Abstract
It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL®). We implemented a panning strategy against two different overlapping peptides, both containing the ‘FKELF’ epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the ‘FKELF’ epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.
References
Abele, M.C., Valente, G., Kerim, S., Navone, R., Onesti, P., Chiusa, L., Resegotti, L., and Palestro, G. (1997). Significance of cell proliferation index in assessing histological prognostic categories in Hodgkin's disease. An immunohistochemical study with Ki67 and MIB-1 monoclonal antibodies. Haematologica82, 281–285.Search in Google Scholar
Endl, E. and Gerdes, J. (2000). The Ki-67 protein: fascinating forms and an unknown function. Exp. Cell Res.257, 231–237.10.1006/excr.2000.4888Search in Google Scholar
Fonatsch, C., Duchrow, M., Rieder, H., Schluter, C., and Gerdes, J. (1991). Assignment of the human Ki-67 gene (MK167) to 10q25-qter. Genomics11, 476–477.10.1016/0888-7543(91)90163-9Search in Google Scholar
Garzetti, G.G., Ciavattini, A., Goteri, G., De Nictolis, M., Stramazzotti, D., Lucarini, G., and Biagini, G. (1995). Ki67 antigen immunostaining (MIB 1 monoclonal antibody) in serous ovarian tumors: index of proliferative activity with prognostic significance. Gynecol. Oncol.56, 169–174.10.1006/gyno.1995.1026Search in Google Scholar
Gerdes, J., Schwab, U., Lemke, H., and Stein, H. (1983). Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int. J. Cancer31, 13–20.10.1002/ijc.2910310104Search in Google Scholar
Gerdes, J., Dallenbach, F., Lennert, K., Lemke, H., and Stein, H. (1984a). Growth fractions in malignant non-Hodgkin's lymphomas (NHL) as determined in situ with the monoclonal antibody Ki-67. Hematol. Oncol.2, 365–371.10.1002/hon.2900020406Search in Google Scholar
Gerdes, J., Lemke, H., Baisch, H., Wacker, H.H., Schwab, U., and Stein, H. (1984b). Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J. Immunol.133, 1710–1715.10.4049/jimmunol.133.4.1710Search in Google Scholar
Hall, P.A., Crocker, J., Watts, A., and Stansfeld, A.G. (1988). A comparison of nucleolar organizer region staining and Ki-67 immunostaining in non-Hodgkin's lymphoma. Histopathology12, 373–381.10.1111/j.1365-2559.1988.tb01952.xSearch in Google Scholar
Jadayel, D.M., Lukas, J., Nacheva, E., Bartkova, J., Stranks, G., De Schouwer, P.J., Lens, D., Bartek, J., Dyer, M.J., Kruger, A.R., and Catovsky, D. (1997). Potential role for concurrent abnormalities of the cyclin D1, p16CDKN2 and p15CDKN2B genes in certain B cell non-Hodgkin's lymphomas. Functional studies in a cell line (Granta 519). Leukemia11, 64–72.Search in Google Scholar
Jaye, D.L., Nolte, F.S., Mazzucchelli, L., Geigerman, C., Akyildiz, A., and Parkos, C.A. (2003). Use of real-time polymerase chain reaction to identify cell- and tissue-type-selective peptides by phage display. Am. J. Pathol.162, 1419–1429.10.1016/S0002-9440(10)64275-7Search in Google Scholar
Key, G., Becker, M.H., Baron, B., Duchrow, M., Schluter, C., Flad, H.D., and Gerdes, J. (1993a). New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope. Lab. Invest.68, 629–636.Search in Google Scholar
Key, G., Petersen, J.L., Becker, M.H., Duchrow, M., Schluter, C., Askaa, J., and Gerdes, J. (1993b). New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections. J. Clin. Pathol.46, 1080–1084.10.1136/jcp.46.12.1080Search in Google Scholar
Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., and Virnekas, B. (2000). Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol.296, 57–86.10.1006/jmbi.1999.3444Search in Google Scholar
Kretzschmar, T. and von Ruden, T. (2002). Antibody discovery: phage display. Curr. Opin. Biotechnol.13, 598–602.10.1016/S0958-1669(02)00380-4Search in Google Scholar
Kubbutat, M.H., Key, G., Duchrow, M., Schluter, C., Flad, H.D., and Gerdes, J. (1994). Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein). J. Clin. Pathol.47, 524–528.10.1136/jcp.47.6.524Search in Google Scholar
McCafferty, J., Griffiths, A.D., Winter, G., and Chiswell, D.J. (1990). Phage antibodies: filamentous phage displaying antibody variable domains. Nature348, 552–554.10.1038/348552a0Search in Google Scholar
Meggetto, F., al Saati, T., Cohen-Knafo, E., Roubinet, F., Selves, J., Bouche, G., Key, G., Gerdes, J., and Delsol, G. (1992). Production of a monoclonal antibody (IND.64) identifying a cell cycle-associated antigen using spleen cells from nude mice bearing Ichikawa tumour. J. Pathol.168, 187–196.Search in Google Scholar
