Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy

Abstract

Background: Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. Methods: A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. Results: Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. Conclusions: Results showed efficient production of the bsAb in E. coli for future large-scale purification.

Immunovirotherapy: The role of antibody based therapeutics combination with oncolytic viruses

“…As an examples for this type of combination, Bahrololoumi et al constructed a bsAb (antiHN scFv/antiCD16 scFv) and a trispecific antibody(antiHN scFv/IL-15/antiCD16 scFv) to bind to the haemagglutinin neuraminidase (HN), a viral protein that is expressed on the surface of the NDV infected tumor cell, and the CD16 activating receptor on the surface of the NK cells for redirecting NK cells toward the tumor cells ( 201 , 202 ). NDV-Ulster is a non-lytic strain of NDV that was used to inflame the tumoral microenvironment in this study, and also in NDV-based autologous tumor cell vaccines for stimulating the immune response in the patient’s body ( 93 , 178 , 203 , 204 ).…”