Synthesising new solutions for small molecule antibodies

Toni Rogers and David Bunka, Aptamer Group discuss how newer antibody alternative technologies are demonstrating significant developments, offering novel solutions to improve success, decrease project timelines and produce innovative low-cost assays to small molecule targets across many sectors.

Small molecules are ubiquitous in our environment, from toxins and heavy metals to vitamins, hormones and drugs of abuse. The ability to detect and quantify these chemicals is crucial to drug discovery and development and is being pursued for therapeutic drug monitoring applications to personalise the drug dosage to the patient. Yet the development of antibodies that can specifically bind small molecule targets to enable these diagnostics remains challenging.

The pharmaceutical industry, in particular, has traditionally been dominated by small molecules. Used to treat a variety of diseases and conditions, the small size and physicochemical profiles of these drugs offers diversity in their mechanism of action. They have been used as enzyme inhibitors, allosteric modifiers, and to effectively target extracellular receptors, cytosolic and nuclear proteins and biomarkers of the central nervous system. Despite their perceived limitations and the rise of large molecule biologics, such as antibody therapeutics, small molecule drugs remain the major component of an ever-expanding therapeutic toolbox. Over the period from 2010-2017 the US FDA approved a total of 262 new molecular entities, with 76% being small molecules and only a quarter being biologics.¹ However, as biologics are costly to manufacture and standardise, the revenue generated from these therapeutic modalities appears significantly greater, with a rise of 70% in sales revenue from biologics between 2011 and 2017.²

Currently the main method for analysing and monitoring small molecule drugs and their metabolites is mass spectrometry (MS). This is a costly and labour-intensive solution. Analysis of small molecules with MALDI-TOF MS remains challenging but can be readily detected with electrospray ionisation (ESI) mass spectrometry.³ However, as ESI MS is less tolerant to the presence of contaminants within samples, it often requires the coupling of liquid chromatography separation before MS. Advances in these systems to increase throughput have made use of solid-phase extraction for desalting rather than liquid chromatography. The Agilent RapidFire system further automates sample aspiration, solid-phase extraction desalting and injection steps to deliver cycling times in the region of 10 seconds.^{4, 5} Drawbacks of this approach include the common requirement for time-consuming and potentially complex sample pre-treatment procedures, and the high-cost and large footprint of equipment with skilled staff to carry out this analysis, which often requires centralised facilities unavailable to smaller laboratories or clinics.^{6, 7}

The ability to use antibody-based assays, such as ELISA and biosensors, in place of MS analysis could reduce the cost, increase the speed, and allow these tests to be performed at the point-of-need/point-of-care for more rapid drug development processes and improved patient outcomes.^{7, 8} As small molecules are limited in the number of binding epitopes available for antibody pair binding, the majority of small molecule detection is performed with competitive assays. However, these rely on a negative readout and often suffer sensitivity issues. Generating high-affinity molecules such as antibodies to these small molecule targets is essential to deliver quick and sensitive detection for targets, such as drugs and their metabolites, markers of disease or metal ions, to monitoring human health.⁹

From rodent to recombinant

To generate antibodies via traditional methods, the antigen is injected into an animal and the resulting antibodies, generated as part of the immune response, are extracted. Making antibodies to small molecules is not so simple. Due to their size, small molecules are non-immunogenic, meaning no immune response is raised and so no antibodies generated (Fig.1). Antibody developers circumnavigate this problem, by conjugating the small molecule to a carrier protein, in which case it is termed a hapten (half antigen). Carrier proteins, such as keyhole limpet hemocyanin and bovine serum albumin, are conjugated to multiple copies of the small molecule. The presence of the larger protein molecule raises an immune response in the injected animal, with a portion of the generated antibodies specific to the hapten.¹⁰

