Site Selective Antibody-Oligonucleotide Conjugation via Microbial Transglutaminase

Abstract

Nucleic Acid Therapeutics (NATs), including siRNAs and AntiSense Oligonucleotides (ASOs), have great potential to drug the undruggable genome. Targeting siRNAs and ASOs to specific cell types of interest has driven dramatic improvement in efficacy and reduction in toxicity. Indeed, conjugation of tris-GalNAc to siRNAs and ASOs has shown clinical efficacy in targeting diseases driven by liver hepatocytes. However, targeting non-hepatic diseases with oligonucleotide therapeutics has remained problematic for several reasons, including targeting specific cell types and endosomal escape. Monoclonal antibody (mAb) targeting of siRNAs and ASOs has the potential to deliver these drugs to a variety of specific cell and tissue types. However, most conjugation strategies rely on random chemical conjugation through lysine or cysteine residues resulting in conjugate heterogeneity and a distribution of Drug:Antibody Ratios (DAR). To produce homogeneous DAR-2 conjugates with two siRNAs per mAb, we developed a novel two-step conjugation procedure involving microbial transglutaminase (MTGase) tagging of the antibody C-terminus with an azide-functionalized linker peptide that can be subsequently conjugated to dibenzylcyclooctyne (DBCO) bearing oligonucleotides through azide-alkyne cycloaddition. Antibody-siRNA (and ASO) conjugates (ARCs) produced using this strategy are soluble, chemically defined targeted oligonucleotide therapeutics that have the potential to greatly increase the number of targetable cell types.

Keywords: antibody-siRNA conjugate (ARC); antisense oligonucleotides; copper-less click; microbial transglutaminase; monoclonal antibodies; oligonucleotide therapeutics; siRNA.

Figure 1

MTGase site specifically labels engineered anti-CD33 mAb. (a) Structure of mAb-KF peptide conjugate. Conjugated lysine is highlighted in blue and fluorescein is highlighted in green; (b) UV protein stained SDS-PAGE gel of unconjugated mAb and mAb-KF. Molecular weight markers are in kilodaltons (kDa). HC—heavy chain, LC—light chain, HC-KF—heavy-chain-KF conjugate, MTGase—microbial transglutaminase; (c) FITC epifluorescence image of the same SDS-PAGE gel. KF—free KF peptide; (d) Flow cytometry of mAb-KF stained THP1 (CD33-positive) and Jurkat (CD33-negative) cells. Following spin column cleanup, mAb-KF conjugate was directly diluted in stain buffer at the indicated ratios: blue—1:10,000, green—1:1,000, orange—1:100. Unstained cells are represented in gray. All plots are represented as percent maximum cell count.

Figure 2

Conjugation and purification of mAb-KN₃ conjugate. (a) Structure of mAb-KN₃ peptide conjugate. Conjugated lysine is highlighted in blue and conjugatable azidolysine is highlighted in red; (b) UV protein stained SDS-PAGE gel of unconjugated mAb and mAb-KN₃. Molecular weight markers are in kilodaltons. HC—heavy chain, LC—light chain, HC-KN₃—heavy-chain-KN₃ conjugate, MTGase—microbial transglutaminase; (c) SEC analysis of the crude mAb-KN₃ conjugation reaction. mAb-KN₃ – soluble mAb-KN₃ conjugate, MTGase—microbial transglutaminase enzyme, Peptide—unconjugated KN₃ peptide. (d) SEC purification of mAb-KN₃ conjugate. Gray boxes indicate the elution volumes of collected fractions (6–9).

Figure 3

Conjugation and purification of ARC. (a) Structure of mAb-KN₃-DBCO-TEG-siRNA conjugate. Conjugated lysine is highlighted in blue. Conjugated triazole (azidolysine origin) is highlighted in red. Conjugated DBCO moiety is highlighted in green; (b) SEC analysis of the crude ARC conjugation reaction. UV absorbance at 260 nm is traced in red and absorbance at 280 nm is traced in blue; (c) SEC purification of ARC. Gray boxes indicate the elution volumes of collected fractions (5–7); (d) UV protein stained SDS-PAGE gel of unconjugated mAb, mAb-KN₃ and ARC. Molecular weight markers are in kilodaltons. HC—heavy chain, LC—light chain, HC-KN₃—heavy-chain-KN₃ conjugate, HC-KN₃-siRNA–heavy-chain-KN₃-siRNA conjugate.

Figure 4

ARC Binding to THP1 Cells. Flow cytometry of mAb and ARC stained THP1 (CD33-positive) cells. Following SEC purification, mAb/ARC were applied to cells at 25 nM in FACS buffer. Secondary FITC labeled antibody was diluted 1:200 in FACS buffer. Unstained cells are represented in gray. Secondary FITC labeled antibody only is a solid black line. Anti-CD33 mAb is represented by a blue line. Anti-CD33 ARC is represented by a red line. All plots are represented as percent maximum cell count.