(523a) Modulating Antibody/Antigen Affinity By Triggered Assembly and Disassembly of an Artificially Split Protein M | AIChE

(523a) Modulating Antibody/Antigen Affinity By Triggered Assembly and Disassembly of an Artificially Split Protein M


Gaynor, A. - Presenter, University of Delaware
Kim, H., University of Delaware
Chen, W., University of Delaware
While antibodies are the fastest growing therapeutic drug class in the US, promising high efficacy for patients and profits from drug companies, many antibody therapies suffer from off-target activity. While typically occurring in a fraction of patients, promiscuous antibody interactions are associated with treatment-related complications and costly failures in clinical trials. Therefore, a generalizable means of restricting antibody therapies from functioning outside of their intended target areas is greatly needed.

Herein, we propose that Protein M (ProtM)—a recently discovered antibody binding domain found on the surface of Mycoplasma genitalium—might meet this demand. ProtM binds with high affinity to the variable light chain (VL) of human IgG, and its C-terminus is able to interact loosely with the complimentary determining region (CDR) of those antibodies. By imposing a steric barrier between the CDR and its respective target, ProtM is able to reduce greatly antibody/antigen affinity. To demonstrate ProtM’s ability to modulate this affinity, we cloned artificial C-terminal truncations to identify a ProtM variable capable of antibody binding without antigen-blocking activity. We then employed this variant to engineer a protease-responsive antibody binding switch. To achieve this, we ligated the ProtM truncation to the remainder of its C-terminus (pMCT) using Sortase A (SrtA) to activate ProtM activity, and we inserted a thrombin cut site to allow for C-terminus removal to deactivate the full-length ligation product. To test our switch, we used an MBP/anti-MBP dot blot as a model system: MBP was dotted onto a nitrocellulose membrane and then mixed with anti-MBP antibody pre-incubated with different ProtM combinations. The truncation product showed a vastly reduced ability to inhibit antibody binding, and pMCT showed no inhibition. However, the truncation ligated to pMCT returned to full-length activity. Upon cleavage with thrombin, ProtM once again lost its ability to inhibit antigen recognition. Because ProtM operates similarly on a wide vary of different antibodies, this work represents a first step towards a generalizable, universal approach to reduce off-target antibody activity in therapies.