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Engineering alternative scaffold proteins for conditional targeting

Time: Fri 2023-11-17 10.15

Location: Webinar: 697 5953 6894, Kollegiesalen, Brinellvägen 8

Language: English

Subject area: Biotechnology

Doctoral student: Malin Jönsson , Proteinvetenskap, Science for Life Laboratory, SciLifeLab

Opponent: Professor Mark Howarth, University of Cambridge, Storbritannien

Supervisor: Professor Sophia Hober, Centrum för Bioprocessteknik, CBioPT, Science for Life Laboratory, SciLifeLab, Albanova VinnExcellence Center for Protein Technology, ProNova, Proteinteknologi

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QC 2023-10-26

Abstract

Engineering naturally occurring proteins enables us to customize affinity domains according to our specific needs, tailoring them to become an important tool in a wide array of applications limited merely by our creativity. One of nature’s ways to regulate protein activity is by creating a functional change through alteration of a protein’s tertiary structure upon interaction with metal ions. Inspired by this elegant solution, this thesis has focused on engineering calcium-regulated affinity proteins using two different strategies and protein scaffolds.

The first strategy revolved around designing and selecting a calcium- binding motif that can render the inherent target affinity of a naturally occurring protein domain to be turned on or off depending on whether calcium is present or not. The subject of this part of the thesis was one of the immunoglobulin-binding domains derived from Streptococcal Protein G. A library of various loops with prerequisites for attracting calcium was inserted between the IgG-binding surfaces of the domain prior to performing cell display selections aimed for rendering the inherent target interaction dependent on the presence of calcium. Successful selections resulted in a calcium-dependent version of the IgG-binding protein and its structure could be solved using NMR. A deeper investigation of the incorporated structural calcium-dependency could explain the underlying mechanisms giving rise to the functional on-and-off switch in target affinity and show how it derived from the evolutionary selection pressures applied.

The second strategy included the creation of a combinatorial library based on a calcium-dependent protein scaffold, derived from Staphylococcal Protein A, for development of small calcium-regulated affinity – CaRA –imolecules with novel target specificities. Mimicking the multifaceted usefulness of naturally occurring metalloproteins, this second part of the thesis aimed at performing phage display selection campaigns towards a diverse set of targets relevant for various applications from bioprocessing (e.g. scFv) to biological therapies (e.g. TNFa, IL-23, EGFR). When evaluating the binding properties in the presence and absence of calcium, all discovered CaRA variants display calcium-dependent binding and target affinities in the nanomolar range.

Engineering conditional binding can enhance the potential of next generation therapies in several ways. When used as calcium-dependent affinity ligands, it enables mild purification at neutral pH of therapeutic antibodies and antibody fragments that was previously limited by harsh acidic elution conditions. Reducing the risk of aggregated product by eluting at neutral pH would result in improved safety as well as the possibility to manufacture a greater repertoire of antibody formats. Furthermore, the conferred calcium- dependency of the CaRA scaffold can be used in a therapeutic approach envisioned to result in increased tissue penetration due to its small size and improved intracellular delivery by taking advantage of the existing calcium- gradient across the endosomal membrane of cells. This could lead to higher therapeutic efficacy by enabling lower doses or dosing frequency, further advancing a more patient-friendly future.

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