Selection of anti-cancer-associated gene single-chain variable fragments derived from gastric cancer patients using ribosome display

Xin,Lin; Cao,Jiaqing; Liu,Chuan; Zeng,Fei; Cheng,Hua; Hu,Xiaoyun; Zhu,Peiqian; Shao,Jianghua

doi:10.3892/mmr.2013.1502

Selection of anti-cancer-associated gene single-chain variable fragments derived from gastric cancer patients using ribosome display

Authors:
- Lin Xin
- Jiaqing Cao
- Chuan Liu
- Fei Zeng
- Hua Cheng
- Xiaoyun Hu
- Peiqian Zhu
- Jianghua Shao
View Affiliations

Affiliations: Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330006, P.R. China
Published online on: May 31, 2013 https://doi.org/10.3892/mmr.2013.1502
Pages: 631-637

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The aim of this study was to construct a human single-chain variable fragment (scFv) gene library for gastric cancer, from which human anti-cancer-associated gene (CAGE) scFvs are selected. Human lymphocytes were isolated from the peripheral blood of 10 gastric cancer patients and whole human heavy and light chain genes were cloned by reverse transcription-polymerase chain reaction (RT-PCR). VH and VL were rearranged randomly by splicing by overlap extension PCR. The ribosome complexes were enriched against the recombinant CAGE protein conjugated to magnetic beads. scFv antibodies were evaluated by western blot analysis, and affinity constants in a solution of antigen-antibody complexes were determined by enzyme-linked immunosorbent assay. An scFv library was constructed using the peripheral blood lymphocytes. The expressed scFv proteins from the ternary ribosome complexes were analyzed by western blot analysis and the affinity [equilibrium dissociation constant (KD)] of scFv for CAGE was determined to be 7.6x10-8 M. The ribosome display technique is efficient for selecting a fully human antibody fragment from a patient-derived gene pool.

