Expression of recombinant anti‐colorectal cancer large single‐chain monoclonal antibody in insect cells

Abstract

Colorectal cancer is the third most commonly diagnosed cancer in the world. Monoclonal antibody (mAb) CO17‐1A recognizes the tumor‐associated antigen GA733‐2, a cell surface glycoprotein highly expressed in colorectal carcinoma cells which is applicable for preventing and curing colorectal cancer. In this study, we tried to produce a new recombinant anti‐colorectal cancer large single chain (lsc) mAb based on mAb CO17‐1A in the baculovirus‐insect cell protein expression system. Two kinds of recombinant lsc mAbs were generated where variable light chain (VL) and heavy chain (HC) of mAb CO17‐1A were fused together by an interchain linker. The only difference between the two mAbs is based on fusion of an ER retention signal (KDEL) at its C‐terminus of HC. Polymerase chain reaction analysis verified the presence of both recombinant genes in the bacmid for generating viral expression vectors in insect cells. Western blot confirmed the expression of lsc mAbs in baculovirus‐infected insect cells. Cell enzyme linked immunosorbent assay (ELISA) showed that the mAbs from cell lysates bound to SW480 and SW620 human colorectal cancer cells. These results indicate that the baculovirus insect expression system can produce anti‐colorectal lsc mAb recognizing human colorectal cancer cells.

Expression, glycosylation and function of recombinant anti‐colorectal cancer mAb CO17‐1A in SfSWT4 insect cells

“…Thus, the baculovirus system is efficient for production of the multiple recombinant complex proteins such as monoclonal antibodies with regard to the feasible quantity and purity (Song et al 2010). Thus, this expression system may prove to be a valuable tool for the production of therapeutic glycoproteins including monoclonal antibodies (Hu 2005;Hervas-Stubbs et al 2007;Ahn et al 2008;Shao et al 2012). Thus, this expression system may prove to be a valuable tool for the production of therapeutic glycoproteins including monoclonal antibodies (Hu 2005;Hervas-Stubbs et al 2007;Ahn et al 2008;Shao et al 2012).…”

“…The baculovirus system produces large amounts of recombinant proteins with posttranslational processing in eukaryotic cells achieving proper assembly and folding of glycoproteins, which are essential for biological activities, stability and longevity (Kitts 1996;Jarvis 2003;Hu 2005;Ahn et al 2008;Geisler et al 2008). Thus, this expression system may prove to be a valuable tool for the production of therapeutic glycoproteins including monoclonal antibodies (Hu 2005;Hervas-Stubbs et al 2007;Ahn et al 2008;Shao et al 2012). The present data demonstrate that HC and LC chains of mAb CO17-1A can be expressed in insect cells and assemble to be biologically functional with insect specific glycosylation in SfSWT4 cells, which β(1,4)-galactosyltransferase, α(2,6)-sialyltransferase, N-acetylglucosaminyltransferase I, N-acetylglucosaminyltransfer-ase II, sialic acid synthase and CMP-sialic acid synthetase.…”

“…Monoclonal antibodies (mAb), which induce the destruction of cancer cell are being continuously developed specific safe, and stable biomolecules for cancer therapy, and most new antibody formats have demonstrated utility within this field (Brekke & Sandlie 2003;Shao et al 2012). The mAb CO17-1A, which recognizes the tumor associated antigen GA733 is a glycoprotein that plays an important role in the treatment of micrometastases and the recurrence of colorectal cancer.…”

In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

“…CO17‐1AK bacmid mixed with cell fectin reagent was inoculated to Spodoptera frugiperda (Sf9) cells (Invitrogen) to generate passage 1 (P 1 ) baculovirus as per Shao et al . (). The P 3 baculovirus was generated from P 2 baculovirus, which was obtained from P 1 baculovirus as described by Park et al .…”

“…Monoclonal antibodies (mAbs) have been continuously developed as biomolecules for cancer immunotherapy for decades (Brekke & Sandlie ; Shao et al . ). Currently, most anti‐cancer mAbs are stably produced in mammalian cells (Palmberger et al .…”