FROM ANTIBODY-DRUG CONJUGATES TO MASKED ANTIBODIES: BIOPHYSICAL INSIGHT FOR THE RATIONAL DESIGN OF FUTURE THERAPIES
type: CreativeWork
description: Monoclonal antibodies have become a major class of therapeutics over the past twenty-five years and, during this time, many successful antibody scaffolds have been based on wild-type humanized and human sequences of the immunoglobulin G isotype 1 (IgG1) subclass. In the past, several mutations have been engineered into these scaffolds, to optimise biological/physical properties or to allow site-specific conjugation. In the first project, the thermodynamic, thermal and kinetic stabilities of two sets of mutations were measured: one was a triple mutation in the CH2 domain (L234F/L235E/P331S), and the other had an additional substitution in the CH3 domain, S442C, to enable site-specific conjugation of a cytotoxic payload. Overall, results showed that the order of the domains in increasing stability were CH2, CH3 and Fab. The triple mutation was found to destabilize the CH2 domain, while the substitution in the CH3 domain did not affect stability significantly. Antibody-drug conjugates have been one of the most actively developed classes of drugs in the past fifteen years combining the strengths of large and small molecule therapeutics. Recently, strategies involving the insertion of a cysteine and maleimide linkers to achieve site-specific conjugation have been developed in order to attain highly controlled drug to antibody ratios. One such antibody scaffold, Fc-C239i, that formed an unexpected disulfide bridge during manufacture, was characterized. A combination of mass spectrometry and biophysical techniques were used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody. Alternative strategies to the use of maleimide linkers have recently been developed, e.g. DVP and tetraDVP linkers, by the Spring Group in the Department of Chemistry. The impact of the conjugation of DVP and tetraDVP-linkers on the stability and dynamics of the antibody trastuzumab was investigated. Results showed t…
abstract: Monoclonal antibodies have become a major class of therapeutics over the past twenty-five years and, during this time, many successful antibody scaffolds have been based on wild-type humanized and human sequences of the immunoglobulin G isotype 1 (IgG1) subclass. In the past, several mutations have been engineered into these scaffolds, to optimise biological/physical properties or to allow site-specific conjugation. In the first project, the thermodynamic, thermal and kinetic stabilities of two sets of mutations were measured: one was a triple mutation in the CH2 domain (L234F/L235E/P331S), and the other had an additional substitution in the CH3 domain, S442C, to enable site-specific conjugation of a cytotoxic payload. Overall, results showed that the order of the domains in increasing stability were CH2, CH3 and Fab. The triple mutation was found to destabilize the CH2 domain, while the substitution in the CH3 domain did not affect stability significantly. Antibody-drug conjugates have been one of the most actively developed classes of drugs in the past fifteen years combining the strengths of large and small molecule therapeutics. Recently, strategies involving the insertion of a cysteine and maleimide linkers to achieve site-specific conjugation have been developed in order to attain highly controlled drug to antibody ratios. One such antibody scaffold, Fc-C239i, that formed an unexpected disulfide bridge during manufacture, was characterized. A combination of mass spectrometry and biophysical techniques were used to understand how the additional disulfide bridge forms, interconverts, and changes the stability and structural dynamics of the antibody. Alternative strategies to the use of maleimide linkers have recently been developed, e.g. DVP and tetraDVP linkers, by the Spring Group in the Department of Chemistry. The impact of the conjugation of DVP and tetraDVP-linkers on the stability and dynamics of the antibody trastuzumab was investigated. Results showed t…
author: Orozco, Carolina
language: eng
keywords: antibody-drug conjugates, masked antibodies, protein folding, protein stability, pro-antibodies, mass spectrometry
license: https://www.rioxx.net/licenses/all-rights-reserved/
date published: 2021-09-29
date modified: 2026-07-22T22:24:20Z
provider: Cambridge University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: University of Cambridge

PropertyValue
property ID: DOI
value: 10.17863/CAM.82744