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研究生:楊慧敏
研究生(外文):Hui-Ming Yang
論文名稱:藉由噬菌體展現基因庫去篩選和識別MUC18和EB病毒核蛋白一型(EBNA-1)的單股變異區域抗體
論文名稱(外文):Selection And Characterization of Human Single-Chain Fv Antibodies Against MUC18 and Epstein-Barr VirusNuclear Antigen-1 (EBNA-1) from a Phage Display Library
指導教授:林榮寵
學位類別:碩士
校院名稱:慈濟大學
系所名稱:微免暨分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:64
中文關鍵詞:互補決定區域架構區EB病毒核蛋白抗原一型細胞表面的黏附分子MUC18人類單股變異區域抗體的噬菌體基因庫
外文關鍵詞:CDRFRhuman single-chain Fv (scFv) phage librarycell surface adhesion molecules MUC18Epstein-Barr virus nuclear antigen 1
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許多具有先天人類單股變異區域抗體的噬菌體基因庫被用來尋找會識別腫瘤相關抗原的人類單株抗體,此方法是利用在大腸桿菌E. coli中,被純化合成的蛋白質做為抗原去篩選。細胞表面的黏附分子MUC18會大量表現在一些不同種類的人類腫瘤細胞上;EB病毒核蛋白抗原一型-EBNA-1會出現在所有EB病毒相關的腫瘤;因此,我們挑選MUC18和EBNA-1做為實驗的抗原蛋白質。經過五到六次的篩選、擴增、援救(rescue)後,利用酵素免疫分析方法(ELISA)去測試噬菌體的專一性;在82株辨認MUC18的單株抗體中,74株表現高專一性地結合MUC18(90 %);在辨識EBNA-1的30株單株抗體中,有14株表現高專一性(47 %);這些單株抗體篩選的特異性標準是根據酵素免疫分析方法的讀值A405大於0.5。在西方墨點法(Western blot)的分析中,辨識MUC18的單股變異區域抗體會辨認表現在許多腫瘤細胞上的MUC18,這些腫瘤細胞包含HeLa(人類子宮頸癌細胞)、鼻咽癌細胞、黑色素瘤和乳癌細胞。同樣地,辨識EBNA-1的單股變異區域抗體只會辨認被EB病毒感染的陽性細胞,但西方墨點法辨識的EBNA-1區域卻非常弱;理由可能是因為EBNA-1的單股變異區抗體只經過五次的篩選,而MUC18的單股變異區抗體卻經過六次的篩選。藉由BstNI限制酶切割的片段圖(BstNI fingerprinting)和核苷酸序列分析知道辨識MUC18的單株抗體群呈現單一辨認MUC18的單股變異區域,而辨識EBNA-1的單株抗體群則呈現七種會辨認EBNA-1的單股變異區域。由DNA序列依序推論出的蛋白質序列,已確認抗體結合抗原的架構區( fragment region )和互補決定區域( complementarity determining region )。從DNA序列推論出會辨認MUC18的單股變異區域的蛋白質序列,呈現單一的輕鏈和重鏈的變異區域組合,這表示絕大部分分離出的單株抗體利用相同或極為相近的變異區域去辨識MUC18,這也反映出分離出的單株抗體具有高專一性,並且全部的單株抗體只會辨認單一的抗原區域。在所挑選出會辨識EBNA-1的單株抗體中,雖有不同的BstNI限制酶切割的片段圖,以及不同的核苷酸序列和不同的胺基酸序列,但只有11株單株抗體的輕鏈某些變異區域被發現,這表示這些會辨認EBNA-1的單株抗體只有使用輕鏈的變異區域去結合抗原。我們嘗試將會辨認MUC18專一性的抗體由噬菌體分離出來,此游離的抗體在酵素免疫分析中,呈現陽性反應,但在西方墨點法中,我們無法使用游離的抗體去辨認MUC18。以上這些結果可提供一個新的前導,做為未來進一步地在疾病診斷上的發展和高專一性地辨識腫瘤的免疫治療法。這些被挑選出的高專一性變異區域抗體可用來結合毒殺性高的物質,如:radionuclides、毒性物質、化學治療物質,去毒殺表現MUC18和EBNA-1的腫瘤細胞。
A large naïve human single-chain Fv (scFv) phage library was used to search for human monoclonal antibodies against tumor-associated antigens by panning with the purified recombinant proteins expressed in E. coli. The cell surface adhesion molecules MUC18, which is overexpressed in several types of human
tumors and an Epstein-Barr virus (EBV) nuclear antigen ENBA-1, which is expressed in all EBV-associated tumors, were selected for this study. After five to six rounds of selection-amplification-rescue, 74 of 82 monoclones of MUC18 (90 %) and 14 of 30 (47 %) of EBNA-1 analyzed bound selectively to MUC18 and EBNA-1 in a phage ELISA. The criteria used to assess the specificity of each monoclone was robust binding to the antigen with ELISA reading at A405 >
