Monoclonal antibodies offer promise in the fight against a wide range of diseases, including COVID-19. The length of the discovery process is a barrier to emergency use, however, and poses a commercial risk for pharmaceutical companies. Multidisciplinary innovation could step up the pace, says Verity Jackson.

The current coronavirus pandemic provides an urgent example of the need for faster development of active pharmaceutical products. Monoclonal antibodies (mAbs) in particular have demonstrated considerable value in the last decade: currently six out of the ten best-selling drugs in the world are mAbs – with a combined revenue of over $50 billion p.a. This means that the discovery of high-quality mAbs for therapeutic and diagnostic use is more important than ever, and time to market is crucial for both therapeutic impact and economic success.

This blog covers

– The promise of monoclonal antibodies
– The evolution of antibody screening workflows
– Droplet technology for single-cell encapsulation
– Improving prediction of therapeutic potential
– Future antibody screening technologies 

The promise of monoclonal antibodies

Therapeutic mAbs are one of the fastest growing classes of drugs due to their promise for better specificity and safety. This has led to the approval of over 100 mAbs for therapeutic use since the mid-1980s, primarily for cancers and immunological diseases. 

The average therapeutic antibody discovery research project takes around 4.5 years, followed by another year of preclinical testing before a lead mAb candidate will enter Phase 1A clinical trials [1], meaning substantial acceleration of the process would be needed to meet the emergency needs of a pandemic. Indeed, the first wave of the Ebola epidemic in 2014 saw a similar flurry of therapeutic mAb discovery efforts as we are now seeing for COVID-19, but by the time the drugs were ready to use, the outbreak had begun to wane [2].  

Additionally, from a commercial perspective, the speed of drugs to the market is crucial for pharmaceutical companies. With the best-selling mAb drug, Humira, generating around $20 billion in revenue per year [3], the economic rewards of being even one month earlier to market can be large, providing additional motivation for pharma companies to accelerate these workflows.  

The evolution of antibody screening workflows

At the same time as accelerating mAb discovery processes, pharma and biotech companies are beginning to address more challenging antigens, such as G-protein-coupled receptors (GPCRs) and ion channels, which reside in cell membranes.  

These classes of protein have traditionally been highly druggable, representing the targets of ~40% of small molecule drugs, but have proven recalcitrant to the development of effective mAb therapeutics. The hunt for these rare antibodies can involve screening over 100 million cells, each expressing a single type of unique antibody, which must be quickly and accurately reduced to a subset of cells secreting antibodies with desired properties for more detailed downstream analysis. This process is known as primary screening. 

Antibody screening technologies are continuously evolving to balance the competing needs of increased screening scale and depth, whilst reducing costs, timelines and the risk of later-stage failures in the antibody discovery workflow. The ongoing challenge that is driving change in antibody screening workflows is the ability to collect as much predictive data about an antibody’s therapeutic potential as early as possible in the screening campaign. This is requiring multidisciplinary innovation in hardware, cell systems, reagents and assay formats. 

Microfluidic encapsulation of cells for screening workflows using droplets in oil.

From hybridoma to B-cell screening

One of the biggest changes that has reduced timescales associated with antibody screening is the introduction of primary B-cell screening as an addition to conventional hybridoma technology.  

Hybridomas are produced by fusing primary B-cells with immortal cancer cell lines and are notoriously slow to generate, taking weeks or months to enter primary screening before any information on the antibodies they produce is obtained.  

In contrast, primary B-cells can be rapidly isolated from immunised animals or sera from convalescent patients. The limited lifespan of these cells has placed increased pressure on the speed of workflows to ensure all necessary data about the antibodies is extracted before the B-cell dies.  

Single-cell droplet technology enables ultra-high throughput screening

The gradual switch from hybridoma technology to screening individual primary B-cells has also paved the way to substantial changes in antibody screening workflow formats, which have traditionally been carried out in microwell plates or, more recently, using flow cytometry.  

Microfluidic droplet biology technologies offer opportunities to achieve ultra-high throughputs with greatly reduced footprints and plasticware consumption. These technologies have enabled a step change in throughput of single cell analytics, and antibody screening is no exception with companies such as 10X Genomics and Sphere Fluidics offering proprietary reagents, hardware and software in this space.  

In addition, therapeutic companies such as HiFiBio are both developing and leveraging these technologies to mine deep into the immune repertoire in search of drug candidates [4]. Here individual B-cells, along with assay reagents for antibody detection, are enclosed in tiny aqueous droplets of approximately 100 picolitre (that is, 1/10,000,000th of a millilitre).  

This substantially reduced assay volume allows detectable levels of antibody to accumulate in a short timeframe, maintaining viability of the enclosed B-cell. Single cell workflows have also enabled antibody screening from rare cell types, such as B-memory cells, which naturally produce antibodies with higher affinity and specificity.  

Functional Assays to Improve Prediction of Therapeutic Potential

Obtaining numerous pieces of high-quality data about each antibody early in the screening pipeline is crucial for scientists to make an informed decision about which antibody candidates to take forward.  

Historically, primary screening of antibodies has relied on antigen-binding data to determine whether an antibody candidate should be proceeded. The past few years have seen these studies migrate from detecting antibody binding to an immobilised fragment of target antigen on a surface (microplate or bead) to detecting binding of candidate antibodies to genetically modified “reporter” cell lines expressing the antigen of interest [5]. This approach allows antibody binding to be screened in a more biologically relevant environment – with intact surface receptors embedded in a lipid bilayer.  

This move has been enabled by the development of no-wash cell-binding immunoassays and related high-performance cytometry instrumentation for screening antibody binding to cells. Early proof-of-principle demonstrations have shown promise for similar set-ups in microfluidic droplets, but challenges remain both in terms of the hardware, assay chemistries and readouts [6]. 

However, the ability of an antibody to function therapeutically relies on a multitude of factors and cannot be predicted based on simple antigen-binding data. For example, an antibody may have to activate or inhibit an intracellular signalling pathway or open or close an ion channel to exert its therapeutic effect. For this reason, primary screening increasingly relies on complex multiplexed fluorescent readouts – for example, simultaneously reporting on cell viability, antigen-binding specificity and activation of a specific signalling pathway (phenotypic readout), an approach being pioneered commercially by Swiss Federal Institute of Technology spin-out, VELabs. By conducting these types of assay during primary screens, antibodies that bind their target antigens without exerting a functional effect can be eliminated early, allowing more detailed secondary screens and functional testing to focus on a smaller pool of candidates. 

What does this mean for future antibody screening technologies?

The requirements for antibody screening workflows are pushing the boundaries of current technologies in terms of throughput, assay readout and plex. Single cell technologies have already proven hugely enabling in both antibody screening and the cell therapy space and innovations there could migrate across industry. One such example could be ultra-fast cell sorting for screening of display libraries [7] or isolation of rare antibody producing cells, such as memory B-cells. The robust and flexible implementation of functional assays in ultra-high throughput formats, such as droplets, is certainly a challenge that will be resolved only by multidisciplinary effort, including innovations in reagents, assay design and cellular systems working in synergy with bespoke hardware for fluid and cell handling and detection.

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Verity Jackson