Merz, H., Lange, K., Gaiser, T., Muller, A., Kapp, U., Bittner, C., Harder, S., Siebert, R., Bentz, M., Binder, T., et al. (2002). Characterization of a novel human anaplastic large cell lymphoma cell line tumorigenic in SCID mice. Leuk. Lymphoma43, 165–172.10.1080/10428190210193Search in Google Scholar
Nolte, M., Werner, M., Nasarek, A., Bektas, H., von Wasielewski, R., Klempnauer, J., and Georgii, A. (1998). Expression of proliferation associated antigens and detection of numerical chromosome aberrations in primary human liver tumours: relevance to tumour characteristics and prognosis. J. Clin. Pathol.51, 47–51.10.1136/jcp.51.1.47Search in Google Scholar
Ohta, M., Iwaki, T., Kitamoto, T., Takeshita, I., Tateishi, J., and Fukui, M. (1994). MIB1 staining index and scoring of histologic features in meningioma. Indicators for the prediction of biologic potential and postoperative management. Cancer74, 3176–3189.Search in Google Scholar
Pavlik, P., Siegel, R.W., Marzari, R., Sblattero, D., Verzillo, V., Collins, C., Marks, J.D., and Bradbury, A. (2003). Predicting antigenic peptides suitable for the selection of phage antibodies. Hum. Antibodies12, 99–112.Search in Google Scholar
Pluckthun, A. and Pack, P. (1997). New protein engineering approaches to multivalent and bispecific antibody fragments. Immunotechnology3, 83–105.10.1016/S1380-2933(97)00067-5Search in Google Scholar
Rauchenberger, R., Borges, E., Thomassen-Wolf, E., Rom, E., Adar, R., Yaniv, Y., Malka, M., Chumakov, I., Kotzer, S., Resnitzky, D., et al. (2003). Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J. Biol. Chem.278, 38194–38205.10.1074/jbc.M303164200Search in Google Scholar PubMed
Reineke, U., Kramer, A., and Schneider-Mergener, J. (1999). Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis. Curr. Top. Microbiol. Immunol.243, 23–36.10.1007/978-3-642-60142-2_2Search in Google Scholar PubMed
Reynolds, G.M., Rowlands, D.C., and Mead, G.P. (1995). Detection of Ki67 antigen by a new sheep polyclonal antiserum. J. Clin. Pathol.48, 1138–1140.10.1136/jcp.48.12.1138Search in Google Scholar PubMed PubMed Central
Rose, D.S., Maddox, P.H., and Brown, D.C. (1994). Which proliferation markers for routine immunohistology? A comparison of five antibodies. J. Clin. Pathol.47, 1010–1014.10.1136/jcp.47.11.1010Search in Google Scholar PubMed PubMed Central
Schluter, C., Duchrow, M., Wohlenberg, C., Becker, M.H., Key, G., Flad, H.D., and Gerdes, J. (1993). The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins. J. Cell Biol.123, 513–522.10.1083/jcb.123.3.513Search in Google Scholar PubMed PubMed Central
Scott, I.S., Morris, L.S., Bird, K., Davies, R.J., Vowler, S.L., Rushbrook, S.M., Marshall, A.E., Laskey, R.A., Miller, R., Arends, M.J., and Coleman, N. (2003). A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer. J. Pathol.201, 187–197.10.1002/path.1444Search in Google Scholar
Shi, S.R., Chaiwun, B., Young, L., Imam, A., Cote, R.J., and Taylor, C.R. (1994). Antigen retrieval using pH 3.5 glycine-HCl buffer or urea solution for immunohistochemical localization of Ki-67. Biotech. Histochem.69, 213–215.Search in Google Scholar
Shi, S.R., Gu, J., and Turrens, J. (2000) Development of the antigen retrieval technique: philosophical and theoretical bases. In: Antigen Retrieval Techniques: Immunohistochemistry & Molecular Morphology, S.R. Shi, J. Gu, and C. Taylor, eds. (Natick, USA: Eaton Publishing).Search in Google Scholar
Smith, G.P. (1985). Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science228, 1315–1317.10.1126/science.4001944Search in Google Scholar
Sompuram, S.R., Vani, K., Messana, E., and Bogen, S.A. (2004). A molecular mechanism of formalin fixation and antigen retrieval. Am. J. Clin. Pathol.121, 190–199.10.1309/BRN7CTX1E84NWWPLSearch in Google Scholar
Tordsson, J., Abrahmsen, L., Kalland, T., Ljung, C., Ingvar, C., and Brodin, T. (1997). Efficient selection of scFv antibody phage by adsorption to in situ expressed antigens in tissue sections. J. Immunol. Methods210, 11–23.10.1016/S0022-1759(97)00165-8Search in Google Scholar
Van Ewijk, W., de Kruif, J., Germeraad, W.T., Berendes, P., Ropke, C., Platenburg, P.P., and Logtenberg, T. (1997). Subtractive isolation of phage-displayed single-chain antibodies to thymic stromal cells by using intact thymic fragments. Proc. Natl. Acad. Sci. USA94, 3903–3908.10.1073/pnas.94.8.3903Search in Google Scholar PubMed PubMed Central
©2006 by Walter de Gruyter Berlin New York
