Template-Catalyzed, Disulfide Conjugation of Monoclonal Antibodies Using a Natural Amino Acid Tag

Abstract

The high specificity and favorable pharmacological properties of monoclonal antibodies (mAbs) have prompted significant interest in re-engineering this class of molecules to add novel functionalities for enhanced therapeutic and diagnostic potential. Here, we used the high affinity, meditope-Fab interaction to template and drive the rapid, efficient, and stable site-specific formation of a disulfide bond. We demonstrate that this template-catalyzed strategy provides a consistent and reproducible means to conjugate fluorescent dyes, cytotoxins, or "click" chemistry handles to meditope-enabled mAbs (memAbs) and memFabs. More importantly, we demonstrate this covalent functionalization is achievable using natural amino acids only, opening up the opportunity to genetically encode cysteine meditope "tags" to biologics. As proof of principle, genetically encoded, cysteine meditope tags were added to the N- and/or C-termini of fluorescent proteins, nanobodies, and affibodies, each expressed in bacteria, purified to homogeneity, and efficiently conjugated to different memAbs and meFabs. We further show that multiple T-cell and Her2-targeting bispecific molecules using this strategy potently activate T-cell signaling pathways in vitro. Finally, the resulting products are highly stable as evidenced by serum stability assays (>14 d at 37 °C) and in vivo imaging of tumor xenographs. Collectively, the platform offers the opportunity to build and exchange an array of functional moieties, including protein biologics, among any cysteine memAb or Fab to rapidly create, test, and optimize stable, multifunctional biologics.

Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells

“…10 Through a series of biophysical studies, we have markedly improved the affinity of the interaction and developed this system to conjugate memAbs with imaging agents, toxins, and other biologics, either through mechanically interlocked meditopes 12 or through template-catalyzed disulfide functionalization. 13 To create a universal CAR T cell using this meditope technology, we have simply replaced the antigen recognition domain with a meditope peptide and used memAbs to define the specificity. Akin to adding a bike rack to a car to carry one or more bikes to a specific site, we have coined the meditope-bearing 'receptor' as a Fabrack.…”

“… 10 Through a series of biophysical studies, we have markedly improved the affinity of the interaction and developed this system to conjugate memAbs with imaging agents, toxins, and other biologics, either through mechanically interlocked meditopes 12 or through template-catalyzed disulfide functionalization. 13 …”

Refining the Quality of Monoclonal Antibodies: Grafting Unique Peptide-Binding Site in the Fab Framework

“…Once engineered, meditope binding site acts effectively as a unique and highly specific but innocuous receptor site on the mAb. As such, there are multiple applications made available with this technology[6,7].…”

Peptide-Directed Photo-Cross-Linking for Site-Specific Conjugation of IgG

“…11,12 In the last two decades, various alternative approaches have been developed to achieve more controlled modifications. These approaches include enzyme-mediated conjugation reactions of sortase A 13 or transglutaminase; 14 and chemical reactions based on the thiol group of the substituted cysteine, 15,16 the N-terminal amine group of the peptide, 17 or bioorthogonally reactive groups generated by either enzymatic conversion 18 or from site-specifically incorporated amino acid analogues. 19,20 While these methods have been proven powerful for generating antibody conjugates, the majority of them require amino acid sequence changes, which can affect the biological and physicochemical properties of an antibody.…”

“…Conjugation of antibody to various cargos, such as small molecules, , proteins, − nucleic acids, and synthetic materials, , has applications in therapeutics and diagnostics. , Two proteinous residues of cysteine and lysine have been widely used as conjugation sites in antibodies, but these methods usually result in unwanted heterogeneous products. , In the last two decades, various alternative approaches have been developed to achieve more controlled modifications. These approaches include enzyme-mediated conjugation reactions of sortase A or transglutaminase; and chemical reactions based on the thiol group of the substituted cysteine, , the N-terminal amine group of the peptide, or bioorthogonally reactive groups generated by either enzymatic conversion or from site-specifically incorporated amino acid analogues. , While these methods have been proven powerful for generating antibody conjugates, the majority of them require amino acid sequence changes, which can affect the biological and physicochemical properties of an antibody. Additionally, some of these methods consist of a lengthy series of chemical reaction steps.…”