This approach has yielded some successes: anti-digitoxigenin, anti-nitrophenol, anti-biotin and anti-fluorescein are workhorse antibodies in biochemistry, as specific binders for the detection of covalently modified proteins.¹¹Some of these antibodies, such as anti-fluorescein, are able to bind both the protein-conjugated and unconjugated small molecule,¹²though the majority only bind to the conjugated form. Many of these antibodies now exhibit good affinity for their small molecule target, with reported K_D values in the nanomolar range.¹³ Though this is often only achievable following extensive, stringent in vitro selection from immune libraries and affinity maturation.^{11, 13-15}

Despite these successes, the failure rate for anti-hapten antibodies in discovery remains high. It is estimated that 50-75% of all anti-hapten antibody development programmes fail to deliver reagents with sufficient affinity or specificity. Unfortunately, the use of carrier molecules often leads to the developed antibodies being specific to the hapten-carrier molecule linker region rather than to the desired hapten. Conjugation of the small molecule to a carrier protein also limits the available epitopes for antibody binding, which result in variation in the performance of the antibody in binding the conjugated target compared to when free in solution, with much lower affinity for the free molecule in solution.¹⁶

Over the past two decades, the method of choice for antibody generation for commercial applications has changed from relying heavily on animal-derived hybridomas to selection from recombinant antibody libraries.¹⁷ Increasingly, there is an awareness of the improved product consistency, the ability to add stringent selection conditions to modulate binding kinetics, the freedom to target a more diverse range of molecules and the reduction of animal usage by employing in vitro libraries.^18-20

Antibody alternatives: the next big thing for small molecules

ScFv and Fab fragments

Antibody fragments, such as scFv and Fab fragments, represent for many a proven antibody alternative as they are derived from the traditional antibody scaffold. They can easily be made into recombinant libraries, overcoming the problems of relying on an animal hosts, and where the Fc domain is not required for effector functions, such as in diagnostic assays, manufacture is simpler and cheaper.¹⁹

The development of sensitive affinity reagents to estradiol-17β has been a goal within the diagnostics industry, as the currently available assays do not offer sufficient sensitivity for accurate detection of low levels of this hormone. This small molecule target is still being pursued with a range of antibody alternatives, with recent data suggesting through extensive affinity maturation of scFv’s, these reagents may be approaching the required sensitivity for diagnostic tests.²¹ Further scFV to small molecules that have been developed via this strategy include cotinine²², cortisol²³, and Δ⁹-tetrahydrocannabinol (THC)²⁴, all of which are diagnostically relevant. However, the process of affinity maturation takes months of expensive development work, which has been a problem in the development of traditional antibodies to targets. Additionally, as many of these antibody fragments still rely on intra-domain disulphide bonds for stability, they need expensive mammalian cells for their production.²⁵

An alternative range of affinity reagents engineered to overcome the antibody-associated problems of cost, long developmental lead times, complexity in scale-up and design, and limited targeting through reliance upon the immune system, include protein scaffolds and aptamers. While many of these protein scaffolds have focused on human protein targets to support therapeutic development, a limited number have shown potential for binding to small molecules.^{11, 26}

Anticalins

Anticalins are an antibody alterative platform commercialised by Pieris Pharmaceuticals.²⁷ Based upon the lipocalin proteins, this scaffold comprises a beta-barrel structure that is capable both of binding protein molecules with the variable end loop regions and encapsulating small molecules within the barrel structure (Fig. 2).¹¹ Despite the development of high affinity anticalins to proof-of-concept small molecule targets, such as fluorescein and digitoxigenin,¹¹Pieris have yet to bring a product to market using their small molecule Anticalin binders.

Affimers

Similar to Anticalins, Affimers are a protein scaffold affinity reagent developed by Avacta Life Science, based upon the Cystatin protein family of cysteine protease inhibitors (Fig.2).^{28, 29}  Early work showed the development of an Affimer to the anti-fungal agent posaconazole,³⁰ though no specific affinities were reported and no further progress with this binder appears to have been made. Recently Avacta adopted an alternative strategy to small molecule detection, with the development of Affimers targeting a small molecule-antibody complex. This has been exemplified with the hormone estradiol, where current competitive assays yielding a negative readout could be converted to a gain-of-signal for simple readout and incorporation onto point-of-care diagnostics, such as lateral flow devices.²⁶