0.5. In Western blot analyses, the MUC18 scFv fragment antibodies recognized the MUC18 molecules expressed in a variety of tumor cells including HeLa, NPC, melanoma, and breast cancer cell lines. Similarly, EBNA-1 scFv fragment
antibodies detected the EBNA-1 molecules only in EBV-positive cells, but the
intensity of bands was very weak. The reason for weak detection could be
attributed to the low titers of the scFv antibodies, which were obtained after five rounds of selection, in contrast to MUC18 that had gone through six rounds of selection. Diversity analyses of these antigen-selective individual clones by BstNI fingerprinting and nucleotide sequencing revealed a single distinct scFv fragment of MUC18 clones and 7 distinct scFv fragments of EBNA-1 clones. Protein sequences were deduced from the DNA sequences and fragment
regions (FR) and complementarity determining regions (CDR) were also
identified. The deduced amino acid sequences from the nucleotide sequences of the MUC18 clones analyzed also revealed a single pattern of both light and heavy chains of V-gene. It appears that most of the MUC18 scFv clones isolated
use identical or very closely related V-gene sequences. This may reflect that the isolated clones were highly specific and all recognized a single epitope. Despite the variations in BstNI fingerprintings, nucleotide sequences, and deduced amino acid sequences of 11 EBNA-1 binding clones, only the light chain of the V-gene was detected. These results indicated that EBNA-1 binding clones use only VL sequences. The isolation of soluble scFv fragment phages specific for MUC18 was attempted. Even though we could detect by ELISA the soluble forms of scFv antibodies for MUC18, we were unable to detect the MUC18
molecules by Western blot using the soluble scFv antibodies. These results provide a lead for further development of diagnostic assays and selective tumor targeting immunotherapy. Thus, the scFv fragment phage antibodies selected from the phage display library can be used to deliver highly cytotoxic moieties such as radionuclides, toxin, or chemotherapeutic agents to the tumors expressing MUC18 and EBNA-1.
誌謝……………………………………………………………………I
Abstract………………………………………………………………1-2
中文摘要……………………………………………………………..3-4
Introduction…………………………………………………………..5-9
Materials & Methods…………………………………………….…10-15
Results……………………………………………………………...16-21
Discussion………………………………………………………….22-25
Acknowledgements…………………………………………………...26
References………………………………………………………….27-33
Legends to figures………………………………………………….34-38
Tables………………………………………………………………39-43
Figures……………………………………………………………..44-56
Appendix……………………………………………………….…..57-60
1. Shawler, D. L., R. M. Bartholomew, L. M. Smith, and R. O. Dillman. 1985. Human immune response to multiple injections of murine monoclonal IgG. J.
Immunol. 135: 1530-1535.
