AJICAP: Affinity Peptide Mediated Regiodivergent Functionalization of Native Antibodies

Abstract

Supportinginformation and the ORCID identification number(s) for the author(s) of this article can be found under: https://doi.

Site-Selective Lysine Acetylation of Human Immunoglobulin G for Immunoliposomes and Bispecific Antibody Complexes

“…MALDI-TOF MS analysis showed a molecular weight increase and approximately 50% of the heavy chain was modified in 15 m and 80% in 1 h (assuming that the modified heavy chain has a similar ionization property to the unmodified) (Figure b). Atezo-TAMRA showed binding interaction with a purified extracellular, soluble region of PD-L1 (Figure S10), consistent with the notion that modification of the Fc region of antibodies does not affect the fragment antigen-binding region (Fab region). − Similarly, Cy5-labeled Atezo (Atezo-Cy5) fluorescently labeled PC-12 cells that overexpressed a GFP-labeled PD-L1 (PD-L1-GFP) (Figure c). We also synthesized TAMRA-labeled trastuzumab (Tras-TAMRA; trastuzumab, Tras, is a HER2-specific mAb) and labeled the surface of HER2+ (SK-OV-3) cells (Figure d), suggesting that fluorescent labeling did not jeopardize the binding capability of the antibodies.…”

“…Breaking the disulfide bond − or modifying the surface glycans created reactive groups with activity above the basal level for conjugation reactions. Or, proximity-induced (affinity-guide or ligand-directed) reactions can realize site-selective modification based on the local confinement of the reactivity; examples include a glycan-directed tosyl reaction, light-activated benzoyl-phenylalanine reaction driven by IgG–protein G interaction, IgG conjugation reaction through the 4-fluorophenyl carbamate lysine driven by IgG–FB protein (the B domain from Staphylococcus protein A) interaction, IgG–peptide conjugation through a cross-linker, , and site-specific modification of asparagine-79 by a hexarhodium metallopeptide catalyst driven by the IgG–peptide interaction . Notwithstanding, these reactions still hardly match the post-translational protein modifications catalyzed by dedicated enzymes in nature: either a bulky stub is left on the protein, a protein partner with a sophisticated unnatural amino acid is required, or the reaction fails to achieve single-residue resolution.…”

“…3,4 To achieve site selectivity, chemists have sought to activate existing chemical groups on the antibodies through various methods. Staphylococcus protein A) interaction, 14 IgG−peptide conjugation through a cross-linker, 15,16 and site-specific modification of asparagine-79 by a hexarhodium metallopeptide catalyst driven by the IgG−peptide interaction. 17 Notwithstanding, these reactions still hardly match the post-translational protein modifications catalyzed by dedicated enzymes in nature: either a bulky stub is left on the protein, a protein partner with a sophisticated unnatural amino acid is required, or the reaction fails to achieve single-residue resolution.…”

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

“… 62 This same peptide, together with other previously known peptides such as the original FcIII and the minimized Z domain described above, also forms the basis of the AJICAP technology, described by Yamada et al This technology is based on the functionalization of native antibodies with thiol groups through the use of these affinity reagents, which then allows for further conjugation of cytotoxic payloads. 63 The same group later also describes the use of the AJICAP technology for gram-scale synthesis of stable and homogeneous ADCs. 64 …”

Manipulating the Click Reactivity of Dibenzoazacyclooctynes: From Azide Click Component to Caged Acylation Reagent by Silver Catalysis

“…Therefore, harnessing caging/decaging process on DBCOs, we successfully achieved site-specific modification of a commercial antibody drug trastuzumab using a DBCO-tagged Fc-binding peptide (16) as shown in Figure 6. The cyclic peptide was previously reported [24] to interact with Fc domain near the K249. We incubated DBCOpeptide 16 with trastuzumab in a pH 7.4 buffer, allowing the peptide to approach the Fc domain when DBCO was caged.…”