Aptamers

Aptamers are short, single-stranded synthetic RNA and DNA molecules selected from libraries. They form specific 3D structures that allow them to recognise and bind relevant molecules.⁹As they are manufactured via solid-phase synthesis, there is no requirement for cellular systems for their production, offering simpler scale-up and more cost-effective production processes.^{31, 32} Many companies are actively pursuing aptamer development to small molecule targets, including amino acids,³³ bacterial quorum sensing molecules,³⁴ antibiotics,^{35 b}and chemotherapeutics,^{7, 8, 35}  to develop diagnostic and bioanalytical assays.

An emerging development in this field is the focus on transitioning biosensor platforms to point-of-care offerings. Studies showing sensitivities within the clinical concentration range for antibiotics and chemotherapeutics have been demonstrated on these platforms. A further benefit of this strategy is the need for only one affinity reagent, removing the arduous process of detecting small molecules via affinity reagent pairs, such as in sandwich ELISA. This could rapidly speed assay development and revolutionise the detection and quantification of small molecules.

Nonetheless, a recent survey found that only 25% of all aptamers have been developed to small molecule targets.³⁶  This low success rate has been attributed in large part to technical challenges that affect selection and characterisation.⁹

Moving to solution/Cutting the links on immobilised targets

While these advances in antibody alternatives have reduced costs and increased the speed of development, issues remain in targeting small molecules. In all the processes employed by the various affinity technologies, there is a requirement to immobilise the small molecule during selection or bind the small molecule to another affinity reagent for immobilisation. Both of these strategies reduce the number of epitopes available for binding on the small molecule target by any new affinity reagent and increase the potential of targeting other moieties, such as the linker region for immobilisation, rather than the small molecule itself.^{36, 37} Target immobilisation during selection is known to reduce the affinity of the binders to the small molecule in solution,^{36, 37}and in most cases it is the unbound form of the small molecule drug that needs to be quantified in patient samples during drug development and for therapeutic drug monitoring purposes.

Aptamer Group has recently developed a novel high-throughput selection method for its small molecule Optimers. Optimers are next-generation aptamers, optimised for increased affinity, assay conditions and improved manufacturability profiles. The new selection method for small molecule binders is based upon immobilisation of the aptamer rather than the small molecule target (Fig.3). In this method, the aptamer is bound to a compatible linker region on a bead, with the small molecule present in solution. High affinity binding of the aptamer to the small molecule is required to overcome the interaction with the linker region, ensuring the selection of binders to small molecule targets with the required high affinity for the target in solutions, absent from many of the available alternative selection strategies. The current success rate for this selection method to small molecules is in excess of 80%, and the reagents generated via this platform are showing excellent performance. Binders to the chemotherapeutics, imatinib and irinotecan, were recently validated to FDA standards for critical reagents in bioanalytical assays.^7,8 There is potential to develop these platforms into rapid point-of-care diagnostics to personalise drug doses for improved patient outcomes and reduced healthcare costs.

This displacement method is also being applied to generate single-reagent ELISA and LFD assays with positive readouts to simplify and increase the speed of diagnostic assay development.

Developing anti-hapten antibodies has long been a challenging issue with high failure rates ultimately hindering the development and monitoring of small molecule drugs. The turn towards alternative affinity technologies and the use of novel selection methods will drive the acceleration of discovery and development across the life science industry, with the use of high-affinity single-reagent assays leading to a step-change in the efficiency of small molecule detection and monitoring.

Volume 22, Issue 2 – Spring 2021

About the Authors

Toni Rogers graduated from the University of York with an MChem in Chemistry. After joining Aptamer Group as a Selection Scientist, she has progressed to Principal Scientist within the selection team for small molecule aptamers and works to refine the selection system for small molecule targets.

David Bunka is the Chief Technical Officer for Aptamer Group with over 20 years of experience developing nucleic acid aptamers. He leads the development and production teams at Aptamer Group using high-throughput automated selection methods for the development of Optimer reagents for use in research, diagnostics and as therapeutics.

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