2. Balls, M., A. M. Goldberg, J. H. Fentem, C. L. Broadhead, R. L. Burch, M. F. Festing, J. M. Frazier, C. F. Hendriksen, M. Jennings, M. D. yan der Kamp, D. B. Morton, A. N. Rowan, C. Russell, W. M. Russell, H. Spielmann, M. L. Stephens, W. S. Stokes, D. W. Straughan, J. D. Yager, J. Zurol, and B. F. van Zutphen. 1995. The three Rs: the way forward: the report and recommendations of ECVAM Workshop 11. Altern Lab Anim. 23: 838-866.
3. Junghans, R. P., T. A. Waldmann, N. F. Landolfi, N. M. Avdalovic, W. P. Schneider, and C. Queen. 1990. Anti-Tac-H, a humanized antibody to the interleukin 2 receptor with new features for immunotherapy in malignant and immune disorders. Cancer Res. 50:1495-1502.
4. Vaughan, T. J., J. K. Osbourn, and P. R. Tempest. 1998. Human antibodies by design. Nat Biotechnol. 16: 535-539.
5. Lobuglio, A. F., R. H. Wheeler, J. Trang, A. Hayens, K. Rogers, E. B. Harvey, L. Sun, J. Ghrayeb, and M. B. Khazaeli. 1989. Mouse/human chimeric monoclonal antibody in man: kinetics and immune response. Proc. Natl. Acad. Sci. USA. 86: 4220-4224.
6. Elliott, M. J., R. N. Maini, M. Feldmann, J. R. Kalden, C. Antoni, J. S. Smolen, B. Leeb, F. C. Breedveld, J. D. Macfarlane, H. Bijl, et al. 1994. Randomised double-blind comparison of chimeric monoclonal antibody to tumour necrosis factor (cA2) versus placebo in rheumatoid arthritis. Lancet. 344: 1105-1110.
7. Jones, P. T., P. H. Dear, J. Foote, M. S. Neuberger, and G. Winter. 1986. Replacing the complementarity-determining regions in a human antibody with those from a mouse. Nature. 321: 522 -525.
8. Foote, J., and G. Winter. 1992. Antibody framework residues affecting the conformation of the hypervariable loops. J. Mol. Biol. 224: 487-499.
9. Mclaughlin, P., A. J. Grillo-Lopez, B. K. Link, R. Levy, M. S. Czuczman, M. E. Williams, M. R. Heyman, I. Bence-Bruckler, C. A. White, F. Cabanillas, V. Jain, A. D. Ho, J. Lister, K. Wey, D. Shen, and B. K. Dallarie. 1998. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J. Clin. Oncol. 16: 2825-2833.
10. Pegram, M. D., A. Lipton, D. F. Hayes, B. L. Weber, J. M. Baselga, D. Tripathy, D. Baly, S. A. Baughman, T. Twaddell, J. A. Glaspy, and D. J. Slamon. 1998. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J. Clin. Oncol. 16: 2659-2671.
11. Vincenti, F., B. Nashan, and S. Light. 1998. Daclizumab: outcome of phase III trials and mechanism of action. Transplant. Proc. 30: 2155-2158.
12. Bruggemann, M., and M. S. Neuberger. 1996. Strategies for expressing human antibody repertoires in transgenic mice. Immunol. Today. 17: 391-397.
13. Winter, G., A. D. Griffiths, R. E. Hawkins, and H. R. Hoogenboom. 1994. Making antibodies by phage display technology. Annu. Rev. Immunol. 12: 433-455.
14. Gavilondo, J. V., and J. W. Larrick. 2000. Antibody engineering at the millennium. Biotechniques. 128 -132, 134-136, 138 passim.
15. Smothers, J. F., S. Henikoff, and P. Carter. 2002. Affinity selection from biological libraries. Science. 298: 621-622.
16. Tan, W. S., G. H. Tan, K. Yusoff, and H. F. Seow. 2005. A phage-displayed cyclic peptide that interacts tightly with the immunodominant region of hepatitis B surface antigen. J. Clin. Virol. 34: 35-41.
