Production of an anti-TM4SF5 monoclonal antibody and its application in the detection of TM4SF5 as a possible marker of a poor prognosis in colorectal cancer

Park,Byoung Kwon; Park,Jae-Young; Kim,Te Ha; Kim,Dongbum; Wu,Guang; Gautam,Avishekh; Maharjan,Sony; Lee,Su In; Lee,Younghee; Kwon,Hyung-Joo; Choi,Kyung Chan

doi:10.3892/ijo.2018.4385

Production of an anti-TM4SF5 monoclonal antibody and its application in the detection of TM4SF5 as a possible marker of a poor prognosis in colorectal cancer

Authors:
- Byoung Kwon Park
- Jae-Young Park
- Te Ha Kim
- Dongbum Kim
- Guang Wu
- Avishekh Gautam
- Sony Maharjan
- Su In Lee
- Younghee Lee
- Hyung-Joo Kwon
- Kyung Chan Choi
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Affiliations: Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea, Department of Pathology, Hallym University Sacred Heart Hospital, Chuncheon 24253, Republic of Korea, Department of Microbiology, College of Medicine, Hallym University, Chuncheon 24252, Republic of Korea, Department of Biochemistry, College of Natural Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea
Published online on: April 26, 2018 https://doi.org/10.3892/ijo.2018.4385
Pages: 275-285

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Abstract

The cell surface transmembrane 4 superfamily member 5 protein (TM4SF5) has been implicated in various human cancers. Immunization with a peptide vaccine targeting human TM4SF5 has been shown to exert prophylactic and therapeutic effects against the development of hepatocellular carcinoma and colon cancer in mouse models. In this study, we developed a novel monoclonal antibody (mEC2‑CF) targeting a cyclic epitope of TM4SF5 and evaluated its reactivity to TM4SF5 in colorectal cancer (CRC) cells and cancer tissues. The isotype of mEC2‑CF was IgG2a and the antibody specifically recognized the cyclic peptide, based on ELISA. The antibody recognized recombinant TM4SF5 overexpressed in 293F cells, irrespective of N‑glycosidase F treatment. The antibody was internalized into the cytosol after binding to the surface of TM4SF5‑expressing CRC cells, suggesting that this antibody may be useful in therapeutics. In addition, we evaluated TM4SF5 expression in the tissues of patients with CRC patients to determine its prognostic significance. TM4SF5 expression was assessed by immunohistochemistry using mEC2‑CF and tissue microarray blocks of 204 primary CRC samples. The overall rate of TM4SF5 overexpression in the samples (immunohistochemical score >4) was 27.0% (55 of 204). The increased expression of TM4SF5 was significantly associated with a shorter survival rate (P=0.0014) and a worse disease‑free survival (P=0.0483) of patients with CRC. No association was observed between TM4SF5 expression and clinicopathological characteristics, apart from tumor depth of invasion (P=0.027). These results suggest that our novel antibody can be used to detect endogenous and recombinant TM4SF5, and that TM4SF5 may be a possible marker for the poor prognosis of patients with CRC.

