Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies

Abstract

The baculovirus vector expression system is considered a useful insect cell‐based platform for large‐scale production of recombinant biopharmaceutical glycoproteins. The GA733 antigenic protein is a 40 kDa cell surface glycoprotein that is overexpressed in human colorectal carcinoma. Three GA733‐expressing baculovirus vectors were constructed: (i) GA733 fused to the Fc fragment of human immunoglobulin G (GA733‐Fc); (ii) GA733‐Fc tagged with the endoplasmic reticulum (ER) retention signal KDEL (GA733‐FcK); and (iii) GA733‐FcK without the signal peptide [GA733‐FcK (w/o SP)]. These three recombinant proteins were expressed in Fall Armyworm (Spodoptera frugiperda) Sf9 insect cells. GA733‐Fc purified from insect culture medium (CM) and cell lysate (CL) (GA733I‐FcCM and GA733I‐FcCL, respectively), and GA733‐FcK and GA733‐FcK (w/o SP) purified from CL [GA733I‐FcCL and GA733I‐FcKCL (w/o SP), respectively] were injected intraperitoneally into BALB/c mice [three times at 1 μg, with or without an adjuvant (aluminum hydroxide)]. Surface plasmon resonance (SPR) analysis (involving a chip with the immobilized GA733 antigen) showed that, in general, the serum samples from mice immunized with the adjuvant yielded stronger SPR signals than did those from mice immunized without an adjuvant. The serum samples from mice after the second injection yielded stronger signals than did those from mice before the second injection. The group immunized with GA733I‐FcKCL (glycosylated with an oligomannose) showed the strongest SPR signals. The SPR signals from the GA733I‐FcKCL group were stronger than or similar to those from mice immunized with mammalian‐cell‐expressed GA733‐Fc (GA733M‐Fc). Thus, the baculovirus vector expression system can be used to produce immunogenic cancer glycoprotein.

Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

“…, ; Lee et al . ). BEVS has many advantages, including scalability using high‐density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post‐translational modifications (Deparis et al .…”

“…, ; Lee et al . ). Accurate viral stock titration is crucial for determining the multiplicity of infection (MOI) value, which represents the total number of virions added per cell during infection (Neutra et al .…”

“…The baculovirus expression vector system (BEVS) has been proven useful for the production of recombinant proteins in eukaryotic insect cells (Deparis et al 2003;Song et al 2010;Park et al 2011Park et al , 2014Park et al , 2015Lee & Ko 2014;Kim et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005).…”

“…BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005). In the present study, we used the BEVS technology to express diverse recombinant proteins, including anti-colorectal cancer monoclonal antibody CO17-1A (Song et al 2010;Park et al 2011Park et al , 2014Lee & Ko 2014 and anti-cancer vaccine candidate GA733-Fc (Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016).…”

“…Optimization of infection conditions influences the expression of recombinant proteins, such as the monoclonal antibody CO17-1A (Park et al 2011), which recognizes the colorectal antigen GA733-2 (gastrointestinal tumorassociated antigen 2) (Strassburg et al 1992;Zaloudik et al 2002;Lu et al 2012;Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). Accurate viral stock titration is crucial for determining the multiplicity of infection (MOI) value, which represents the total number of virions added per cell during infection (Neutra et al 1992;Mulvania et al 2004;Janakiraman et al 2006) and ultimately determines recombinant protein expression (Radford et al, 1997).…”