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Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

ABSTRACT: Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.

SUBMITTER: Dobson CL

PROVIDER: S-EPMC5171805 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Dobson Claire L CL Devine Paul W A PW Phillips Jonathan J JJ Higazi Daniel R DR Lloyd Christopher C Popovic Bojana B Arnold Joanne J Buchanan Andrew A Lewis Arthur A Goodman Joanne J van der Walle Christopher F CF Thornton Peter P Vinall Lisa L Lowne David D Aagaard Anna A Olsson Lise-Lotte LL Ridderstad Wollberg Anna A Welsh Fraser F Karamanos Theodoros K TK Pashley Clare L CL Iadanza Matthew G MG Ranson Neil A NA Ashcroft Alison E AE Kippen Alistair D AD Vaughan Tristan J TJ Radford Sheena E SE Lowe David C DC

Scientific reports 20161220

Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes revers ...[more]

PMID: 27995962

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Project description:Near-infrared (NIR) fluorophores show superior in vivo imaging properties than visible-light fluorophores because of the increased light penetration in tissue and lower autofluorescence of these wavelengths. We have recently reported that new NIR cyanine dyes containing a novel C4'-O-alkyl linker exhibit greater chemical stability and excellent optical properties relative to existing C4'-O-aryl variants. In this study, we synthesized two NIR cyanine dyes with the same core structure and charge but different indolenine substituents: FNIR-Z-759 bearing a combination of two sulfonates and two quaternary ammonium cations, and FNIR-G-765 bearing a combination of two sulfonates and two guanidines, resulting in zwitterionic charge with distinct cationic moieties. In this study, we compare the in vitro and in vivo optical imaging properties of monoclonal antibody (mAb) conjugates of FNIR-Z-759 and FNIR-G-765 with panitumumab (pan) at antibody-to-dye ratios of 1?:?2 or 1?:?5. One-to-five conjugation of pan-to-FNIR-G-765 was not successful due to aggregate formation during the conjugation reaction. Conjugates of both dyes to pan (2?:?1) demonstrated similar quenching capacity, stability, and brightness in target cells in vitro. However, FNIR-Z-759 conjugates showed significantly lower accumulation in the mouse liver, resulting in higher tumor-to-liver ratio. Thus, FNIR-Z-759 conjugates appear to have superior in vivo imaging characteristics compared with FNIR-G-765 conjugates, especially in the abdominal region. Moreover, from a chemistry point of view, mAb conjugation with FNIR-Z-759 has an advantage over FNIR-G-765, because it does not form aggregates at high dye-to-mAb ratio. These results suggest that zwitterionic cyanine dyes are a superior class of fluorophores for conjugating with mAbs for fluorescence imaging applications due to improving target-to-background contrast in vivo. However, zwitterionic cyanine dyes should be designed carefully, as small changes to the structure can alter in vivo pharmacokinetics of mAb-dye conjugates.

Dataset Information

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

Publications

Engineering the surface properties of a human monoclonal antibody prevents self-association and rapid clearance in vivo.

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