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- Materials and Methods
CD98 is known as a cell surface antigen expressed in proliferating normal tissues and in almost all tumor cells. Although the function of CD98 is not yet fully elucidated, it is suggested that CD98 is concerned functionally in lymphocyte activation, cell proliferation, and malignant transformation. Monoclonal antibody against human CD98 heavy chain (h.c.), termed HBJ127, shows inhibition of lymphocyte activation and tumor cell growth in vitro. These observations suggest that the epitope recognized by HBJ127 may be crucial for CD98 function. In the present study, the authors investigated the epitope recognized by HBJ127 using a phage display random heptapeptide library. Approximately 2.4 × 104-fold amplification of eluted phage titer was obtained after three rounds of panning of the phage library against HBJ127. Seven different heptapeptide sequences were isolated from 30 randomly selected clones of the post-panning phage population. A homology search using ClustalW identified the peptide sequence corresponding to 442AFS444 of human CD98 h.c. It was also found that 443F is a human-specific amino acid by comparing sequences of human, rat, and mouse origin. Reduced reactivity of HBJ127 was detected against the phenylalanine-substituted peptide but not detected against the alanine or serine-substituted one. It has been identified that HBJ127 reacts only with human species and the HBJ127 epitope position is predicted in 418–529 of human CD98 h.c. From these results and observations, it was estimated that 442AFS444 of human CD98 h.c. may be the HBJ127 epitope. Moreover, 443F may be critical for the binding of HBJ127 against human CD98 h.c. (Cancer Sci 2007; 98: 1696–1700)
CD98/GP125 is a heterodimeric protein with a relative molecular mass of 125-kDa consisting of a glycosylated 85-kDa heavy chain (h.c.) and a non-glycosylated 40-kDa light chain, which are disulfide-linked.(1) Analyses of human CD98 cDNA have revealed that CD98 is a type II transmembrane glycoprotein(2) that is disulfide-linked to a non-glycosylated light chain of a member of the permease family.(3) CD98 was identified originally as a cell surface antigen associated with lymphocyte activation defined by the 4F2 monoclonal antibody (mAb),(1) and is expressed in proliferating normal tissues(4) and in almost all tumor cells.(5) These findings suggest that CD98 is involved functionally in lymphocyte activation, cell proliferation and malignant transformation. In fact, a mAb against CD98, termed HBJ127, which was originally raised against T24 human bladder cancer,(5) inhibited tumor cell growth(6) and lymphocyte proliferation.(7) Furthermore, CD98 h.c. cDNA-transfected murine fibroblasts showed various malignant phenotypes.(8)
Phage display, originally developed by Smith,(9) is the procedure for expression of proteins or peptides of interest on the surface of filamentous phage M13 as a fusion protein with viral coat proteins. Biopanning, which is characteristic for phage display, made it easy to screen and select the positive clones because several rounds of reaction between the phage display library and the target molecule can concentrate the population of positive clones in the library. This technique is now widely used for isolation of recombinant antibody fragments,(10–15) for identification of receptor ligands,(16–18) and for epitope mapping of mAb.(19–21)
Because HBJ127 inhibits CD98 function, the epitope recognized by this mAb may play an important role in expressing CD98 function. In the present study, the authors tried to identify the epitope sequence recognized by HBJ127 using a phage display random peptide library. Three rounds of biopanning resulted in 2.4 × 104-fold amplification of the HBJ127-bound phage titer. Sequence analysis of 30 clones of the HBJ127-bound phage revealed that these clones consisted of seven individual sequences. ClustalW analysis obtained seven different heptapeptide sequences and a human CD98 h.c. amino acid sequence revealed that the epitope recognized by HBJ127 might be 443AFS445 in CD98 h.c. The authors concluded that 444F might be crucial for HBJ127 binding to CD98 h.c. because the reactivity of HBJ127 against synthetic peptide with point mutation (444F to A) was greatly reduced compared with the peptide of the original sequence.
