Sequencing Technology for Antibody Drug Discovery

In the intricate landscape of antibody drug discovery, the choice of sequencing technology emerges as a pivotal determinant in the quest for uncovering potent therapeutic candidates. Among the array of sequencing methods available, each technology boasts distinct attributes that hold the potential to reshape our understanding of antibodies. This article delves into the merits of Sanger sequencing, Illumina platforms (MiSeq and NextSeq), PacBio sequencing, and Oxford Nanopore sequencing, while unraveling their tailored applications within the context of antibody characterization.

Quality control of recombinant antibody repertoires by high-throughput sequencing. (Parola et al., 2018)

Sanger Sequencing: A Beacon of Accuracy

The venerable Sanger sequencing method, renowned for its precision, unveils sequences with unwavering accuracy and extended read lengths ranging from 500 to 1000 base pairs. Its forte lies in the scrutiny of immunoglobulin G (IgG), Fab, and single-chain variable fragments (scFv). Though throughput remains modest, Sanger sequencing emerges as the paragon for meticulously dissecting a curated selection of sequences. It is the go-to choice for endeavors that demand impeccably high-quality results, such as validating lead antibody candidates or deciphering the intricacies of specific clones.

Illumina Sequencing: A Fusion of Throughput and Depth

The diverse portfolio of Illumina sequencing platforms, exemplified by MiSeq and NextSeq, harmonizes the delicate balance between throughput and sequence coverage, rendering them tailored for diverse antibody characterization goals. MiSeq, boasting paired-end reads of 2 x 250 base pairs, emerges as the workhorse for capturing the essence of variable heavy (VH) and variable light (VL) chain analysis, or embracing the entirety of single-domain antibodies (sdAbs).

In contrast, NextSeq, powered by 2 x 150 base pair reads, stands as the torchbearer for scrutinizing specific domains of an antibody—namely, the complementarity-determining region of the heavy chain (CDR-H3) and its adjacent precincts. The amplified throughput of NextSeq, relative to MiSeq, capacitates the analysis of expanded sample sets, augmenting the depth of insights derived.

PacBio Sequencing: Pioneering Full-Length Insights

Harnessing the prowess of PacBio sequencing, underpinned by reads spanning approximately 10 to 25 kilobases, the exploration of antibody sequences at full length becomes a reality. This technology shines in its ability to seamlessly unravel the intricacies of scFv and antibodies of substantial length. PacBio's magnetic charm extends to multivalent antibody constructs, such as nanobodies, facilitating the holistic sequencing of variable regions sans the complexities of assembly. It's worth noting that while PacBio delivers accuracy through elongated reads, considerations around throughput and cost per base should be acknowledged in the decision-making process.

Oxford Nanopore Sequencing: A Glimpse of the Future

The avant-garde Oxford Nanopore sequencing, with its striking read lengths scaling up to 2 megabases, tantalizes the realm of genomics. However, within the niche of antibody sequencing, its adoption is yet to attain significant momentum. The technology's nascent error rates and precision challenges temper its prevalence in antibody characterization endeavors. As nanopore technology evolves, its potential to revolutionize antibody sequencing looms on the horizon, promising enhanced accuracy and expanded applications.

Advantages of Sequencing Technology in Antibody Discovery

• Enhanced Diversity Analysis: Sequencing technology enables the in-depth analysis of antibody repertoires, providing insights into the diversity and complexity of the immune response against a specific antigen. This comprehensive view aids in the identification of rare and potent antibodies.
• Rational Design: The sequence data obtained through sequencing technologies allows researchers to perform rational design of antibodies. By analyzing sequence motifs and structural information, researchers can engineer antibodies with improved binding affinity, stability, and therapeutic properties.
• Faster Discovery: High-throughput sequencing accelerates the antibody discovery process. Researchers can quickly screen large libraries of antibody sequences to identify candidates with desired properties, reducing the time and resources required for drug development.
• Reduced Dependence on Animal Models: Sequencing technology reduces the reliance on animal immunization, making antibody discovery more ethically and logistically feasible.

In the dynamic domain of antibody drug discovery, the strategic selection of sequencing technology assumes a paramount role. Whether the pursuit is the meticulous validation of antibody contenders, comprehensive analysis of repertoires, or the unveiling of full-length sequences, each technology becomes a piece of the puzzle. As innovation propels these technologies forward, the arsenal at researchers' disposal flourishes, leading to deeper insights into antibody structure, functionality, and therapeutic potency. By weaving the strengths of sequencing technologies into the fabric of antibody characterization, researchers journey towards the forefront of antibody drug discovery, propelled by the synergistic marriage of knowledge and technology.

Reference:

1. Parola, Cristina, Daniel Neumeier, and Sai T. Reddy. "Integrating high‐throughput screening and sequencing for monoclonal antibody discovery and engineering." Immunology 153.1 (2018): 31-41.