Mechanistic Insights to Drive Rational Design of Masked Antibodies using FabALACTICA®

Scientists at the University of Cambridge, Astra Zeneca and collaborators undertook mechanistic studies to investigate antibody masking and better understand the parameters involved and their relative impact. FabALACTICA proved to be a valuable tool in this study, allowing the authors to generate antibody subunits. Using the Fab region, as opposed to the full-length antibody, allowed for both higher resolution SEC-MALS experiments to be performed, and supported structural determination of the complexes.

Monoclonal antibodies (mAbs) have become a major class of therapeutics and are proven as effective cancer therapies. Despite the clear benefits, they can also cause side effects, mainly due to on-target, off tumour activity. Strategies have emerged to improve the selectivity of therapeutic antibodies at the tissue level, by masking the paratope of antibodies, allowing them to be injected as an inactive, pro-drug that get unmasked at the site of activity.
The scientists in this study investigated the importance of different parameters by engineering masked antibodies based on the well-characterized therapeutic antibody trastuzumab. Trastuzumab is used to treat HER2-positive breast cancer, but it is also known to provoke cardiotoxicity in some cases. Four potential masks were tested, including three anti-idiotypic antibody fragments fused to trastuzumab, to prevent binding to HER2, as well as an antibody fragment that was not specific to trastuzumab, to probe the effect of steric hindrance. A wide range of biophysical techniques, including SEC-MALS, HDX-MS and X-ray crystallography were used to probe and identify the location of binding sites on the antibody.
Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) was used to understand to what extent the masks interact with the CDRs of trastuzumab. In these experiments, FabALACTICA proved to be a useful tool in the generation of isolated Fab domains. FabALACTICA is a cysteine protease that digests human IgG1 at one specific site above the hinge, generating intact Fab and Fc fragments. Digesting the antibody into smaller fragments was critical in this study as it allowed the investigators to achieve higher resolution on the SEC-MALS, generated subunits which were of optimal mass for the analytical tools available and subsequently allowed for more confident data interpretation.

Prior to initiation of this study, no high-resolution structure of trastuzumab in complex with any of the tested masks was available. Determination of a trastuzumab-mask complex was, therefore, a key goal of this study, especially considering that localization of the binding site of the mask of a key parameter that the authors wanted to assess. FabALACTICA was again a valuable tool in these experiments, as a crystal structure of the trastuzumab Fab region, generated following digestion using FabALACTICA, in complex with one of the potential masks was solved. This also enabled the scientists to assess the possibility of using HDX-MS as a tool to accurately identify a binding site.
In summary, this work revealed a number of parameters in need to be optimized to successfully engineer an effective affinity-based mask. The work also highlights the effectiveness and utility of enzymatic tools such as FabALACTICA to allow for middle-level biophysical characterisation. Digesting the antibody to generate isolated Fab regions allowed for higher resolution SEC-MALS to be generated, than would be possible at the intact level. It also facilitated the structural characterisation through simplification of the trastuzumab molecule to achieve a solved structure in complex with one of the tested masks.


Orozco et al., 2022. Mechanistic insights into the rational design of masked antibodies. mAbs.









FabALACTICA – Above hinge digestion of human IgG1