Eliminating Bottlenecks in mAb Drug Discovery

Twist Biopharma’s approach to antibody discovery incorporates phage display to generate massive antibody libraries, and high-throughput surface plasmon resonance technology to accelerate screening

0
Twist Biopharma employees
Twist Biopharma employs synthetic DNA libraries against almost any antigen. The company creates large antibody libraries, which are then screened with high-throughput SPR technology from Carterra to find candidate antibodies within weeks.

All monoclonal antibody (mAb) development projects begin with a target—a receptor, protein, or gene that participates in a biological pathway of interest. After sequencing, cloning, and producing the target antigen, investigators raise a mAb library against it using hybridoma methods, B-cell cloning, or our preferred method, phage display.

Twist Biopharma, a division of Twist Bioscience, chose phage display because it allows the construction of libraries against almost any antigen, eliminates the need for immunization and hybridoma protocols, and can be made to express human or human-like antibodies. The company’s lab in South San Francisco has created several libraries against G-protein-coupled receptors, protein targets that are intimately associated with cell membranes and therefore notoriously difficult to drug.

Next comes screening, where hundreds to thousands of antibodies that bind to the target are identified. Those hits undergo various characterization steps where the team assesses expressibility (the ease with which the antibodies can be produced in suitable cell lines) and developability or druggability (the degree to which the antibodies can satisfy toxicology, formulation, off-target activity, manufacturing, and other requirements).

Hits are ranked according to binding strength, but whenever possible, the company tries to learn more about the nature of the mAb–target interaction. Advances in label-free workflows, such as Carterra’s high-throughput surface plasmon resonance (HT-SPR™) technology, enable large-scale antibody screening and greater understanding of the antibody’s mechanism of action.

Traditional methods

Endpoint ELISA assays followed by an orthogonal biophysical method like SPR is a suitable first-pass mAb characterization workflow. But even automated ELISA assays are laborious and suffer from false positives and insufficient sensitivity to detect potent, low-abundance hits.

Flow cytometry is sometimes used for screening intact cells, but flow methods work best for detecting discrete cellular events (for example, the presence of a surface marker) and less well for affinity and kinetics. Also, flow methods are expensive and pose yield and purity problems in the processing of rare cells.

SPR, a label-free optical phenomenon that measures biomolecular interactions in real time, provides accurate measures of concentration, kinetics, and affinity for a wide range of proteins and small molecules in almost any combination. SPR uses refractive index changes to quantify interactions between ligands and antibodies immobilized on a gold surface.

SPR is currently the gold standard for characterizing antibody–antigen interactions. Commercial SPR systems are capable of full antibody and antibody fragment characterization, kinetics and affinity determinations, binding site mapping, specificity determinations, and other studies.

Epitope binning

Reducing two analytical steps to one is always desirable, particularly when the single method might provide more information than the two combined methods it replaces. Epitope binning by SPR, which screens and characterizes the epitope landscape of a panel of antibodies in a single step, would be an ideal replacement for conventional ELISA-based binning except for standard SPR’s inherent lack of parallelism and multiplexity.

Carterra’s Epitope
Carterra’s Epitope™ software provides an intuitive way of navigating epitope binning results for tens of thousands of protein–protein interactions. In a binning network plot, the nodes represent the antibodies (identified by number), the chords indicate the pairwise blocking relationships, and the envelopes (or bins) group antibodies with the same competition profile.

In a typical antibody binning study, hundreds of mAbs that bind to a target are tested in a pairwise fashion for their ability to simultaneously bind the target, and the antibodies are then grouped by binding location. Conventional binding assays give an affinity readout but cannot distinguish among the antibodies or groups of antibodies that favor specific epitopes.

Epitope binning is useful on many levels. It can enable the pursuit of several candidate mAbs simultaneously or sequentially, thus expanding the intellectual property value of an antibody project. Binning also permits the exclusion of classes of mAbs that target less-desirable epitopes. Combining competitive binding information with kinetic data enables the ranking of individual entries within families according to their binding strength.

HT-SPR

Over the years, SPR has been refined for greater automation and better integration into mAb discovery and development workflows. A relatively recent development of massively parallel, high-throughput SPR (HT-SPR) enables the power of real-time label-free binding data to be paired with multiplexing capabilities sufficient for today’s screening projects.

By screening tens of thousands of interactions in a single experiment, HT-SPR eliminates the compromises inherent in older methods.

As the first HT-SPR protein analysis platform, Carterra’s automated LSA instrument fully integrates into mAb discovery workflows. The LSA combines microfluidics with HT-SPR detection to deliver up to a hundred times the data of conventional SPR systems in 10% of the time, a 1000-fold improvement in throughput. The system also requires just 1% of the sample input of traditional SPR-based methods, has low consumable costs, and eliminates ELISA’s requirements for multiple liquid- and plate-handling systems.

In contrast with standard SPR, a one-to-one analysis platform, HT-SPR enables one-to-many affinity interaction studies via a microarray format. It allows researchers to compare the binding of antibody library hits to homologous antigens from different species, which could take up to 10 times longer using conventional SPR analysis.

HT-SPR in action

At a recent symposium, scientists from Boehringer Ingelheim described experiments that simultaneously assessed epitope diversity and binding kinetics for a large antibody library. They demonstrated how the Carterra LSA platform enabled the fast and optimized selection of antibody panels for mechanism-of-action studies, while simultaneously providing a crucial evaluation of antibody-generation technologies.

In another presentation, a group from Bristol-Myers Squibb reported how capturing the full diversity of large antibody panels is critical to finding the optimal therapeutic candidate and using resources efficiently.

They concluded that HT-SPR exploits the full epitope diversity generated by different transgenic animals and different antibody-generation techniques while facilitating the rapid selection of antibody panels for mechanistic studies.

Conclusion

Phage display is a powerful method for generating massive mAb libraries, but having millions of test antibodies presents challenges as well as opportunities. Until recently, the analytical technology did not exist to take full advantage of phage display. Conventional SPR, currently the gold standard for kinetics, affinity, and specificity measurements, is a low-throughput method that fails to meet the demands of modern drug discovery.

In Twist Biopharma’s laboratories, the Carterra LSA platform has been critical to consolidating mAb workflows and enabling us to deliver quality results to our customers.

 

Aaron Sato, PhD, is CSO at Twist Biopharma, a division of Twist Bioscience, based in South San Francisco, CA. Twist Bioscience utilizes its silicon-based DNA synthesis platform to generate DNA and DNA-based products for the development of diagnostics, therapeutics, bio-based chemicals, food, DNA-based data storage, and leverages this platform through Twist Biopharma specifically for antibody drug discovery.

This site uses Akismet to reduce spam. Learn how your comment data is processed.