17. Pasqualini, R., and E. Ruoslahti.1996. Organ targeting in vivo using phage display peptide libraries.
Nature. 380: 364-366.
18. Laakkonen, P., K. Porkka, J. A. Hoffman, and E. Ruoslahti. 2002. A tumor-homing peptide with a targeting specificity related to lymphatic vessels. Nat. Med. 8: 751-755.
19. Odermatt, A., A. Audige, C. Frick, B. Vogt, B. M. Frey, F. J. Frey, and L. Mazzucchelli. 2001. Identification of receptor ligands by screening phage-display peptide libraries ex vivo on microdissected kidney tubules. J. Am. Soc. Nephrol. 12: 308-316.
20. Wang, B., Y. B. Chen, O. Ayalon, J. Bender, and A. Garen. 1999. Human single-chain Fv immunoconjugates targeted to a melanoma-associated chondroitin sulfate proteoglycan mediate specific lysis of human melanoma cells by natural killer cells and complement. Proc. Natl. Acad. Sci. USA. 96: 1627-1632.
21. Melani, C., M. Figini, D. Nicosia, E. Luison, V. Ramakrishna, G. Casorati, G. Parmiani, Z. Eshhar, S. Canevari, and M. P. Colombo. 1998. Targeting of interleukin 2 to human ovarian carcinoma by fusion with a single-chain Fv of antifolate receptor antibody. Cancer Res. 58: 4146-4154.
22. Marks, J. D., H. R. Hoogenboom, T. P. Bonnert, J. McCafferty, A. D. Griffiths, and G. Winter. 1991. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222: 581-597.
23. Sui, J., Y. He, X. Jiang, S. Dubel, Z. Han, and Z. Song. 2004. A single chain antibody obtained by cell panning of antibody phage inhibits homoaggregation of human leukemia cells. Hum Antibodies. 13: 111-118.
24. Lehmann, J. M., B. Holzmann, E. W. Breitbart, P. Schmiegelow, G. Riethmüller, and J. P. Johnson.
1987. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and a protein with a molecular weight of 76,000. Cancer Res. 47: 841-845.
25. Shih, I. M., T. L. Wang, and W. H. Westra. 1996. Diagnostic and biological implications of Mel-CAM expression in mesenchymal neoplasms. Clin Cancer Res. 2: 569-575.
26. Lehmann, J. M., G. Riethmüller, and J. P. Johnson.1989. MUC-18, a marker of tumor progression in human melanoma, shows sequence similarity to the neural cell adhesion molecules of the immunoglobulin superfamily. Proc. Natl. Acad. Sci. USA. 86: 9891-9895.
27. Pourquie, O., C. Corbel, J. L. Caer, J. Rossier, and N. M. Le Douarin. 1992. BEN, a surface glycoprotein of the immunoglobulin superfamily, is expressed in a variety of developing systems. Proc. Natl. Acad. Sci. USA. 89: 5261-5265.
28. Cunningham, B. A., J. J. Hemperly, B. A. Murray, E. A. Prediger, R. Brackenbury, and G. M. Edelman. 1987. Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing. Science. 236: 799-806.
29. Fearon, E. R., K. R. Cho, J. M. Nigro, S. E. Kern, J. W. Simons, J. M. Ruppert, S. R. Hamilton,
A. C. Preisinger, G. Thomas, K. W. Kinzler, and B. Vogelstein. 1990. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science. 247: 49-56.
30. Shih, I. M. 1999. The role of CD146 (Mel-CAM) in biology and pathology. J. Pathol. 189: 4-11.
31. Bardin, N., V. Francès, G. Lesaule, N. Horschowski, F. George, and J. Sampol. 1996. Identification of the S-Endo 1 endothelial-associated antigen. Biochem. Biophys. Res. Commun. 218: 210-216.
32. Sers, C., K. Kirsch, U. Rothbacher, G. Riethmüller, and J. P. Johnson. 1993. Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains. Proc. Natl. Acad. Sci. USA. 90: 8514-8518.
