Design of an Expression System for Rapid Production of Tri-Functional Antibody Substitution of Hybrid Hybridoma Technology

  • Mohammad Reza Dehghani Mail Department of Molecular Medicine, School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
  • Reza Ahangari Cohan Department of Pilot Nanobiotechnology, New Technologies Research Group, Pasteur Institute of Iran, Tehran, Iran.
  • Kobra Omidfar Biosensor Research Center, Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences, Tehran, Iran.
  • Mohammad Hossein Ghahremani Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran. AND Department of Pharmacology-Toxicology, School of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.
Insilico, Tri-functional antibody, Cancer, Immunotherapy, Biotechnology


Tri-functional antibodies, as an effective novel tumor targeting agents, are composed of anti-CD3 rat IgG2b and an anti-tumor antigen antibody. We have intended to develop a novel drug system to induce the apoptosis of HER2-expressing tumor cells, activate and engage cytotoxic T lymphocytes, natural killer cells, and macrophages. In addition, the drug can  inhibit programmed death ligand 1 (PDL-1)-expressing tumor cells. We have designed a mammalian vector system suitable to express the trifunctional antibody composed of antiHER2×human-CD80: human-IgG1Fc antibody. This antibody contains four chains of anti-HER2/CL, anti-HER2/CH1-3, B7.1/CL, and B7.1/CH1-3 within the human IgG1 framework and so the vector system will simultaneously express the four chains. The amino acid/nucleotide sequences datasets were retrieved from the GenBank, UniProtKB and PDB databases. The heavy and light chains variable domain framework regions and complementarity determining regions of scA21 antibody were determined by IMGT/V-QUEST and Paratome software. The amino acid sequences of tri-functional antibody chains manually were assembled and converted to DNA sequences by sequence manipulation suite software. The adapting codon usage of these DNA sequences was performed by JCAT software. Finally, the secondary structures of obtained RNAs from the DNAs were individually analyzed by RNAfold program. The Prodigy equilibrium dissociation constant, a ratio of koff/kon, between the antibody and its antigen for hantiHER2VHCH-HER2, hantiHER2VLCL-HER2, CD80CH-CD28, and CD80CL-CD28 were equal to 3.60E-10, 3.10E-9, 1.10E-8 and 2.70E-10; respectively. These findings were confirmed the composition and nano-molar affinity of the respective constructs. A single specific no-cloning expression vector, pHuchiTriomAb, was designed in silico with a desirable length of 8.292 Kbps and bidirectional expression potential for the four chimeric antibody chains. This construct was designed, and gene codon usage adapted to be expressed within CHO cells and secreted a trifunctional antibody into the cell culture medium by interleukin 21 signal peptide.This robust expression system for rapid production of tri-functional antibody has been designed to substitute hybrid hybridoma technology (quadroma and trioma) with the facilitated purification of the trifunctional antibody from the antibody variants.

Author Biography

Mohammad Reza Dehghani, Department of Molecular Medicine, School of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran.
Molecular Medicine Department


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How to Cite
Dehghani MR, Ahangari Cohan R, Omidfar K, Ghahremani MH. Design of an Expression System for Rapid Production of Tri-Functional Antibody Substitution of Hybrid Hybridoma Technology. Acta Med Iran. 56(3):140-150.