Transformative Antibody Technology Brings Speed and Flexibility to Bioanalytical Assay Development

Tech Snapshot® captures today’s cutting-edge tools and technologies that will help drive drug discovery tomorrow. This installment was written by Bio-Rad, a global leader in custom recombinant antibody generation.

Development of pharmacokinetic (PK) and anti-drug-antibody (ADA) ligand binding assays for large molecule biologics is complex and time consuming. Having confidence in the critical antibody reagents you use and trusting your results are vitally important. In this article, we introduce how antibody phage display and SpyTag technologies have been combined to make TrailBlazer Antibodies, which are transforming the design and optimization of bioanalytical assays.

What Makes a First-Class Assay?
Understanding what to measure, and knowing whether it is being measured accurately, are critical to the bioanalytical scientist. Biological samples not only contain the drug of interest, but also other factors that could interfere in the assay, such as the drug target, soluble receptors, or serum matrix proteins. Antibody capture and detection reagents must be highly specific to the drug, and not cross-react with these interfering factors. In the special case when the biotherapeutic is a human or humanized monoclonal antibody, the reagent must differentiate between the drug antibody and the naturally occurring immunoglobulins (Ig) present in excess in the sample. The selectivity and sensitivity of an assay are both crucial to success.

The complexities of different drug modalities and sample types, and consequently, the need to select the most appropriate reagents and technology platform, mean that a ‘one size fits all’ approach for bioanalytical assays is not possible. Each assay needs to be fully characterized, have a sufficiently robust method that enables transfer between labs, and generate trustworthy and easily interpretable data. Once the assay has been approved for use, a sustainable and reproducible supply of critical antibody reagents is needed throughout the lifetime of the study.

Find the Answers with a Library of 45 Billion Antibodies and a 14 Amino Acid Peptide
The careful generation and characterization of recombinant antibody tools helps address the challenges of bioanalytical assay development. Bio-Rad generates custom recombinant antibodies using Human Combinatorial Antibody Libraries (HuCAL®) and a proprietary method of phage display with automated high-throughput processes.

HuCAL technology is proven and well published and has been used by our team to generate antibodies for research and diagnostic applications since 2004. The structural diversity of the human antibody repertoire is represented in the HuCAL PLATINUM® library by seven heavy chain and six light chain variable region genes, which give rise to 42 master framework combinations. Highly diverse genetic cassettes, built by trinucleotides and encoding the complementarity determining regions (CDRs) of the antibody binding sites, are combined with these frameworks, creating antibody genes that code for some 45 billion unique antibodies in Fab format. Screening of HuCAL is performed in vitro, enabling the successful selection of antibodies against challenging targets such as drug-target complexes, neo-epitopes, ADC toxins, or chimeric antigen receptors. As the sequence of any selected antibody is known, it is possible to reproduce the genes synthetically if needed. This sequence back up secures the future supply of the antibody, and recombinant production methods ensure a high level of consistency between batches.

The in vitro guided selection method enables selection of inhibitory and noninhibitory anti-idiotypic antibodies, and drug target complex-specific binders, all of which are essential capture and detection reagents for ligand binding assays to measure free, total, or bound drug (Figure 1). An antibody that detects the drug only when in complex with its target is an especially valuable reagent for a PK antigen capture ELISA when the drug is a monovalent Fab antibody, and a bridging format is not possible. Selection is carried out on the drug in the presence of isotype subclass-matched antibodies and human serum as blocking reagents, to avoid enrichment of specificities that bind to other regions of the antibody drug or serum matrix proteins, and ensure the final antibodies have the desired specificity. Being fully human in sequence, the inhibitory anti-idiotypic antibodies are also ideal as a surrogate positive control in ADA assays.

Fig. 1. Three different types of anti-biotherapeutic antibody.

To expand the potential of the system even further, we have incorporated SpyTag technology into the HuCAL phage display platform, resulting in the generation of antibodies with completely new versatility. SpyTag technology is based on the SpyTag peptide and SpyCatcher protein, which are derived from the fibronectin-binding protein (FbaB) of Streptococcus pyogenes (Spy). The FbaB protein contains an intrachain isopeptide bond between the sidechains of a lysine and an aspartic acid within the Ig-like collagen adhesin domain. The SpyTag peptide incorporates the aspartic acid residue, and the SpyCatcher has the lysine residue; when the SpyTag and SpyCatcher are mixed, the isopeptide bond is formed between these amino acids. This reaction occurs spontaneously with high yield in diverse conditions of pH, temperature, and buffer. Following optimization of the two components (SpyTag2 and SpyCatcher2), the reaction time was shortened from hours to minutes and has subsequently been decreased even further (SpyTag3 and SpyCatcher3).