View Figures

View References

1	Cho B, Lim Y, Lee DY, Park SY, Lee H, Kim WH, Yang H, Bang YJ and Jeoung DI: Identification and characterization of a novel cancer/testis antigen gene CAGE. Biochem Biophys Res Commun. 292:715–726. 2002. View Article : Google Scholar : PubMed/NCBI
2	Kim Y and Jeoung D: Role of CAGE, a novel cancer/testis antigen, in various cellular processes, including tumorigenesis, cytolytic T lymphocyte induction, and cell motility. J Microbiol Biotechnol. 18:600–610. 2008.
3	Kim Y, Park H, Park D, Lee YS, Choe J, Hahn JH, Lee H, Kim YM and Jeoung D: Cancer/testis antigen CAGE exerts negative regulation on p53 expression through HDAC2 and confers resistance to anti-cancer drugs. J Biol Chem. 285:25957–25968. 2010. View Article : Google Scholar : PubMed/NCBI
4	Por E, Byun HJ, Lee EJ, Lim JH, Jung SY, Park I, Kim YM, Jeoung DI and Lee H: The cancer/testis antigen CAGE with oncogenic potential stimulates cell proliferation by up-regulating cyclins D1 and E in an AP-1- and E2F-dependent manner. J Biol Chem. 285:14475–14485. 2010. View Article : Google Scholar : PubMed/NCBI
5	Caballero OL and Chen YT: Cancer/testis (CT) antigens: potential targets for immunotherapy. Cancer Sci. 100:2014–2021. 2009. View Article : Google Scholar : PubMed/NCBI
6	Filpula D: Antibody engineering and modification technologies. Biomol Eng. 24:201–215. 2007. View Article : Google Scholar : PubMed/NCBI
7	Dreier B and Plückthun A: Ribosome display: a technology for selecting and evolving proteins from large libraries. Methods Mol Biol. 687:283–306. 2011. View Article : Google Scholar : PubMed/NCBI
8	Rothe A, Nathanielsz A, Hosse RJ, Oberhäuser F, Strandmann EP, Engert A, Hudson PJ and Power BE: Selection of human anti-CD28 scFvs from a T-NHL related scFv library using ribosome display. J Biotechnol. 130:448–454. 2007. View Article : Google Scholar : PubMed/NCBI
9	Rothe A, Nathanielsz A, Oberhäuser F, von Pogge SE, Engert A, Hudson PJ and Power BE: Ribosome display and selection of human anti-cD22 scFvs derived from an acute lymphocytic leukemia patient. Biol Chem. 389:433–439. 2008. View Article : Google Scholar : PubMed/NCBI
10	Li F, Su P, Lin C, Li H, Cheng J and Shi D: Ribosome display and selection of human anti-placental growth factor scFv derived from ovarian cancer patients. Protein Pept Lett. 17:585–590. 2010. View Article : Google Scholar : PubMed/NCBI
11	He M, Cooley N, Jackson A and Taussig MJ: Production of human single-chain antibodies by ribosome display. Methods Mol Biol. 248:177–189. 2004.PubMed/NCBI
12	He M and Taussig MJ: Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res. 25:5132–5134. 1997. View Article : Google Scholar : PubMed/NCBI
13	Friguet B, Chaffotte AF, Djavadi-Ohaniance L and Goldberg ME: Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 77:305–319. 1985. View Article : Google Scholar : PubMed/NCBI
14	Yan XH and Xu ZR: Production of human single-chain variable fragment (scFv) antibody specific for digoxin by ribosome display. Indian J Biochem Biophys. 42:350–357. 2005.PubMed/NCBI
15	Yoong J, Michael M and Leong T: Targeted therapies for gastric cancer: current status. Drugs. 71:1367–1384. 2011. View Article : Google Scholar : PubMed/NCBI
16	Andratschke M, Luebbers CW, Johannson V, Schmitt B, Mack B, Zeidler R, Lang S, Wollenberg B and Gildehaus FJ: Biodistribution of 131I-labeled anti-CK8 monoclonal antibody in HNSCC in xenotransplanted SCID mice. Anticancer Res. 31:3315–3321. 2011.PubMed/NCBI
17	Kreitman RJ and Pastan I: Antibody fusion proteins: anti-CD22 recombinant immunotoxin moxetumomab pasudotox. Clin Cancer Res. 17:6398–6405. 2011. View Article : Google Scholar : PubMed/NCBI
18	Ricart AD: Antibody-drug conjugates of calicheamicin derivative: gemtuzumab ozogamicin and inotuzumab ozogamicin. Clin Cancer Res. 17:6417–6427. 2011. View Article : Google Scholar : PubMed/NCBI
19	Bernett MJ, Karki S, Moore GL, et al: Engineering fully human monoclonal antibodies from murine variable regions. J Mol Biol. 396:1474–1490. 2010. View Article : Google Scholar : PubMed/NCBI
20	Yamashita M, Katakura Y and Shirahata S: Recent advances in the generation of human monoclonal antibody. Cytotechnology. 55:55–60. 2007. View Article : Google Scholar
21	He M and Khan F: Ribosome display: next-generation display technologies for production of antibodies in vitro. Expert Rev Proteomics. 2:421–430. 2005. View Article : Google Scholar : PubMed/NCBI
22	Zahnd C, Amstutz P and Plückthun A: Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods. 4:269–279. 2007. View Article : Google Scholar : PubMed/NCBI
23	He M and Taussig MJ: Selection of recombinant antibodies by eukaryotic ribosome display. Methods Mol Biol. 484:193–205. 2008. View Article : Google Scholar : PubMed/NCBI
24	Suri A: Cancer testis antigens - their importance in immunotherapy and in the early detection of cancer. Expert Opin Biol Ther. 6:379–389. 2006. View Article : Google Scholar : PubMed/NCBI
25	Cho B, Lee H, Jeong S, Bang YJ, Lee HJ, Hwang KS, Kim HY, Lee YS, Kang GH and Jeoung DI: Promoter hypomethylation of a novel cancer/testis antigen gene CAGE is correlated with its aberrant expression and is seen in premalignant stage of gastric carcinoma. Biochem Biophys Res Commun. 307:52–63. 2003. View Article : Google Scholar : PubMed/NCBI
26	Iwata T, Fujita T, Hirao N, et al: Frequent immune responses to a cancer/testis antigen, CAGE, in patients with microsatellite instability-positive endometrial cancer. Clin Cancer Res. 11:3949–3957. 2005. View Article : Google Scholar : PubMed/NCBI
27	Accardi L and Di Bonito P: Antibodies in single-chain format against tumour-associated antigens: present and future applications. Curr Med Chem. 17:1730–1755. 2010. View Article : Google Scholar : PubMed/NCBI
28	Ullman CG, Frigotto L and Cooley RN: In vitro methods for peptide display and their applications. Brief Funct Genomics. 10:125–134. 2011. View Article : Google Scholar : PubMed/NCBI
29	Lee MS, Kwon MH, Kim KH, Shin HJ, Park S and Kim HI: Selection of scFvs specific for HBV DNA polymerase using ribosome display. J Immunol Methods. 284:147–157. 2004. View Article : Google Scholar : PubMed/NCBI