Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

So, Yangkang; Lee, Kyung Jin; Kim, Deuk-Su; Lee, Jeong‐Hwan; Oh, Doo-Byoung; Hwang, Kyung‐A; Ko, Kinarm; Choo, Young‐Kug; Ko, Kisung

doi:10.1007/s11240-012-0249-z

Cited by 26 publications

(34 citation statements)

References 37 publications

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“…Many plant-derived recombinant pharmaceutical proteins have been produced in transgenic tobacco plant and were extracted directly from harvested leaves because this plant is well established in the expression of a non-food/feed crop, and its high biomass yields and rapid scalability for molecular biofarming (Ko et al, 2005;Lee et al, 2013;Lu et al, 2012;So et al, 2012). Tobacco has been widely adopted as a model system by several biotech companies (Accardi and Di Bonito, 2010;Twyman et al, 2003).…”

Section: Resultsmentioning

confidence: 99%

“…Another advantage of plant-system is that recombinant vaccine candidates can be expressed in edible plant organ tissues (Frutos et al, 2008;Ma et al, 2003;Matsuda and Kubota, 2010). To this day, various horticultural crops, along with tobacco and Arabidopsis thaliana (Arabidopsis), have been used in bioreactors for producing vaccines (Lu et al, 2012), antibodies (Lee et al, 2013;So et al, 2012), and various other pharmaceutical proteins (Stoger et al, 2005). In general, recombinant biopharmaceutical proteins are expressed and accumulated in soluble forms in plant biomass, thus, produced total soluble protein (TSP).…”

Section: Introductionmentioning

confidence: 99%

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Comparison of total soluble protein in various horticultural crops and evaluation of its quantification methods

Song

Kim²,

Kim

et al. 2015

Hortic. Environ. Biotechnol.

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Various horticultural crops have been considered as potential heterologous expression systems for plant-made recombinant pharmaceutical proteins. However, there is little information concerning the total soluble protein (TSP) levels of major horticultural crops. Ten major horticultural crops-Chinese cabbage, broccoli, garlic, onion, cabbage, bunching onion, cucumber, zucchini, radish, and carrot, along with tobacco and Arabidopsis, were selected to investigate the TSP levels in their individual tissues. In tobacco, SDS-PAGE assay showed that leaf tissues had stronger protein bands than stem tissues, and freshly harvested samples had slightly stronger band density than the -70°C frozen samples, suggesting that fresh leaf should be used to measure the total soluble proteins without any protein loss or degradation. Bicinchoninic acid (BCA) protein assay revealed that among various horticultural crops, garlic (41.4 mg·g -1 FW), broccoli (21.9 mg·g -1 FW), and Chinese cabbage (11.9 mg·g -1 FW) had the highest TSP levels suggesting that these horticultural crops could be good candidates for plant molecular biofarming to produce highly valuable recombinant proteins. The inner clove tissue in garlic, the flower tissue in broccoli, and the green leaf tissue in Chinese cabbage showed the strongest protein band density as compared to other tissues. The TSP of Arabidopsis tissues was quantified by SDS-PAGE, BCA, and Nano-drop methods. In general, the middle leaf tissue showed the highest TSP levels. To evaluate TSP levels of various horticultural crops, these three different methods were compared. The correlation and regression analyses between SDS-PAGE and BCA, and SDS-PAGE and Nano-drop suggested that there were significant correlations between SDS-PAGE and BCA protein assays as compared to SDS-PAGE and Nano-drop assays, indicating that BCA assay is reliable to quantify TSP levels. In conclusion, the TSP levels varied depending on the horticultural crops and their tissue types, and BCA assay could be applied to quantify the TSP.Additional key words: BCA protein assay, nano-drop, protein quantification method, SDS-PAGE, total soluble protein (TSP)Hort.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of total soluble protein in various horticultural crops and evaluation of its quantification methods

Song

Kim²,

Kim

et al. 2015

Hortic. Environ. Biotechnol.

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“…These results suggest that the ER retention signal KDEL retains GA733-Fc in the ER without secretion to CM (in the absence of the signal peptide, the default secretion pathway was not followed). Attachment of KDEL has been used to enhance production of recombinant proteins with oligomannose glycan structure in animal, plant and insect cells (Henderson et al 1996;Schouten et al 1996;Lee et al 2013;So et al 2013;Kim et al 2015;Lim et al 2015). Oligomannose can enhance an immune response because of the interaction between oligomannose of glycoproteins and the mannose receptor on macrophages and dendritic cells.…”

Section: Discussionmentioning

confidence: 99%

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

Lee

Song

et al. 2015

Entomological Research

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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.

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“…; So et al . ). KDEL is an endoplasmic reticulum (ER) retention signal that can be used to retrieve proteins from a subsequent compartment of the secretory pathway and return them to the ER (Munro & Pelham ; Pelham , ; Ellgaard et al .…”

Section: Introductionmentioning

confidence: 97%

“…In a previous study, a pFastBacDual vector was employed to carry the gene encoding the light and heavy chains of mAb CO17-1A under the control of the P10 (PP10) and polyhedrin promoters (PPH), respectively. The recombinant antibodies were processed, modified and targeted to their appropriate subcellular locations, including secretion to intracellular spaces (Ko et al 2003;Xing et al 2009;So et al 2013). KDEL is an endoplasmic reticulum (ER) retention signal that can be used to retrieve proteins from a subsequent compartment of the secretory pathway and return them to the ER (Munro & Pelham 1987;Pelham 1988Pelham , 1990Ellgaard et al 1999).…”

Section: Introductionmentioning

confidence: 99%

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

Lee

Hwang

Park

et al. 2014

Entomological Research

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The anti‐breast cancer monoclonal antibody (mAb) BR55 was expressed in the baculovirus–insect cell expression system, which is advantageous because of its high production capacity, cell culture flexibility and glycosylation capability. The baculovirus–insect cell expression system was successfully established for production of mAb BR55 and mAb BR55 fused with the KDEL (Lys–Asp–Glu–Leu) endoplasmic reticulum (ER) retention signal (mAb BR55K). The heavy chain (HC) and light chain (LC) genes of mAb BR55 were cloned under the control of the polyhedrin (PPH) and P10 promoters, respectively, in the pFastBacDual vector. The antibody gene‐expression cassettes carrying both the HC and LC genes were transferred into a bacmid in Escherichia coli (DH10Bac). The bacmid carrying the expression cassettes was transfected into Sf9 insect cells to generate baculovirus expressing mAb BR55 and BR55K. Western blot analysis confirmed the expression of mAb BR55 and BR55K in baculovirus‐infected insect cells. Cell direct enzyme linked immunosorbent assay (ELISA) showed that both mAbs from insect cell lysates or cell culture medium bound to MCF‐7 human breast cancer cells. Both mAb BR55 and BR55K were successfully purified using a Protein A affinity column. Collectively, these results suggest that the anti‐breast cancer mAb BR55 can be expressed, properly assembled and purified from the baculovirus expression system, which can serve as an alternative system for antibody production.

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Glycomodification and characterization of anti-colorectal cancer immunotherapeutic monoclonal antibodies in transgenic tobacco

Cited by 26 publications

References 37 publications

Comparison of total soluble protein in various horticultural crops and evaluation of its quantification methods

Comparison of total soluble protein in various horticultural crops and evaluation of its quantification methods

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

Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system

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