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1	World Health Organization: GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012. http://globocan.iarc.fr/Pages/fact_sheets_cancer.aspx. Retrieved April, 2015.
2	Wilkinson N and Scott-Conner CE: Surgical therapy for colorectal adenocarcinoma. Gastroenterol Clin North Am. 37:253–267. 2008. View Article : Google Scholar : PubMed/NCBI
3	Goodwin RA and Asmis TR: Overview of systemic therapy for colorectal cancer. Clin Colon Rectal Surg. 22:251–256. 2009. View Article : Google Scholar :
4	Müller-Pillasch F, Wallrapp C, Lacher U, Friess H, Büchler M, Adler G and Gress TM: Identification of a new tumour-associated antigen TM4SF5 and its expression in human cancer. Gene. 208:25–30. 1998. View Article : Google Scholar : PubMed/NCBI
5	Lee SA, Lee SY, Cho IH, Oh MA, Kang ES, Kim YB, Seo WD, Choi S, Nam JO, Tamamori-Adachi M, et al: Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma. J Clin Invest. 118:1354–1366. 2008. View Article : Google Scholar : PubMed/NCBI
6	Lee SA, Ryu HW, Kim YM, Choi S, Lee MJ, Kwak TK, Kim HJ, Cho M, Park KH and Lee JW: Blockade of four-transmembrane L6 family member 5 (TM4SF5)-mediated tumorigenicity in hepatocytes by a synthetic chalcone derivative. Hepatology. 49:1316–1325. 2009. View Article : Google Scholar : PubMed/NCBI
7	Kwon S, Kim D, Park BK, Cho S, Kim KD, Kim YE, Park CS, Ahn HJ, Seo JN, Choi KC, et al: Prevention and therapy of hepatocellular carcinoma by vaccination with TM4SF5 epitope-CpG-DNA-liposome complex without carriers. PLoS One. 7:e331212012. View Article : Google Scholar : PubMed/NCBI
8	Kwon S, Kim D, Park BK, Wu G, Park MC, Ha YW, Kwon HJ and Lee Y: Induction of immunological memory response by vaccination with TM4SF5 epitope-CpG-DNA-liposome complex in a mouse hepatocellular carcinoma model. Oncol Rep. 29:735–740. 2013. View Article : Google Scholar
9	Kwon S, Kim YE, Kim D, Park BK, Wu G, Kim TH, Choi SH, Kim DS, Kwon HJ and Lee Y: Prophylactic effect of a peptide vaccine targeting TM4SF5 against colon cancer in a mouse model. Biochem Biophys Res Commun. 435:134–139. 2013. View Article : Google Scholar : PubMed/NCBI
10	Wu YB, Huang YS, Xu YP, Sun YF, Yu DL, Zhang XQ, Long X, Zhu SQ, Zhou JL and Xu JJ: A high level of TM4SF5 is associated with human esophageal cancer progression and poor patient survival. Dig Dis Sci. 58:2623–2633. 2013. View Article : Google Scholar : PubMed/NCBI
11	Wright MD, Ni J and Rudy GB: The L6 membrane proteins - a new four-transmembrane superfamily. Protein Sci. 9:1594–1600. 2000. View Article : Google Scholar : PubMed/NCBI
12	Detchokul S, Williams ED, Parker MW and Frauman AG: Tetraspanins as regulators of the tumour microenvironment: Implications for metastasis and therapeutic strategies. Br J Pharmacol. 171:5462–5490. 2014. View Article : Google Scholar
13	Yáñez-Mó M, Barreiro O, Gordon-Alonso M, Sala-Valdés M and Sánchez-Madrid F: Tetraspanin-enriched microdomains: A functional unit in cell plasma membranes. Trends Cell Biol. 19:434–446. 2009. View Article : Google Scholar : PubMed/NCBI
14	Jung O, Choi S, Jang SB, Lee SA, Lim ST, Choi YJ, Kim HJ, Kim DH, Kwak TK, Kim H, et al: Tetraspan TM4SF5-dependent direct activation of FAK and metastatic potential of hepatocarcinoma cells. J Cell Sci. 125:5960–5973. 2012. View Article : Google Scholar : PubMed/NCBI
15	Kim HJ, Kwon S, Nam SH, Jung JW, Kang M, Ryu J, Kim JE, Cheong JG, Cho CY, Kim S, et al: Dynamic and coordinated single-molecular interactions at TM4SF5-enriched microdomains guide invasive behaviors in 2- and 3-dimensional environments. FASEB J. 31:1461–1481. 2017. View Article : Google Scholar : PubMed/NCBI
16	Lee SA, Kim YM, Kwak TK, Kim HJ, Kim S, Ko W, Kim SH, Park KH, Kim HJ, Cho M, et al: The extracellular loop 2 of TM4SF5 inhibits integrin alpha2 on hepatocytes under collagen type I environment. Carcinogenesis. 30:1872–1879. 2009. View Article : Google Scholar : PubMed/NCBI
17	Kwon S, Choi KC, Kim YE, Ha YW, Kim D, Park BK, Wu G, Kim DS, Lee Y and Kwon HJ: Monoclonal antibody targeting of the cell surface molecule TM4SF5 inhibits the growth of hepatocellular carcinoma. Cancer Res. 74:3844–3856. 2014. View Article : Google Scholar : PubMed/NCBI
18	Kim YE, Kwon S, Wu G, Kim D, Park BK, Park JA, Choi KC, Kim DS, Kwon HJ and Lee Y: Therapeutic effect of a TM4SF5-specific monoclonal antibody against colon cancer in a mouse model. Oncotarget. 5:8402–8415. 2014. View Article : Google Scholar :
19	Wu G, Kim D, Park BK, Park S, Ha JH, Kim TH, Gautam A, Kim JN, Lee SI, Park HB, et al: Anti-metastatic effect of the TM4SF5-specific peptide vaccine and humanized monoclonal antibody on colon cancer in a mouse lung metastasis model. Oncotarget. 7:79170–79186. 2016. View Article : Google Scholar : PubMed/NCBI
20	Kim D, Kwon S, Ahn CS, Lee Y, Choi SY, Park J, Kwon HY and Kwon HJ: Adjuvant effect of liposome-encapsulated natural phosphodiester CpG-DNA. BMB Rep. 44:758–763. 2011. View Article : Google Scholar : PubMed/NCBI
21	Park BK, Choi SH, Kim YE, Park S, Lee Y, Lee KW and Kwon HJ: Monoclonal antibodies against the human respiratory syncytial virus obtained by immunization with epitope peptides and CpG-DNA-liposome complex. Monoclon Antib Immunodiagn Immunother. 34:101–109. 2015. View Article : Google Scholar : PubMed/NCBI
22	Park BK, Lee SI, Lee Y and Kwon HJ: Effect of a monoclonal antibody against human relaxin-2 on cancer cell growth inhibition. Appl Biol Chem. 59:739–746. 2016. View Article : Google Scholar
23	Yokoyama WM: Production of monoclonal antibodies. Curr Protoc Cytom 2006. Appendix 3:Appendix 3J. 2006. View Article : Google Scholar
24	Yokoyama WM, Christensen M, Santos GD and Miller D: Production of monoclonal antibodies. Curr Protoc Immunol. 74:1–25. 2006.
25	Kim D, Kwon S, Rhee JW, Kim KD, Kim YE, Park CS, Choi MJ, Suh JG, Kim DS, Lee Y, et al: Production of antibodies with peptide-CpG-DNA-liposome complex without carriers. BMC Immunol. 12:292011. View Article : Google Scholar : PubMed/NCBI
26	Kwon S, Kim YE, Park JA, Kim DS, Kwon HJ and Lee Y: Therapeutic effect of a TM4SF5-specific peptide vaccine against colon cancer in a mouse model. BMB Rep. 47:215–220. 2014. View Article : Google Scholar :