, or region, on a target of interest. Specific epitope binding typically enables specific target detection. Cross-reactivity occurs when an epitope is conserved between two, typically related, targets. Species cross-reactivity occurs when an epitope is conserved between species, such as human and mouse or human and canine. While 100% homology does not need to exist between the targets for cross-reactivity, a high degree of homology is required at the binding epitope. In a study of several ophthalmic antibody drugs, variation of a single amino acid within the binding epitope significantly reduced human VEGF-A antibody binding in mouse and rat models. Human VEGF-B antibody bound in human, mouse, and rat models, with identical sequences at the binding site for human and rat. The mouse epitope differed by a single amino acid that did not affect binding. A difference of four amino acids within a 12 amino acid epitope yielded no species cross-reactivity for the human TNFα antibody.6 Cross-reactivity occurs with both antibody and aptamer affinity reagents.
Because aptamer selection is a controlled, in vitro process, selection can be designed to amplify aptamer sequences that bind to a target of interest (positive target) while discarding aptamer sequences that bind to related molecules (negative targets), yielding highly selective aptamers.
One drawback to the use of antibodies in therapeutics is immunogenicity – generation of an immune response to the antibody drug and production of anti-drug antibodies. Anti-drug antibodies (ADA) can induce unwanted reactions, alter drug pharmacokinetics, or reduce treatment efficacy by neutralizing drug activity. While the shift to humanized or fully human antibodies has reduced immunogenicity of antibody drugs, it has not been eliminated. Several factors can affect immunogenicity including drug impurities, excipients, dosing regimen, disease type and stage, target cell type, and combinational therapies. In a recent study released by Pfizer, high incidence of ADAs was seen in 27% of patients for human/humanized antibody drugs targeting T cells, compared to 4% for drugs targeting B cells.3
Small DNA and RNA aptamers are inherently non-immunogenic, as demonstrated in several recent in vivo studies involving aptamers.12,19 They are suitable for in vivo use without extensive modification.
Heterophilic Antibodies, Anti-Animal Antibodies, and Rheumatoid Factor
Antibody interference is a common problem in antibody-based detection assays. There are several classes of endogenous antibodies that can cause interference. Heterophilic antibodies are thought to be naturally-occurring antibodies with weak binding to multiple targets, generating a minor degree of assay interference. Anti-animal antibodies, such as HAMA (human anti-mouse antibodies), are produced following acute or extended exposure to animal antibodies through therapeutics or physical contact with animals themselves. Rheumatoid factor refers to antibodies that bind to the Fc region of IgG antibodies. They are common in patients with rheumatoid arthritis and other autoimmune disorders. Interference occurs when these endogenous antibodies interact with the capture and/or detecting antibody in an assay. Interference from these types of endogenous antibodies can either lead to a false negative result (binding of target is blocked by binding of interfering antibody) or a false positive result (interfering antibody forms a bridge between capture and detecting antibody without binding of target), depending upon the interaction.4,15.
The use of DNA or RNA aptamers eliminates potential interference from endogenous antibodies.
Antibody / Aptamer Production
Production of monoclonal antibodies is a complex, time-consuming biological process. Fed-batch and perfusion culture processes have been developed to maximize yields and accelerate the bioproduction process14, but culture contamination remains a concern, as an entire batch of material can be compromised. Purification to isolate the antibody of interest and remove residual host cell impurities, DNA, viruses, and other contaminants is another critical step.17 Batch-to-batch consistency in antibody expression and purification is an ongoing challenge in diagnostic and therapeutic applications.
Because aptamers are chemically synthesized, there is no risk of biological contamination and greater batch-to-batch consistency.
Long-Term Access to Antibody / Aptamer Reagents
Antibody hybridomas are typically cryo-preserved in liquid nitrogen. Hybridomas can be maintained over a period of years, but some are genetically unstable. Because non-producing clones tend to overtake producing ones, hybridoma cells can stop producing antibody over time.11 Failure of a liquid nitrogen tank can mean the loss of a precious hybridoma, so off-site and redundant storage of hybridomas is recommended.
Aptamers are chemically synthesized, so only information regarding the aptamer sequence and any modifications is required to maintain long-term access to the aptamer that was originally produced and validated.
