Biol. Chem., Vol. 387, pp. 995–1003, July 2006 • Copyright  by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2006.123 2006/126 Article in press - uncorrected proof Isolation and comparative characterization of Ki-67 equivalent antibodies from the HuCAL  phage display library Tiantom Jarutat 1, *, Christian Frisch 2 , Cora Nickels 1 , Hartmut Merz 1 and Achim Knappik 2 1 Department of Pathology, University Hospital of Schleswig-Holstein, Campus Lu ¨ beck, Ratzeburger Allee 160, D-23538 Lu ¨ beck, Germany 2 Morphosys AG, Division Antibodies-by-Design, Lena- Christ-Str. 48, D-82152 Planegg/Martinsried, Germany * Corresponding author e-mail: jarutat@patho.uni-luebeck.de 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 equiv- alent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in for- malin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and char- acterization of new monoclonal Ki-67 equivalent antibod- ies 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’ epi- tope. ELISA screening of randomly picked phage anti- body 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. Substitu- tional 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 rec- ognition of Ki-67 antigen in FFPE tissue. Keywords: epitope analysis; immunohistochemistry; Ki-67 antigen; monoclonal antibodies; phage display; tissue fixation. Introduction Antibodies against Ki-67 antigen are routinely used in histology as a tool to assess the proliferation index of tissues. In pathology, Ki-67 first showed its value as a disease marker in the classification of non-Hodgkin’s lymphomas (Gerdes et al., 1984a; Hall et al., 1988). Ki- 67 staining indices have proven to be of importance for the histological grading and prediction of clinical out- come of various diseases and malignancies, including meningioma (Ohta et al., 1994), ovarian cancer (Garzetti et al., 1995), Hodgkin’s lymphoma (Abele et al., 1997), liver tumors (Nolte et al., 1998) and colorectal cancer (Scott et al., 2003). Ki-67 antigen is named after the first monoclonal anti- body recognizing this antigen: ‘Ki-67’. Ki-67 was origi- nally isolated by Gerdes et al. (1983) by immunizing mice with a nuclear preparation of L428 cells. In frozen sec- tions, immunohistochemistry with Ki-67 stains only cell nuclei of proliferating cells in M, G1/2 and S-phase (Gerdes et al., 1984b). Later, several Ki-67 equivalent monoclonal and polyclonal antibodies were isolated, some of which show reactivity in formalin-fixed paraffin- embedded (FFPE) tissue sections following heat-induced epitope retrieval (Key et al., 1993a,b; Reynolds et al., 1995). They were raised by immunization of rabbits or mice with either recombinant protein or with peptides (leading to clones ‘MIB1-3’ and rabbit polyclonal Ki-67 serum) (Key et al., 1993a,b) or Ichikawa tumor-cell graft (leading to clone IND.64) (Meggetto et al., 1992). In par- ticular, the monoclonal Ki-67 equivalent MIB-1 has been proven to give reliable results in FFPE tissues and is thus routinely used in histology (Rose et al., 1994). The gene locus for the Ki-67 antigen (MKI67) lies on chromosome 10 (Fonatsch et al., 1991). The mRNA tran- script consists of 15 exons with a total length of approx- imately 12.5 kb. A shorter isoform lacking exon 7 is also translated (Schluter et al., 1993). Sixteen repeat regions were found in exon 13, nine of them containing a 66-bp consensus ‘Ki-67 motif’ sup- posedly recognized by Ki-67 and equivalent antibodies (Key et al., 1993a). Epitope analysis of Ki-67 and equivalent antibodies indicated that the repetitive amino acid sequence FKEL(F) is recognized by Ki-67 (FKEL) and MIB1 (FKELF) (Kubbutat et al., 1994) (Figure 1A). Phage antibody display technology was introduced by McCafferty et al. (1990) as a modification of phage dis- play, originally devised by Smith (1985). Phage antibody display libraries usually contain a population of antigen- binding variable fragments of antibodies in the form of either single-chain Fv or Fab with separately expressed V(L) and V(H) chains. The synthetic human combinatorial antibody library (HuCAL  ) was constructed from consensus sequences of seven human V(L) and V(H) regions with engineered, highly diverse CDR-cassettes (Knappik et al., 2000; Rau- chenberger et al., 2003). The library version HuCAL GOLD  , which was used in this study, contains approx- imately 2 =10 10 members in the Fab format (Kretzschmar and von Ruden, 2002). Recombinant antibody technology based on large, naı¨ve, antibody gene libraries combined with selection technologies such as phage display provides an inter- esting alternative to traditional immunization of animals, since the technology results in monoclonal specificities in a short period of time, is not limited by tolerance Brought to you by | Nanyang Technological University Authenticated Download Date | 5/20/15 8:45 PM