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

Site-selective acetylation of a single lysine residue in a protein that reaches a lysine acetyltransferase's accuracy, precision, and reliability is challenging. Here, we report a peptide-guided, proximity-driven group transfer reaction that acetylates a single lysine residue, Lys 248, of the fragment crystallizable region (Fc region) in the heavy chain of the human Immunoglobulin G (IgG). An Fc-interacting peptide bound with the Fc domain and positioned a phenolic ester close to Lys 248, which induced a nucleophilic reaction and resulted in the transfer of an acetyl group to Lys 248. The acetylation reaction proceeded to a decent yield under the physiological condition without the need for deglycosylation, unnatural amino acids, or catalysts. Along with acetylation, functional moieties such as azide, alkyne, fluorescent molecules, or biotin could also be site-selectively installed on Lys 248, allowing IgG's further derivatization. We then synthesized an antibody-lipid conjugate and constructed antibody-conjugated liposomes (immunoliposomes), targeting HER2-positive (HER2+) cancer cells. We also built a bispecific antibody complex (bsAbC) covalently linking an anti-HER2 antibody and an anti-CD3 antibody. The bsAbC showed in vitro effector-cell-mediated cytotoxicity at nanomolar concentrations. Compared with bispecific antibodies (bsAbs), bsAbCs are constructed based on native IgGs and contain two antigen-binding sites to each antigen, twice that of bsAbs. Altogether, this work reports a method of site-selective acetylation of native antibodies, highlights a facile way of site-selective IgG functionalization, and underscores the potential of bsAbCs in cancer immunotherapy.

Site-selective antibody-lipid conjugates for surface functionalization of red blood cells and targeted drug delivery.

Displaying antibodies on carrier surfaces facilitates precise targeting and delivery of drugs to diseased cells. Here, we report the synthesis of antibody-lipid conjugates (ALCs) through site-selective acetylation of Lys 248 in human Immunoglobulin G (IgG) and the development of antibody-functionalized red blood cells (immunoRBC) for targeted drug delivery. ImmunoRBC with the HER2-selective antibody trastuzumab displayed on the surface (called Tras-RBC) was constructed following a three-step procedure. First, a peptide-guided, proximity-induced reaction transferred an azidoacetyl group to the ε-amino group of Lys 248 in the Fc domain. Second, the azide-modified IgG was subsequently conjugated with dibenzocyclooctyne (DBCO)-functionalized lipids via strain-promoted azide-alkyne cycloaddition (SPAAC) to result in ALCs. Third, the lipid portion of ALCs was then inserted into the cell membranes, and IgGs were displayed on red blood cells (RBCs) to construct immunoRBCs. We then loaded Tras-RBC with a photosensitizer (PS), Zinc phthalocyanine (ZnPc), to selectively target HER2-overexpressing cells, release ZnPc into cancer cells following photolysis, and induce photodynamic cytotoxicity in the cancer cells. This work showcases assembling immunoRBCs following site-selective lipid conjugation on therapeutic antibodies and the targeted introduction of PS into cancer cells. This method could apply to the surface functionalization of other membrane-bound vesicles or lipid nanoparticles for antibody-directed drug delivery.

Site-Selective Tyrosine Reaction for Antibody-Cell Conjugation and Targeted Immunotherapy.

Targeted immunotherapies capitalize on the exceptional binding capabilities of antibodies to stimulate a host response that effectuates long-lived tumor destruction. One example is the conjugation of immunoglobulins (IgGs) to immune effector cells, which equips the cells with the ability to recognize and accurately kill malignant cells through a process called antibody-dependent cellular cytotoxicity (ADCC). In this study, a chemoenzymatic reaction is developed that specifically functionalizes a single tyrosine (Tyr, Y) residue, Y296, in the Fc domain of therapeutic IgGs. A one-pot reaction that combines the tyrosinase-catalyzed oxidation of tyrosine to o-quinone with a subsequent [3+2] photoaddition with vinyl ether is employed. This reaction installs fluorescent molecules or bioorthogonal groups at Y296 of IgGs or the C-terminal Y-tag of an engineered nanobody. The Tyr-specific reaction is utilized in constructing monofunctionalized antibody-drug conjugates (ADCs) and antibody/nanobody-conjugated effector cells, such as natural killer cells or macrophages. These results demonstrate the potential of site-selective antibody reactions for enhancing targeted cancer immunotherapy.