AbDirect: The Game-Changer in Serological Antibody Discovery

How can we unlock the full potential of human antibodies hidden in complex biological samples?

Building on our previous articles about Abvion's founding mission and the power of protein-level antibody discovery, I'm excited to share the technical backbone that makes it all possible. My name is Sem Tamara, and I'm Head of Discovery Technologies at Abvion. With a PhD from Prof Albert Heck's lab at Utrecht University, our company's birthplace, I've seen firsthand how mass spectrometry transforms antibody discovery. This is the third article in our series – let's explore the proteomics approaches driving innovation in this field.

The Mass Spectrometry Toolbox

Mass spectrometry (MS) is a cornerstone of proteomics – the study of proteins and proteomes. It enables us to identify and sequence proteins by measuring their masses, or more specifically, their mass-to-charge ratios. Such proteomics analyses typically rely on prior knowledge about the protein’s amino acid sequence. The challenge with natural antibodies, however, is that their sequences are unknown beforehand. Therefore, they require technology capable of true de novo sequencing. For antibody discovery, MS provides sequence information at different levels of molecular complexity:

• Bottom-up MS digests proteins into small peptides that can be analyzed with high confidence, offering high throughput and sequence coverage but losing context in complex mixtures.

• Middle-down MS works with bigger fragments (e.g., antibody chains), balancing detail and coverage but lacking information on how these fragments piece together.

• Top-down MS analyzes intact proteins (e.g., intact antibodies), preserving high-level structural information but requiring advanced instrumentation and intricate data analysis.

Solving the Polyclonal Puzzle: The Integrated Approach

While methods in the mass spectrometry toolbox have made tremendous progress, no single one of them is yet able to deal with the complexity of typical endogenous samples by itself. This is because the central challenge in antibody de novo sequencing has always been a frustrating trade-off. Bottom-up proteomics gives you the exact "words" (peptides) but without knowing which "sentence" (antibody molecule) they came from. Top-down proteomics, on the other hand, shows you the full-sentence structures, but often leaves individual words missing or incomplete.

An integrated approach combines bottom-up with top- and middle-down proteomics to ensure high-confidence antibody sequencing.

To tell the full story, an integrated approach therefore combines bottom-up with top- and middle-down proteomics. This enables us to see clearly how the pieces of the puzzle fit together, creating the precision required to address the complexity of real-world samples. It is furthermore well-suited for integration with upfront antibody repertoire screening, a technique we co-developed at Utrecht University. By “fingerprinting” repertoires, this informs us of which antibodies are of most interest, e.g., based on their reactivity or abundance.

In pioneering works like Bondt et al. (2021, Cell Systems) and Peng et al. (2022, Analytical Chemistry), we demonstrated that repertoire profiling and integrative sequencing could be used to identify highly abundant clones dominating sepsis and monoclonal gammopathy repertoires. In parallel, Van Rijswijck et al. (2022, Nature Communications) expanded the repertoire screening approach to monitor the cross-reactivity of individual antibodies in complex responses post-SARS-CoV-2 infection. But what if we could now combine the two to tackle real-world polyclonal sequence discovery?

AbDirect: Our Platform Advancing Integrated MS

At Abvion, we have now converged these pivotal developments in antibody repertoire profiling and integrative de novo sequencing. Building on these fundamental advances, we have created our proprietary platform, AbDirect. It generates potential for a streamlined antibody discovery pipeline that handles high sample complexity while ensuring high-confidence sequencing – all with minimal sample input (e.g., 2 mL of serum).

AbDirect embodies the power of integrated MS, sequencing human antibodies standalone without the need for orthogonal data. It reconstructs complete antibodies, characterizing them down to minute molecular details. By profiling repertoires upfront in their full polyclonal context, we identify hidden gems and ensure precision for therapeutic leads. We are currently applying this technology in pilots for infectious diseases and autoimmunity, partnering with biopharma and academia to advance antibody therapeutics. The advantages shine: AbDirect navigates biological sample complexity to deliver fully functional, novel candidates, setting the stage for scalable discovery campaigns.

AbDirect fuses upfront repertoire screening with integrative de novo sequencing to deliver novel therapeutic candidates from complex polyclonal biofluids.

What’s Next?

Proteomics-based antibody discovery, from bottom-up sequencing to integrated discovery pipelines, is poised to transform the field. In the coming months, we'll unveil our proof-of-concept, showcasing AbDirect's first real-world application to discover highly potent human antibodies inaccessible by conventional workflows.

Join our journey and share what excites you about mass spectrometry in biotech. Follow us on LinkedIn and stay tuned for more!