NEW YORK – While diagnostic test makers have come to rely on antibodies for test development – a whole class of tests, immunodiagnostics, are based on antibodies – issues around them have been known for decades with industry and researchers working to solve them.
Against that backdrop, one firm, Avacta, has developed an alternative to antibodies with a technology called Affimers, and according to company officials, a growing number of diagnostic companies are exploring the use of the reagents.
The Wetherby, UK-based company said it has begun booking revenue from engagements with diagnostic companies, and in its last fiscal year ended July 31, 2019, it reported £1.2 million ($1.5 million) in combined revenue and orders for its Affimer research and diagnostics reagents business, a 130 percent increase over the previous year.
Avacta said it continues to receive orders from global commercial partners for paid technology evaluations and custom projects. It engaged with seven undisclosed diagnostic companies in 2019; four were among the top 10 in vitro diagnostic companies globally. Further, the company's Affimer research and diagnostics reagents group participated in 14 projects with pharma and biotech companies, including with four out of the top 10 largest pharma companies. As part of a deal struck with New England Biolabs, for example, Avacta and NEB are looking to commercializing a product that uses Affimer technology in both life science and diagnostics assays.
Affimer molecules are small proteins that, similar to antibodies, bind to target molecules. The proteins, also called affinity reagents, are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications.
Avacta acquired its technology from Leeds University and UK-based Medical Research Council in 2013, and it provides a basis for two businesses. In addition to leveraging the technology for research and diagnostics, the firm is applying Affimer technology to the development of immuno-oncology therapies. The therapy projects are higher risk but also have the potential to provide higher rewards in the long run than the research and diagnostics projects over their lifetime, Avacta's CEO Alastair Smith said in an interview.
The company anticipates its research and diagnostics reagents business will be profitable in about three years, Smith said.
Affimer reagents are manufactured using phage display, a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them.
David Wilson, Avacta's commercial director of diagnostics, said that the firm's pipeline of Affimer diagnostic assay licensing deals has continued to grow in 2020, and the company is drafting plans to develop its own diagnostic tests using the technology.
Antibodies are a dominant technology throughout in vitro diagnostic testing, he said. They are well understood, and companies have invested in them over many years. "The adoption of an alternative technology is therefore only usually considered for applications where antibodies have limitations and where products such as Affimer reagents can offer technically viable solutions," Wilson said.
Avacta has developed its reagents for many of these applications and they "represent significant diagnostics markets," he said.
The technology eliminates drawbacks such as reagent batch-to-batch variability that stems from the reliance on the response of animals' immune systems in the manufacture of antibodies, according to Avacta. Such batch-to-batch variability in antibody reagents can hamper test development and production, and when that happens, "tests have to be revalidated, which can be expensive," Wilson said.
While pursuing greater adoption of its technology in research and diagnostics, the firm has noticed that some applications are better suited than others for use of Affimers.
"We've learned that Affimers differentiate themselves most strongly in two areas," Wilson said. "The first is the development of anti-idiotypic binders for assays used in drug development and monitoring, and the second is the conversion of competitive assays to more sensitive and more specific sandwich assays."
Sandwich assays are a workhorse of the diagnostics industry with many potential applications, but Avacta sees the most near-term opportunities in their development and use for small-molecule drug development and monitoring.
The approach to developing Affimer reagents — involving use of a high-throughput selection technique that finds reagents that bind to targets of interest from within an extensive library — is the basis of many of the products' benefits. One of the main ones is that "with the selected Affimer in hand, you have its DNA sequence and you can reproduce that with high consistency," according to Smith.
The manufacturing process is also less expensive than that used in traditional antibody manufacturing, he said. Because they are reproducible and consistent from lot to lot, Affimer reagents bring certainty and security to the availability of materials that are so critical for diagnostic tests, and they can be developed more rapidly than antibody reagents, according to Avacta.
The reagents can also be designed to have long-term stability in high-temperature settings, an important requirement of some point-of-care tests.
David Fraser, group product manager at immunoassay development and manufacturing firm BBI Solutions, said in an interview that "over time we will see more of a transition in diagnostics to alternatives to antibodies, especially in the generation of reagents for certain targets that are quite difficult to generate by traditional processes. Affinity reagents can be particularly useful in identifying small molecule drug targets, and there is a need for alternatives to antibodies in that specific area."