33. Pickl, W. F., O. Majdic, G. F. Fischer, P. Petzelbauer, I. Fae, M. Waclavicek, J. Stockl, C. Scheinecker, T. Vidicki, H. Aschauer, J. P. Johnson, and W. Knapp. 1997. MUC18/MCAM (CD146), an activation antigen of human T lymphocytes. J. Immunol. 158: 2107-2115.
34. Johnson, J. P. 1994-1995. Identification of molecules associated with the development of metastasis in
human malignant melanoma. Invasion Metastasis. 14: 123-130.
35. Shih, I. M., M. Nesbit, M. Herlyn, and R. J. Kurman. 1998. A new Mel-CAM (CD146)-specific monoclonal antibody, MN-4, on paraffin-embedded tissue. Mod. Pathol. 11: 1098-1106.
36. Kieff, E, and A. B. Rickinson. 2001. Epstein-Barr virus and its replication. Fields Virology, Fourth Edition. DM Knipe and PM Howley (eds.) Lippincott Williams and Wilkins. Philadelphia, Pennsylvania. 2511-2573.
37. Cahir-McFarland, E. D., D. M. Davidson, S. L. Schauer, J. Duong, and E. Kieff. 2000. NF- B inhibition causes spontaneous apoptosis in Epstein-Barr virus-transformed lymphoblastoid cells.
Proc. Natl. Acad. Sci. USA. 97: 6055-6060.
38. Yates, J., N. Warren, D. Reisman, and B. Sugden. 1984. A cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells. Proc. Natl. Acad. Sci. USA. 81: 3806–3810.
39. Reisman, D., J. Yates, and B. Sugden. 1985. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol. 5: 1822–1832.
40. Chen, M. R., J. M. Middeldorp, and S. Diane Hayward. 1993. Separation of the complex DNA binding domain of EBNA-1 into DNA recognition and dimerization of subdomains of novel structure. J. Virol. 67: 4875–4885.
41. Ambinder, R. F., M. Mullen, Y. N. Chang, G. S. Hayward, and S. Diane Hayward. 1991. Functional domains of Epstein-Barr virus nuclear antigen EBNA-1. J. Virol. 65: 1466-1478.
42. Frappier, L., and M. O’Donnell. 1994. Epstein-Barr nuclear antigen 1 mediates a DNA loop within the latent replication origin of Epstein-Barr virus. Proc. Natl. Acad. Sci. USA. 88: 10875-10879.
43. Snudden, D. K., J. Hearing, P. R. Smith, F. A. Grässer, and B. E. Griffin. 1994. EBNA-1, the major nuclear antigen of Epstein-Barr virus, resembles 'RGG' RNA binding proteins. EMBO J. 13: 4840- 4847.
44. Levitskaya, J., A. Sharipo, A. Leonchiks, A. Ciechanover, and M. G. Masucci 1997. Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein- Barr virus nuclear antigen 1. Proc. Natl. Acad. Sci. USA. 94: 12616–12621.
45. Wrightham, M. N., J. P. Stewart, N. J. Janjua, S. D. V. Pepper, C. Sample, C. M. Rooney, and J. R. Arrand. 1995. Antigenic and sequence variation in the C-terminal unique domain of the Epstein-Barr virus nuclear antigen EBNA-1. Virology. 208: 521-530.
46. Levitskaya, J., M. Coram, V. Levitsky, S. Imreh, P. M. Steigerwald-Mullen, G. Klein, M. G. Kurilla, and M. G. Masucci. 1995. Inhibition of antigen processing by the internal repeat region of the Epstein -Barr virus nuclear antigen-1. Nature. 375: 685-688.
47. Brinkmann, U., L. H. Pai, D. J. Fitzgerald, M. Willingham, and I. Pastan. 1991. B3(Fv)-PE38KDEL, a single-chain immunotoxin that causes complete regression of a human carcinoma in mice. Proc. Natl. Acad. Sci. USA. 88: 8616-8620.
