Abstract
Site-specific modification of antibodies has become a critical aspect in the development of next-generation immunoconjugates meeting criteria of clinically acceptable homogeneity, reproducibility, efficacy, ease of manufacturability, and cost-effectiveness. Using CRISPR/Cas9 genomic editing, we developed a simple and novel approach to produce site-specifically modified antibodies. A sortase tag was genetically incorporated into the C-terminal end of the third immunoglobulin heavy chain constant region (CH3) within a hybridoma cell line to manufacture antibodies capable of site-specific conjugation. This enabled an effective enzymatic site-controlled conjugation of fluorescent and radioactive cargoes to a genetically tagged mAb without impairment of antigen binding activity. After injection in mice, these immunoconjugates showed almost doubled specific targeting in the lung vs. chemically conjugated maternal mAb, and concomitant reduction in uptake in the liver and spleen. The approach outlined in this work provides a facile method for the development of more homogeneous, reproducible, effective, and scalable antibody conjugates for use as therapeutic and diagnostic tools.
Highlights
Monoclonal antibody-based therapeutics have emerged as very effective tools for treatment of a wide-spectrum of diseases and are already mainstays in the treatment of cancer and autoimmune diseases
In order to genetically incorporate Sortase and Flag tags at the C-terminal end of antibody, two sgRNAs were selected in the region near the 3′ end of CH3 heavy chain stop codon. sgRNA 1 demonstrated greater efficacy and was the primary sgRNA used for generaetion of clones
Hybridoma cells were co-transfected with plasmid expressing both sgRNA and Cas[9], and linearized homology-directed repair (HDR) repair plasmid
Summary
Monoclonal antibody (mAb)-based therapeutics have emerged as very effective tools for treatment of a wide-spectrum of diseases and are already mainstays in the treatment of cancer and autoimmune diseases. Despite the huge potential of antibody-drug conjugates, there have been many challenges in the field which have limited its growth and hindered regulatory success. We have developed a strategy to bypass these challenges in mAb modification via site-specific modification of the immunoglobulin gene within the hybridoma itself. Using CRISPR/Cas[9] genomic editing, we have incorporated Sortase (LPETGG) and Flag (DYKDDDDK) tags at the C-terminal end of the CH3 heavy chain region in a mouse monoclonal antibody providing conjugation of cargoes without loss of antibody affinity, while ensuring optimal orientation of the antibody and minimizing steric hindrance or altered conformation of the www.nature.com/scientificreports/. The strategy presented here reduces the time and cost it takes to make genetically encoded modifications to antibodies This is a key development, as conjugatable antibodies have numerous applications in therapeutics and diagnostics
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