Protein-Antibody Conjugates (PACs): A Plug-and-Play Strategy for Covalent Conjugation and Targeted Intracellular Delivery of Pristine Proteins

Abstract

We report here on protein-antibody conjugates (PACs) that are used for antibody-directed delivery of protein therapeutics to specific cells. PACs have the potential to judiciously combine the merits of two prolific therapeutic approaches-biologics and antibody-drug conjugates. We utilize spherical polymer brushes to construct PACs using the combination of two simple and efficient functionally orthogonal click chemistries. In addition to the synthesis and characterization of these nanoparticles, we demonstrate that PACs are indeed capable of specifically targeting cells based on the presence of target antigen on the cell surface to deliver proteins. The potentially broad adaptability of PACs opens up new opportunities for targeted biologics in therapeutics and sensing.

Keywords: antibody-targeted delivery; click chemistry; drug delivery; protein encapsulation; protein-antibody conjugates.

Figure 1.

Protein-Antibody Conjugates - combine the advantages of protein therapeutics and antibody-drug conjugates while addressing their limitations

Figure 2.

Characterization of the particles. a) DLS measurement for different particles. b) FT-IR for functional group transformation. c) TEM image of SiO₂@PHEMA-g-PEG. d) TGA curve of different particles.

Figure 3.

Antibody conjugation and protein encapsulation. a) flow cytometry of the polymer brushes surface staining the targeted CD4+ mT-ALL cells at 4 °C, b) quantified cell surface staining from flow cytometry (PB indicates polymer brush. PB^Ab indicates antibody conjugated polymer brush), c) SDS-PAGE gel for GFP-BA encapsulation with varying polymer brush to GFP-BA ratios, d) Protease stability of GFP under trypsin digestion (PACs are Protein-antibody conjugates, cargo protein: GFP)

Figure 4.

Targeted delivery of GFP to CD4+ mT-ALL cells. a) Flow cytometry for GFP delivery. b) Quantified cytometry data. Concentration of GFP is 4 μg/mL, unless stated otherwise c) Imaging flow cytometry images (GFP-BA concentrations: 2 μg/mL). (PB indicates polymer brush. PB^Ab indicates antibody conjugated polymer brush.)

Figure 5.

Targeted delivery of RNase A. a) Traceless release of RNase A inside cells through a self-immolation process under a reduction condition. b) SDS-PAGE gel for RNase A-BA encapsulation inside polymer brushes with different protein to polymer brush ratios after 24 h reaction. c) RNase A release kinetics under reduction condition. d) activity assay of RNase A. e) cytotoxicity study toward CD4+ mT-ALL cells.

Scheme 1.

Construction of PACs with polymer brush as the vehicle for interior protein encapsulation and surface antibody conjugation.