48. Milenic, D. E., T. Yokota, D. R. Filpula, M. A. Finkelman, S. W. Dodd, J. F. Wood, M. Whitlow, P. Snoy, J. Schlom. 1991. Construction, binding properties, metabolism, and tumor targeting of a single- chain Fv derived from the pancarcinoma monoclonal antibody CC19. Cancer Res. 51: 6363-6371.
49. Yokota, T., D. E. Milenic, M. Whitlow, J. Schlom. 1992. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms. Cancer Res. 52: 3402-3408.
50. Griffiths, A. D., S. C. Williams, O. Hartley, lan M. Tomlinson, P. Waterhouse, W. L. Crosby, R. E. Kontermann, P. T. Jones, N. M. Low, T. John Allison, T. D. Prospero, H. R. Hoogenboom, A. Nissim, J. P. L. Cox, J. L. Harrison, M. Zaccolo, E. Gherardi, and G. Winter. 1994. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13: 3245-3260.
51. Chen, M. R., J. F. Yang, T. Y. Hsu, M. Y. Liu, J. Y. Chen, C. S. Yang. 1996. Use of bacterially expressed GST/EBNA-1 fusion proteins for detection of antibodies in sera from patients with nasopharyngeal carcinoma and healthy donors. Chinese journal of microbiology and immunology. 29: 65-79.
52. Liao, S. K., Y. P. Perng, Y. C. Shen, P. J. Chung, Y. S. Chang, and C. H. Wang. 1998. Chromosomal abnormalities of a new nasopharyngeal carcinoma cell line (NPC-BM1) derived from a bone marrow metastatic lesion. Cancer Genet Cytogenet. 103: 52-58.
53. Menezes, J., W. Leibold, G. Klein, G. Clements. 1975. Establishment and characterization of an Epstein-Barr virus (EBV)-negative lymphoblastoid B cell line (BJA-B) from an exceptional, EBV- genome-negative African Burkitt’s lymphoma. Biomedicine. 22: 276-284.
54. Vaughan, T. J., A. J. Williams, K. P. Jane, K. Osbourn, A. R. Pope, J. C. Earnshaw, J. McCafferty, R. A. Hodits, J. Wilton, and K. S. Johnson. 1996 Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 14: 309-314.
55. Margaret Merchant, A., Z. Zhu, J. Q. Yuan, A. Goddard, C. W. Adams, L. G. Presta, and P. Carter. 1998. An efficient route to human bispecific IgG. Nat Biotechnol. 16: 677-681.
56. Nissim, A., H. R. Hoogenboom, I. M. Tomlinson, G. F. Carol Midgley, D. Lane, and G. Winter. 1994. Antibody fragments from a 'single pot' phage display library as immunochemical reagents. EMBO J. 13: 692-698.
57. Griffiths, A. D., M. Malmqvist, J. D. Marks, J. M. Bye, M. J. Embleton, J. McCafferty, M. Baier, K. Philipp Holliger, B. D. Gorick, N. C. Hughes-Jones, H. R. Hoogenboom, and G. Winter. 1993. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12: 725-734.
58. Yokota, T., D. E. Milenic, M. Whitlow, and J. Schlom. 1992. Rapid tumor penetration of a single- chain Fv and comparison with other immunoglobulin forms. Cancer Res. 52: 3402-3408.
59. Marks, C., and J. D. Marks. 1996. Phage libraries-a new route to clinically useful antibodies. N Engl J Med. 335: 730-733.
60. Schier, R., J. D. Marks, E. J. Wolf, G. Apell, C. Wong, J. E. McCartney, M. A. Bookman, J. S. Huston, L. L. Houston, L. M. Weiner, et al. 1995. In vitro and in vivo characterization of a human anti-c-erbB -2 single-chain Fv isolated from a filamentous phage antibody library. Immunotechnology. 1: 73-81.
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