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- Materials and Methods
In the present study, the phage display random peptide library was used for the identification of the epitope recognized by HBJ127. In general, immunoprecipitation with a specific mAb followed by liquid chromatography–mass spectrometry (LC-MS) analysis has been used for the identification of a recognized antigen. The epitope may be determined by an oligo-peptide array constructed using sequence information of the antigen obtained as above. These procedures seem laborious and time consuming. As for the epitope mapping using the phage display random peptide library, panning of the library against the antibody of interest followed by screening of phage clones from post-panning the library will give phage clones with the antibody binding activity. Sequencing of the peptide-coding region in phage DNA can identify the consensus sequence of antibody-binding clones.
Although the phage display approach is actually simple and easy, this procedure has some disadvantages. Identification of the epitope may be difficult if the sequence information of the antigen of interest is not available, because the homology search of the obtained sequence and the antigen sequence is indispensable for identification of the epitope region on the antigen. The peptide sequence of the most frequent or of the strongest binding to antibody may not be the epitope sequence. In fact, in the present study, the expected epitope-bearing sequence was found in only one of 30 tested clones. Moreover, if the antibody recognizes the conformation-dependent epitope, the actual epitope sequence cannot be identified using this procedure. Combined use of phage display and immunological techniques makes it possible to identify the epitope of the antigen, easily, simply and accurately.
The epitope of HBJ127 was estimated by comparing the isolated heptapeptide sequences and the human CD98 h.c. amino acid sequence using ClustalW. The epitope of HBJ127 lies in between 418 and 529 in the human CD98 h.c. from the study using deletion mutants of the human CD98 h.c. expressed on mouse NIH 3T3 cells.(23) HBJ127 reacts with the human CD98 h.c., not with mouse or rat molecule.(5) The authors’ predicted epitope position (442AFS444) is within 418–529 of the human CD98 h.c. One of the constituents, 443F, is human sequence-specific (mouse and rat sequences have 443L). Decreased reactivity of HBJ127 against the point mutation (F to A)-introduced epitope peptide–BSA conjugate was shown. From these observations and the present findings, we propose that the epitope would be 442AFS444 in human CD98 h.c., and that 443F may be crucial for HBJ127-binding to human CD98 h.c. Binder et al. reported that the epitope of anti-CD25 mAb, dacrizumab, was found to be overlapped with basiliximab, which is also an anti-CD25 mAb, using the mutation-introduced epitope peptides prepared based on the sequences isolated from the phage display peptide library panned against basiliximab.(24) The present procedure for determination of the HBJ127 epitope may be appropriate because a similar approach to the above was used: identification of the epitope-related sequences using the phage display peptide library and confirmation of the predicted epitope sequence using mutation-introduced synthetic peptides.
Antibody medicines are widely used for the treatment of tumors, autoimmune disorders, and infectious diseases. As for tumor therapy, the representatives are transtuzumab and rituximab, which are used for the treatment of c-erb-B2-positive breast cancers and CD20-positive B-cell lymphoma, respectively. As for rheumatoid arthritis therapy, the anti-tumor necrosis factor (TNF)-α mAb, infliximab, is used for treatment in rheumatrex-unresponsive patients. Although antibody medicines show excellent efficacy in the therapy of such diseases as above, they have some disadvantages, for example, repeated injection may be necessary for maintaining efficacy, there is a risk of anaphylaxis, there can be decreased effectiveness due to the anti-antibody response in the hosts, and they are very expensive medicines. The development of new medicines with an efficacy equivalent to the antibody medicines without the shown drawbacks is expected. One candidate is the peptide vaccine prepared based on the epitope sequence of antibody medicines or antibodies with biological activities. Induction of protective immunity would be expected by active immunization of the host with epitope vaccines. Mimotope peptides obtained using the phage display peptide library from cetuximab,(25) rituximab,(26) transtuzumab,(27) and anti-GD2 mAb,(28,29) successfully induced protective immunity in the hosts. Evaluation of the usefulness of a HBJ127-derived epitope peptide immunization for induction of CD98-targeting immunity in the hosts is now in progress.