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Abstract
: 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. 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. 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. Results showed efficient production of the bsAb in E. coli for future large-scale purification.
Highlights
Cancer with approximately 9.6 million (1 out 6) annual deaths, was one of the main causes of death worldwide in 2018 [1]
Construction of the bispecific antibodies (bsAb) (Anti hemagglutinin neuraminidase (HN)- Anti CD16)encoding vector for expression in E. coli and restriction analyses The primary bsAb in pcDNA3.1(-) vector coding for a tetravalent bispecific antibody for secretory expression in mammalian cells was used as the template for developing bsAb coding sequence in bacterial vector
Since for developing a construct without Fc, it was necessary to separate the segment containing-CD16 scFv, the main bsAb-coding pcDNA3.1 plasmid was digested by HindIII and XbaI, and the 833 bp segment was extracted from the gel and cloned in a new pcDNA3.1 plasmid backbone
Summary
Cancer with approximately 9.6 million (1 out 6) annual deaths, was one of the main causes of death worldwide in 2018 [1]. Natural killer (NK) cells are non-B, non-T lymphocytes, and as a part of the innate immune system, they can recognize and kill virus-infected or transformed cells without prior sensitization [2]. 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. 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. Conclusions: Results showed efficient production of the bsAb in E. coli for future large-scale purification
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