SpyTag – the Secret to More Sophisticated Antibodies
The 14 amino acid SpyTag2 peptide is genetically fused to the C-terminus of the recombinant Fab heavy chain. A set of SpyCatchers have been produced, both unconjugated and site-specifically labeled with HRP, biotin, or RPE. Variants include the BiCatcher, where two SpyCatchers are genetically linked to allow formation of a bivalent Fab2, and the FcCatcher, where a SpyCatcher is fused to an immunoglobulin Fc domain, making a full-length Ig-like molecule after the reaction with two SpyTagged Fabs. A Fab antibody with a SpyTag forms a covalent isopeptide bond to the chosen Catcher, enabling site-directed conjugation or fast conversion to bivalent Fab or a full-length Ig-like molecule within an hour (Figure 2).

Fig. 2. A monovalent Fab with SpyTag (center) can be converted within minutes to multiple different formats via formation of a covalent isopeptide bond with the chosen Catcher.

Our SpyCatcher2 and BiCatcher2 have been modified by adding a cysteine residue, which can be used for site-specific conjugation and facilitates a controlled degree of labelling (DOL). Coupling a SpyTagged Fab to a preconjugated Catcher avoids the problem of the label interfering with the antibody-antigen binding site and leads to improved performance in the assay.

Traditionally, recombinant antibodies are converted to different formats by subcloning the gene fragments encoding the antibody variable domains into expression vectors containing the desired elements, such as IgG constant domains, followed by expression and purification. This method is well established but can take several weeks. Using the simple SpyTag-SpyCatcher coupling reaction, monovalent Fab fragments can be converted rapidly into different antibody formats using BiCatchers and FcCatchers. One antibody can be used in alternative formats depending on the application, for instance a monovalent format for intrinsic affinity determination, and a bivalent format as a detection antibody in flow cytometry to take advantage of the avidity effect. The FcCatcher can be coupled to the antibody for those assays that require an Fc domain. Human, mouse, and rabbit FcCatchers are suitable for use with species-specific anti-Fc secondary reagents and can also be used for multiplexing experiments.

Performance in an ADA Assay Using Ig-like Format
Finding the best antibody to act as a reference standard in an anti-drug antibody (ADA) assay is a scenario where conversion to Ig-like format can speed up the assay development process. Several antibodies in monovalent Fab-SpyTag format can be rapidly converted to Ig-like format using the FcCatcher and compared in the assay. This modeling provides data to help the user select one or two appropriate candidates for conversion to fully human IgG for the clinical assays (Figure 3). In instances where ADA isotyping is required, the FcCatchers with different human IgG isotypes offer a quick and cost-effective alternative to full immunoglobulin conversion.

Fig. 3. Performance of high, medium, and low affinity anti-adalimumab antibodies compares well between FcCatcher and IgG formats. Antibody 1, high affinity (red) K_D 0.06 nM, antibody 2, medium affinity (black) K_D 0.2 nM, antibody 3, lower affinity (blue) K_D 11 nM; the three different antibodies were compared in an ADA assay, coupled to FcCatcher (left) and in full-length IgG1 format (right).

Overcome Assay Development Challenges using TrailBlazer Antibodies
When working in a fast-moving, competitive environment, it’s important take advantage of technologies that could make a real difference to achieving your goals. TrailBlazer Antibody custom services offer:

• Rapid generation of highly specific Fab antibodies in less than 3 months
• Isolation of antibodies against virtually any type of antigen
• SpyTag technology incorporated for:
• Rapid assembly of different stable formats
• Site-directed conjugation with consistent DOL
• Sequence-identified antibodies with long-term supply guaranteed

The success of bioanalytical assays is significantly impacted by the quality of the antibodies used. Combining recombinant antibody generation using HuCAL technology with SpyTag and SpyCatchers results in an antibody engineering toolbox that brings unprecedented flexibility to assay design. Having high quality, well-characterized, recombinant antibodies early in the development lifecycle enables the design of a selective and sensitive assay, a guaranteed supply for the duration of the study, and the generation of trustworthy data to support the regulatory approval of the biologic.

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Visit bio-rad-antibodies.com/SpyTag for more information.

HuCAL is a trademark of MorphoSys AG in certain jurisdictions.