Fraser has used Affimers as well as aptamers, another type of affinity reagent, in the development of proof-of-concept tests for undisclosed customers, but he is not affiliated with Avacta.
Because the technology can mitigate some of the drawbacks associated with traditional antibodies, its potential applications are not confined to small-molecule detection, Fraser said.
Affimers open up avenues for designing new types of assays with new targets. "They offer more flexibility than traditional antibodies because you are designing them from scratch," he said. "You can get more control over a test's specificity and sensitivity, and they can provide greater consistency."
The trouble with antibodies
Problems with antibodies are well known. They can be expensive and time consuming to make. They don't exist for all the targets researchers would like to measure, and they offer limited multiplexing capabilities. Some suffer from poor specificity and high background.
However, diagnostic companies that have traditionally used antibodies are in no rush to adopt a new type of reagent, and despite their potential advantages, alternatives to antibody reagents are in the early innings of their adoption.
One of the barriers to their adoption is that diagnostic test developers "have been wedded to antibodies, know the challenges associated with them, and have systems in place to manage those challenges," Fraser said.
Nonetheless, he noted, there are ways to boost the adoption of affinity reagents. Diagnostic test developers are generally aware of alternatives to antibodies but need to be convinced of their clinical utility and better understand what the technology can do for their tests, he said.
Makers of affinity reagents need to show evidence of improved performance and educate potential adopters in the IVD market about alternate options, he said.
Avacta has been gathering such evidence, and its technology has been featured in published research studies.
As part of a study published in eLife and led by Leeds University, for example, investigators described Affimer selection as being faster than antibody production involving animal inoculation. The investigators said that during the study, each selected Affimer coding region was sub-cloned into an E. coli expression vector, and recombinant protein was purified over a further seven days. Without automation, the phage display platform allowed simultaneous screening of up to 24 targets.
Rainer Bischoff, a professor in the department of analytical biochemistry at Netherlands-based University of Groningen, said he and his colleagues have used an Affimer-based liquid chromatography-mass spectrometry (LC-MS) method for quantification of sRAGE (soluble Receptor of Advanced Glycation End-products), a promising biomarker for the detection of chronic obstructive pulmonary disease. They have published their development work in the Journal of Proteome Research.
Bischoff said he believes the study may be the first to demonstrate the application of Affimers in a quantitative LC-MS method, and that it is evidence of the reagents' potential as an alternative to antibodies.
Having control over the reagent was the main driver of his lab's decision to work with Affimer technology, as well as knowing the sequences and binding sites of the reagents, Bischoff said.
Antibody suppliers rarely disclose the DNA sequences and binding sites of their products, which is "a disadvantage because you are working somewhat in the dark and at the mercy of the supplier," he said. Further, using traditional antibody reagents, a test developer or manufacturer may have to do additional work because reference values are altered when the quality of the antibody changes over time, Bischoff added.
Other diagnostic test researchers have described the use of Avacta's Affimers in the development of their assays. For example, in 2018, researchers at the University of Bath and the University of Leeds published a study in Biosensors and Bioelectronics that described an Affimer-functionalized interdigitated electrode-based capacitive biosensor platform for detection and estimation of Her4, a protein tumor biomarker, in undiluted serum.
Separately, an international research group reported in Scientific Reports that it had isolated Affimer non-antibody binding proteins against GPC3, a promising marker for the detection of hepatocellular carcinoma. They used phage display to isolate the Affimer non-antibody binding proteins and developed a sandwich chemiluminescence immunoassay by combining an Affimer with a monoclonal antibody.
With reference to development of its own tests, Avacta "is at the final stages of development and analytical validation of D-dimer and estradiol assays," Wilson said.
The firm anticipates using the estradiol assay to demonstrate the capabilities of Affimer reagents to convert negative-read competition assays for small-molecule targets into positive-read sandwich assays, "improving analytical performance and readability for point-of-care applications," Wilson said. "The D-Dimer assay will demonstrate the high specificity of Affimer reagents and will be available for licensing."