Aptamers vs. Antibodies Table
|Monoclonal Antibody||Aptamer||Aptamer Advantage|
|Development Time* (Delivery of 1st production batch)||~4 – 6 months||~1 – 3 months||Faster development time means faster time to market or publication|
|Development Process||Initial antibody generation requires an immune response in an animal model||Enrichment of an oligonucleotide library through SELEX process||Aptamers can be selected against toxic compounds and non-immunogenic compounds (22)|
|Size||~150 – 170 kDa (IgG)||~12 – 30 kDa (~30-80 nucleotides)||Due to their small size, aptamers can infiltrate tissues and sometimes cells. They also have a higher tendency to be non-toxic and non-immunogenic (20,21,22)|
|Optimal Working Concentration||Varies widely by application||~ 5 to 10 times lower than optimal antibody concentration for some applications||Due to lower molecular weight, there are typically five to ten times more aptamers than antibodies in a solution of the same concentration|
|Minimum Target Size||≥ 600 Daltons||≥ 60 Daltons||Target small molecules|
|Manufacturing Process||In vivo production; Cell culture||Chemical synthesis||Improved batch-to-batch consistency, animal/cell-free process, simple scale-up and purification; Aptamers can be selected under non-physiological conditions; Less costly to manufacture GMP-grade aptamers in large quantities|
|Modification||Antibodies are typically conjugated with one type of signaling or binding molecule||Aptamers can be modified at both the 5’ and 3’ end||Aptamers can be easily modified for attachment and signaling, often during aptamer synthesis|
|Stability||Antibodies are susceptible to high temperatures and pH changes; Denatured antibodies cannot be repaired||Aptamers are fairly stable at ambient temperature and are easily refolded if denatured||Aptamers have a longer shelf life and can be transported at ambient temperature (23)|
|Long-term Availability||Frozen cell stocks must be maintained for monoclonal antibody production||Specific nucleotide sequence (data) is stored for aptamer production||Aptamer sequences are easy to store and transfer to production sites. Sequences are not lost if a freezer goes down.|
*Actual time will vary from one project to another
- Ansar, W., et al. Monoclonal antibodies: a tool in clinical research. Indian Journal of Clinical Medicine. 2013. 4:9-21.
- Braden, M., et al. Examination of Thermal Unfolding and Aggregation Profiles of a Series of Developable Therapeutic Monoclonal Antibodies. Molecular Pharmaceutics. 12(4):1005-1017.
- Davda, J., et al. Immunogenicity of immunomodulatory, antibody-based, oncology therapeutics. Journal for ImmunoTherapy of Cancer. 2019. 7:105.
- Emerson, J.F. et al. Endogenous antibody interferences in immunoassays. Lab Medicine. 2013. 44(1):69-73.
- Gold, L., et al. Aptamers and the RNA World, Past and Present. Cold Spring Harbor Perspectives in Biology. 2012. 4(3) a003582.
- Irani, Y., et al. Species cross-reactivity of antibodies used to treat ophthalmic conditions. 2016. 57(2):586-591.
- Kohler, Georges J.F. and Cesar Milstein. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975. 256(5517):495-7.
- Leslie, Mitch (2018, May 10), “Mini-antibodies discovered in sharks and camels could lead to drugs for cancer and other diseases,” Science, Accessed May 28, 2019. https://www.sciencemag.org/news/2018/05/mini-antibodies-discovered- sharks-and-camels-could-lead-drugs-cancer-and-other-diseases
- Mandal, Ananya. What is an antibody? Medical Life Sciences News. Accessed June 3, 2019. <https://www.news-medical. net/health/What-is-an-Antibody.aspx>
- Monaco, I., et al. Aptamer functionalization of nanosystems for glioblastoma targeting through the blood-brain barrier. Journal of Medicinal Chemistry. 60:4510-4516. 2017.
- Pasqualini, Renata and Wadih Arap. Hybridoma-free generation of monoclonal antibodies. PNAS. 2004. 101(1):257-259.
- Pusuluri, A., et al. Treating tumors at low drug doses using an aptamer-peptide synergistic drug conjugate . Angewandte Chemie International Edition. 2019. 58(5):1437-1441.
- Ryman, Josiah T. and Bernd Meibohm. Pharmacokinetics of monoclonal antibodies. CPT: Pharmacometrics & Systems Pharmacology. 2017. 6:576-588.
- Sargent, Brandy. (2013, June 12), “Perfusion bioreactors -with so much to offer they deserve a closer look,” Cell Culture Dish, accessed May 28, 2019. <https://cellculturedish.com/perfusion-bioreactors-with-so-much-to-offer-they-deserve-a- closer-look/>
- Schwickart, M., et al. Interference in immunoassays to support therapeutic antibody development in preclinical and clinical studies. Bioanalysis. 2014. 6(14):1939-1951. J
- Sheng, Morgan (2015, Dec. 1), “How to get into the brain,” Genentech, Accessed May 23, 2019. https://www.gene.com/ stories/how-to-get-into-the-brain
- Shukla, A.A., Evolving trends in mAb production processes. Bioengineering and Translational Medicine. 2017. 2:58-69.
- Zhang, Y. et al. Aptamer-targeted magnetic resonance imaging contrast agents and their applications. Journal of Neuroscience and Nanotechnology. 2018. 18:3759-3774.
- Zhu, H., et al. Aptamer-PEG-modified Fe3O4@Mn as a novel T1- and T2- dual-model MRI contrast agent targeting hypoxia- induced cancer stem cells. Nature: Scientific Reports. 2016. 6:39245.
- Cao Z, Tong R, Mishra A, Xu W, Wong GCL, Cheng J, Lu Y: Reversible Cell-Specific Drug Delivery with Aptamer-Functionalized Liposomes. Angew. Chem. Int. Ed. 2009, 48:6494-6498.
- Ferreira CSM, Cheung MC, Missailidis S, Bisland S, Gariepy J: Phototoxic aptamers selectively enter and kill epithelial cancer cells. Nucl. Acids Res. 2009, 37:866-876.
- Jayasena, S. D. Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clinical Chemistry. 1999. 45(9):1628-50.
- Sun, H. et al. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy. Molecular Therapy – Nucleic Acids. 2014. 3, e182. Doi: 10.1038/mtna.2014.32.