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Scholarly Works (3 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2024)

The development of engineered biologics as both tools and therapeutic molecules have transformed the scientific landscape tremendously. Due to their increased specificity, potency, bioavailability and decreased immunogenicity, antibodies have become a popular scaffold for drug development. In 2023, antibodies accounted for five of the top ten selling drugs in the US. In addition to their therapeutic purposes, the high specificity of antibodies allows them to be powerful tools for studying and modifying biological systems. Antibodies are now regularly used to modify cellular signaling pathways, block protein interactions, detect a specific antigen of interest, among many other applications.Though our body naturally produces antibodies to fight off infection, the ability to generate synthetic antibodies against a chosen antigen of interest gives scientists the opportunity to expand the scope of druggable targets. A library consisting of up to 1011 fully synthetic antibodies can be displayed on systems such as phage, yeast, or mammalian cells, then screened for binding against an immobilized antigen. If binders are identified from the pool, they are isolated and sequenced to obtain information about the antibody’s amino acid combination for further production and characterization. These antibodies can then be optimized and applied in various formats for downstream biological experiments. Here, I describe three approaches for using antibody and protein engineering to uncover novel information on biological systems at the protein or cellular level. First in chapter 1, we use naïve antibody discovery to identify functional antibody modulators of protein arginine deiminase (PAD4), a protein highly implicated in the development of autoimmune diseases. In chapter 2, we develop a PAD4 fusion protein to target the enzyme to HER2+ expressing cancer cells and identify the substrate scope of PAD4 at the cell surface. In the last chapter, we create an antibody-drug conjugate using site-specific bioconjugation for targeted degradation of challenging cell surface proteins.

    Cover page: Development of antibody-based tools to study PAD4 protein dynamics and cell surface interactions
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2024)
    Unlocking the potential of protein arginine deiminase 4 (PAD4) as a drug target for rheumatoid arthritis requires a deeper understanding of its regulation. In this study, we use unbiased antibody selections to identify functional antibodies capable of either activating or inhibiting PAD4 activity. Through cryogenic-electron microscopy, we characterized the structures of these antibodies in complex with PAD4 and revealed insights into their mechanisms of action. Rather than steric occlusion of the substrate-binding catalytic pocket, the antibodies modulate PAD4 activity through interactions with allosteric binding sites adjacent to the catalytic pocket. These binding events lead to either alteration of the active site conformation or the enzyme oligomeric state, resulting in modulation of PAD4 activity. Our study uses antibody engineering to reveal new mechanisms for enzyme regulation and highlights the potential of using PAD4 agonist and antagonist antibodies for studying PAD4-dependency in disease models and future therapeutic development.
      Cover page: Antibody discovery identifies regulatory mechanisms of protein arginine deiminase 4.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2023)
      In vitro biopanning platforms using synthetic phage display antibody libraries have enabled the identification of antibodies against antigens that were once thought to be beyond the scope of immunization. Applying these methods against challenging targets remains a critical challenge. Here, we present a new biopanning pipeline, RAPID (Rare Antibody Phage Isolation and Discrimination), for the identification of rare high-affinity antibodies against challenging targets. RAPID biopanning uses fluorescent labeled phage displayed fragment antigen-binding (Fab) antibody libraries for the isolation of high-affinity binders with fluorescent activated sorting. Subsequently, discriminatory hit screening is performed with a biolayer interferometry (BLI) method, BIAS (Biolayer Interferometry Antibody Screen), where candidate binders are ranked and prioritized according to their estimated kinetic off rates. Previously reported antibodies were used to develop the methodology, and the RAPID biopanning pipeline was applied to three challenging targets (CHIP, Gαq, and CS3D), enabling the identification of high-affinity antibodies.
        Cover page: Rare antibody phage isolation and discrimination (RAPID) biopanning enables identification of high-affinity antibodies against challenging targets
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