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Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library

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Abstract

Epidermal growth factor-like domain 7 (EGFL7) has been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. The advent of antibody display technology (phage, bacteria, and yeast) led to an enormous revival in the use of antibodies as diagnostic and therapeutic tools for fighting cancer. However, problems with protein folding, posttranslational modification, and codon usage still limit the number of improved antibodies that can be obtained. We describe here the isolation of an EGFL7-specific antibody from a mammalian cell-based full-length antibody display library generated from peripheral blood mononuclear cells of patients with hepatocellular carcinoma. Using a novel vector, contained glycosylphosphatidylinositol anchor and restriction enzyme sites NheI and ClaI, antibody libraries are displayed as whole IgG molecules on the cell surface and screened for specific antigen binding by a combination of magnetic beads and measured by cell ELISA. Anti-EGFL7 antibody was successfully isolated from the library. The mammalian cell-based full-length antibody display library is a great potential application for rapid identification and cloning of human mAbs of targeting hepatocellular carcinoma.

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References

  1. Chowdhury PS, Pastan I (1999) Improving antibody affinity by mimicking somatic hypermutation in vitro. Nat Biotechnol 17:568–572

    Article  PubMed  CAS  Google Scholar 

  2. Ho M, Kreitman RJ, Onda M, Pastan I (2005) In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin. J Biol Chem 280:607–617

    Article  PubMed  CAS  Google Scholar 

  3. Francisco JA, Georgiou G (1994) The expression of recombinant proteins on the external surface of Escherichia coli. Ann N Y Acad Sci 745:372–382

    Article  PubMed  CAS  Google Scholar 

  4. Georgiou G, Stathopoulos C, Daugherty PS, Nayak AR, Iverson BL, Curtiss R 3rd (1997) Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nat Biotechnol 15:29–34

    Article  PubMed  CAS  Google Scholar 

  5. Boder ET, Midelfort KS, Wittrup KD (2000) Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc Natl Acad Sci USA 97:10701–10705

    Article  PubMed  CAS  Google Scholar 

  6. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JM, Graff C, Wiley HS, Wittrup KD (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21:163–170

    Article  PubMed  CAS  Google Scholar 

  7. Leong MK, Chen C, Shar KC, Shiuan D (2007) Selection and characterization of lipase abzyme from phage displayed antibody libraries. Biochem Biophys Res Commun 361:567–573

    Article  PubMed  CAS  Google Scholar 

  8. Aires DSF, Corte-Real S, Goncalves J (2008) Recombinant antibodies as therapeutic agents: pathways for modeling new biodrugs. Biodrugs 22:301–314

    Article  Google Scholar 

  9. Urban JH, Schneider RM, Compte M, Finger C, Cichutek K, Alvarez-Vallina L, Buchholz CJ (2005) Selection of functional human antibodies from retroviral display libraries. Nucleic Acids Res 33:e35

    Article  PubMed  Google Scholar 

  10. Ho M, Nagata S, Pastan I (2006) Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. Proc Natl Acad Sci USA 103:9637–9642

    Article  PubMed  CAS  Google Scholar 

  11. Akamatsu Y, Pakabunto K, Xu Z, Zhang Y, Tsurushita N (2007) Whole IgG surface display on mammalian cells: application to isolation of neutralizing chicken monoclonal anti-IL-12 antibodies. J Immunol Methods 327:40–52

    Article  PubMed  CAS  Google Scholar 

  12. Zhou C, Jacobsen FW, Cai L, Chen Q, Shen WD (2010) Development of a novel mammalian cell surface antibody display platform. MABS 2:508–518

    Article  PubMed  Google Scholar 

  13. Parker LH, Schmidt M, Jin SW, Gray AM, Beis D (2004) The endothelial-cell-derived secreted factor Egfl7 regulates vascular tube formation. Nature 428:754–758

    Article  PubMed  CAS  Google Scholar 

  14. Schmidt M, Paes K, De Mazière A, Smyczek T (2007) EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution. Development 134:2913–2923

    Article  PubMed  CAS  Google Scholar 

  15. Wu F, Yang LY, Li YF, Ou DP, Chen DP, Fan C (2009) Novel role for epidermal growth factor-like domain 7 in metastasis of human hepatocellular carcinoma. Hepatology 50:1839–1850

    Article  PubMed  CAS  Google Scholar 

  16. Huang SF, Liu DB, Zeng JM, Xiao Q, Luo M, Zhang WP, Tao K, Wen JP, Huang ZG, Feng WL (2009) Cloning, expression, purification and functional characterization of the oligomerization domain of Bcr-Abl oncoprotein fused to the cytoplasmic transduction peptide. Protein Expr Purif 64:167–178

    Article  PubMed  CAS  Google Scholar 

  17. Farazi PA, DePinho RA (2006) Hepatocellular carcinoma pathogenesis: from genes to environment. Nat Rev Cancer 6:674–687

    Article  PubMed  CAS  Google Scholar 

  18. Hoogenboom HR (2005) Selecting and screening recombinant antibody libraries. Nat Biotechnol 23:1105–1116

    Article  PubMed  CAS  Google Scholar 

  19. Borrebaeck CA, Wingren C (2011) Recombinant antibodies for the generation of antibody arrays. Methods Mol Biol 785:247–262

    Article  PubMed  CAS  Google Scholar 

  20. Fitting J, Killian D, Junghanss C, Willenbrock S, Murua Escobar H, Lange S, Nolte I, Barth S, Tur MK (2011) Generation of recombinant antibody fragments that target canine dendritic cells by phage display technology. Vet Comp Oncol 9:183–195

    Article  PubMed  CAS  Google Scholar 

  21. Mazor Y, Van Blarcom T, Iverson BL, Georgiou G (2008) E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display. Nat Protoc 3:1766–1777

    Article  PubMed  Google Scholar 

  22. Zhou Y, Chen ZR, Li CZ, He W, Liu S, Jiang S, Ma WL (2010) A novel strategy for rapid construction of libraries of full-length antibodies highly expressed on mammalian cell surfaces. Acta Biochim Biophys Sin 42:575–584

    Article  PubMed  CAS  Google Scholar 

  23. Rothe A, Hosse RJ, Power BE (2006) Ribosome display for improved biotherapeutic molecules. Expert Opin Biol Ther 6:177–187

    Article  PubMed  CAS  Google Scholar 

  24. Gold L (2001) mRNA display: diversity matters during in vitro selection. Proc Natl Acad Sci USA 98:4825–4826

    Article  PubMed  CAS  Google Scholar 

  25. Blaise L, Wehnert A, Steukers MP, van den Beucken T, Hoogenboom HR, Hufton SE (2004) Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene 342:211–218

    Article  PubMed  CAS  Google Scholar 

  26. Jin M, Song G, Carman CV, Kim YS (2006) Directed evolution to probe protein allostery and integrin I domains of 200,000-fold higher affinity. Proc Natl Acad Sci USA 103:5758–5763

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This study was supported by National Key Basic Research Program (973 Program) funded project (2010CB833605), People’s Republic of China. We thank prof. Guo Yajun, International Joint Cancer Institute, The Second Military Medical University, for supplying vector pcDNA3-CH and pcDNA3-CL.

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Correspondence to Guan-Cheng Li.

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Li, F., Liu, YH., Li, YW. et al. Human anti-EGFL7 recombinant full-length antibodies selected from a mammalian cell-based antibody display library. Mol Cell Biochem 365, 77–84 (2012). https://doi.org/10.1007/s11010-012-1245-7

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  • DOI: https://doi.org/10.1007/s11010-012-1245-7

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