The Masking Game: Design of Activatable Antibodies and Mimetics for Selective Therapeutics and Cell Control

Abstract

The high selectivity and affinity of antibody binding have made antibodies all-pervasive tools in therapy, diagnosis, and basic science. A plethora of chemogenetic approaches has been devised to make antibodies responsive to stimuli ranging from light to enzymatic activity, temperature, pH, ions, and effector molecules. Within a single decade, the field of activatable antibodies has yielded marketed therapeutics capable of engaging antigens that could not be targeted with traditional antibodies, as well as new tools to control intracellular protein location and investigate biological processes. Many opportunities remain untapped, waiting for more efficient and generally applicable masking strategies to be developed at the interface between chemistry and biotechnology.

Figure 1

Very diverse molecular engineering strategies have been applied to generate antibodies responsive to a variety of stimuli.

Figure 2

Light-induced activation can be engineered through various mechanisms for therapeutic or cell biology applications. (A) Random nucleophile photocaging enables generating selective T-cell engagers. (B) Genetic encoding of photocaged tyrosine (ONBY) provides selective targeting and cell biology tools such as protein dimerizers. A bispecific nanobody–photobody construct is used as a model dimerizer to recruit GFP from the extracellular space and bring it close to mCherry-EGFR upon UV-light irradiation. (C) A photosensitive variant of the peptide–DNA lock described in section 3.1 could be applied in photopharmacology. (D) Split nanobody assembly via optical Magnets controls protein location to investigate cellular processes. In the confocal images shown, the two split parts of the antibody colocalize with Mito-GFP upon blue light irradiation. (E) Encoding of the LOV domain enables intracellular control of protein location and immunopurification.^, Adapted with permission from refs (, , , and 22). Copyright 2020 Wiley-VCH, and 2019 and 2020 Springer Nature.

Figure 3

Small molecules, peptides, and oligonucleotides can be used to switch antibody binding in diverse applications. (A) A peptide–DNA lock may be used in the construction of molecular logic gates. (B) Chemogenetic antibody activation enables intracellular control of protein location and manipulation of biological systems. (C) scFvs activated with calmodulin-binding peptides could provide useful immunoaffinity purification tools. (D) Rapamycin-activated antibodies may be used to investigate biological systems. (E) Chemical rescue of binding conformation could enable prodrug-activated antibodies. Adapted with permission from refs (, , and 33). Copyright 2020 American Chemical Society and 2020 Springer Nature.

Figure 4

pH sensitivity enhances the therapeutic potential of antibodies. (A) Recycling antibodies have an enhanced half-life and degradation of soluble antigens. Such antibodies may be engineered by introducing histidine residues to induce pH-sensitivity or by evolving Ca²⁺-sensing loops in the CDRs. (B) ADCs with decreased affinity at the lower endosomal pH have enhanced efficacy and selectivity for the tumor tissue. Adapted with permission from refs ( and 57). Copyright 2020 MAbs.

Figure 5

Protease overexpression enables selective antibody activation in diseased tissues. (A) In the probody approach, extending the N-terminus with a mimotope that can be removed by proteolytic cleavage shows great versatility and decreases off-target binding, thereby enhancing antibody circulation time.⁻ The graph schematically represents results from ref (61). (B) Epitope-mimetics and idiotypic masks enable good inactivation efficiency at the expense of transferability to to different antigen specificities.⁻ (C) Approaches relying on the interaction with more conserved regions in the Fv could enable transferability to other antibody specificities and formats.^, (D) Several strategies generally applicable to certain antibody formats have been developed relying on steric hindrance with variable degrees of inactivation efficiency.⁻ (E) Masking via N-terminal coiled-coil domains is efficient and suggests high transferability to other antigen specificities. Adapted with permission from refs ( and 73). Copyright 2013 American Association for the Advancement of Science and 2019 Springer Nature.

Figure 6

Antibody phosphorylation provides selective activation on mice tissues ex vivo. (A) Chemical phosphorylation of a cysteine on an anti-lysozyme nanobody via a dehydroalanine intermediate. (B) Lysozyme-expressing cells implanted in mice brain are selectively stained ex vivo only in response to LPS-induced secretion of SEAP. Adapted with permission from ref (88). Copyright 2014 Nature Communication.

Figure 7

LOCKR system enables recruitment of an effector protein mediated by antigen colocalization. Increasing the number of keys and/or decoys present enables complex Boolean logic operation and enhances target specificity.