Maximizing Diversity and Functionality in Human Single-Domain Antibody Phage Display Libraries

Oscar Mejias Gomez*

*Corresponding author for this work

Research output: Book/ReportPh.D. thesis

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Abstract

Antibodies are essential proteins of our immune system, playing a critical role in defending our bodies against various diseases. These proteins, also known as immunoglobulins, recognize and bind to foreign substances, such as bacteria and viruses, to neutralize or eliminate them from our body. Antibodies are produced by B cells, a type of white blood cell, in response to an antigen and are crucial for the body's ability to fight off infections and diseases. The discovery of the use of antibodies as a form of therapy can be traced back to the late 19th century, when Von Behring and Kitasato observed that certain substances in the serum of infected rabbits could protect and cure uninfected animals from diseases like diphtheria.

In recent years, single-domain antibodies (sdAbs) have emerged as a unique class of immunoglobulins. These small-sized antibody fragments consist of just one functional immunoglobulin domain, making them highly versatile and useful for various applications, such as diagnostics, therapeutics, and research. Their ability to bind epitopes that are not accessible to conventional antibodies also makes them attractive candidates for targeting challenging antigens.

The field of antibody therapy is constantly evolving, and researchers are working on developing new methods to create effective treatments for various diseases. One significant development in this field was the creation of the hybridoma technology in 1975, when César Milstein and Georges Köhler successfully produced the first monoclonal antibodies (mAbs). This involved fusing antibody-producing B cells with immortal cancer cells to create hybrid cells, capable of producing large quantities of identical antibodies (hence "monoclonal"). This breakthrough paved the way for numerous applications, including diagnostics, therapeutics, and research purposes. One notable example of a monoclonal antibody derived from hybridoma technology is rituximab (Rituxan®), which is used for the treatment of non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and rheumatoid arthritis.

Additionally, advancements in molecular biology techniques have enabled the construction of in vitro antibody libraries, which are composed of repertoires of variable genes of immunoglobulins. A cutting-edge technique used to identify and isolate novel antibodies against virtually any antigen is phage display. Developed in 1985 by George P. Smith, this technique uses bacteriophages, viruses that infect bacteria, as a vehicle to display and screen diverse libraries of peptides or antibody fragments, introduced in 1990 by Sir Gregory Winter. Phage display involves inserting the genes encoding for the desired antibody fragments into the bacteriophage's DNA, allowing the phage to display the antibodies on its surface. By exposing the phage-displayed antibody library to a specific antigen, researchers can identify and isolate phages that bind to the target antigen with high affinity and specificity. For phage display to be effective in discovering novel antibodies, it is crucial to have libraries that exhibit both high diversity and high quality. High diversity ensures that the library contains a broad range of antibody sequences, increasing the likelihood of identifying an antibody with the desired characteristics. High-quality libraries are essential for producing functional and properly folded antibodies, which is crucial for their efficacy and stability. One prominent example of a monoclonal antibody discovered using phage display is adalimumab (Humira®), a potent therapeutic agent for the treatment of various autoimmune diseases, including rheumatoid arthritis, psoriasis, and inflammatory bowel diseases.

This thesis focuses on the generation of four human single-domain antibody libraries for phage display, characterized by their high diversity (10 9 ), quality (96% unique clones), and functionality (85,5% in-frame functional clones). The libraries were designed in silico to mimic the aminoacid diversity of human variable regions of the heavy chain of immunoglobulin G (IgG) as well as the CDR length, including 13, 16, 20, and 25 aminoacids. Additional solubility-increasing mutations were introduced in framework regions to ensure the generation of antibodies with high human-like properties, thus reduced immunogenicity, and improved developability properties. The randomization of the complementarity-determining regions (CDRs) within the libraries was performed using state-of-the-art semiconductor-based DNA synthesis, ensuring high accuracy and precision in the generation of diverse antibody sequences, which together with high fidelity cloning techniques ensured maximizing the functional and total diversity of the libraries. To rescue these libraries, a trypsin-sensitive helper phage system was employed, which allows for the efficient removal of “background” phages during the selection process, so that only those binding the desired antigen are carried on in the subsequent rounds of selection. In order to extensively characterize the generated libraries and monitor the biopanning process, Oxford Nanopore Technologies (ONT) sequencing coupled with unique molecular identifiers (UMIs) was introduced. This cutting-edge approach provided a comprehensive and accurate assessment of the libraries’ diversity, clonal distribution, and functional characteristics. Moreover, this method enabled the real-time monitoring of the biopanning process against five adhesion factors of selected clinically relevant Clostridioides difficile strains, a pathogen associated with severe gastrointestinal infections. The libraries were capable of rendering binding sdAbs with a wide range of affinities from high micromolar to low nanomolar.

The results obtained from this PhD thesis not only demonstrate the successful generation of four single-domain antibody libraries of high diversity, quality, and functionality but also showcase the power of phage display and advanced sequencing technologies in the discovery of novel antibody therapeutics. These libraries hold great promise for the development of innovative treatments against a wide range of diseases, including those caused by challenging pathogens like Clostridioides difficile.
Original languageEnglish
Place of PublicationKgs. Lyngby, Denmark
PublisherDTU Bioengineering
Number of pages202
Publication statusPublished - 2023

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  • Protein Science & Signaling Biology

    Mejias Gomez, O. (PhD Student), Chames, P. (Examiner), Spillner, E. H. (Examiner), Goletz, S. (Main Supervisor), Clausen, M. H. (Supervisor) & Kristensen, P. (Supervisor)

    01/09/201931/08/2023

    Project: PhD

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