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en Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy ایمنی شناسی Immunology مقالات اصلی Original Article <div style="text-align: justify;">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.</div> Bispecific Antibody, Escherichia Coli, Immunotherapy, Natural Killer Cell (NK Cell), Newcastle Disease Virus (NDV). 50 57 http://rbmb.net/browse.php?a_code=A-10-348-1&slc_lang=en&sid=1 Mina Bahrololoumi Shapourabadi minabahrololoumi@gmail.com 100319475328460017499 100319475328460017499 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. Farzin Roohvand rfarzin@pasteur.ac.ir 100319475328460017500 100319475328460017500 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. Arash Arashkia a_arashkia@pasteur.ac.ir 100319475328460017501 100319475328460017501 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. Nasir Mohajel n_mohajel@pasteur.ac.ir 100319475328460017502 100319475328460017502 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. Shahriyar Abdoli shahriyaran@gmail.com 100319475328460017503 100319475328460017503 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. Zahra Shahosseini zsh5230@yahoo.com 100319475328460017504 100319475328460017504 No Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran. & Iran University of Medical Sciences, Tehran, Iran. Frank Momburg f.momburg@dkfz-heidelberg.de 100319475328460017505 100319475328460017505 No German Cancer Research Center (DKFZ), Heidelberg, Germany. Mostafa Jarahian m.jarahian@dkfz-heidelberg.de 100319475328460017506 100319475328460017506 No German Cancer Research Center (DKFZ), Heidelberg, Germany. Mohsen Abolhassani mabolhassani@yahoo.com 100319475328460017507 100319475328460017507 No Immunology Department, Pasteur Institute of Iran. Kayhan Azadmanesh azadmanesh@pasteur.ac.ir 100319475328460017508 100319475328460017508 Yes Department of Molecular Virology, Pasteur Institute of Iran, Tehran, Iran.