Tunable modulation of antibody‐antigen interaction by protease cleavage of protein M

Abstract

While immunoglobulins find ubiquitous use in biotechnology as static binders, recent developments have created proantibodies that enable orthogonal switch‐like behavior to antibody function. Previously, peptides with low binding affinity have been genetically fused to antibodies, to proteolytically control binding function by blocking the antigen‐binding site. However, development of these artificial blockers requires panning for peptide sequences that reversibly affect antigen affinity for each antibody. Instead, a more general strategy to achieve dynamic control over antibody affinity may be feasible using protein M (ProtM) from Mycoplasma genitalium, a newly identified polyspecific immunity evasion protein that is capable of blocking antigen binding for a wide range of antibodies. Using C‐terminus truncation to identify ProtM variants that are still capable of binding to antibodies without the ability to block antigens, we developed a novel and universal biological switch for antibodies. Using a site‐specifically placed thrombin cut site, antibody affinity can be modulated by cleavage of the two distinct antibody‐binding and antigen‐blocking domains of ProtM. Because of the high affinity of ProtM toward a large variety of IgG subtypes, this strategy may be used as a universal approach to create proantibodies that are conditionally activated by disease‐specific proteases such as matrix metalloproteinases.

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

“… 62 − 65 (C) Approaches relying on the interaction with more conserved regions in the Fv could enable transferability to other antibody specificities and formats. 66 , 67 (D) Several strategies generally applicable to certain antibody formats have been developed relying on steric hindrance with variable degrees of inactivation efficiency. 68 − 72 (E) Masking via N-terminal coiled-coil domains is efficient and suggests high transferability to other antigen specificities.…”

“…(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 . (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 .…”

“…Easier transferability across some antigen specificities can be achieved with protein M from Mycoplasma genitalium . Chen and co-workers engineered a protease-cleavable version of this protein that recognizes a conserved region in the Fv and extends over the paratope, thus blocking binding in a full-length IgG . Interestingly, this is one of the few approaches that does not require modification of the antibody, which may enable easier transferability to other IgGs and antibody derivatives.…”

Mechanistic insights into the rational design of masked antibodies

“… 4 In the past decade, 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 a pro-drug (i.e., in an inactive form) that gets unmasked when they are in the target region. 5–7 Two main categories of masks have been engineered so far: 1) masks with affinity for the antigen-binding domains, including peptides, 8–10 antibody fragments 11–14 and protein M 15 ; and 2) non-binding masks that rely on steric hindrance to block the antigen-binding site. This second category includes coiled-coil domains, 16 peptide-DNA assemblies, 17 , 18 , 19 and a hinge region.…”

“… 21 The most widely used approach has been activation by hydrolysis with enzymes that are overexpressed in the tumor or organ of interest. 9 , 10 , 11 , 12 , 13 , 16 , 15 , 20 , 22 , 23 , 24 , 25 , 26 Alternatives have also been investigated, 21 such as photo-cleavable linkers, 17 pH-dependent peptide-DNA locks, 18 or pH-sensitive masking peptides. 27 A number of masked antibodies that effectively decrease binding to the target when not at the tumor site and improve therapeutic index (i.e., the ratio of maximal tolerated dose to therapeutic dose) have been successfully engineered.…”