Antibody epitope peptides as potential inducers of IgG antibodies against CD98 oncoprotein

Abstract

An epitope is an antibody-recognition site on a target antigen. As such, active immunization of epitope peptides may induce therapeutic efficacy equivalent to the administration of parent antibody medicines. In the present study, we designed peptides based on the epitope recognized by the tumor-suppresive anti-CD98 monoclonal antibody HBJ127, and investigated their efficacy for induction of antitumor immunity. The immune sera showed reactivity against the corresponding peptide-keyhole limpet hemocyanin (KLH) and peptide-bovine serum abumin (BSA) conjugates, although they did not react with CD98-positive HeLa cells or recombinant CD98 heavy chain. To elucidate whether the epitope peptide failed to induce antitumor immunity or not, we constructed the IgG1, kappa Fab phage display libraries from spleen cells of immunized mice and tried to retrieve CD98-reactive recombinant Fab (rFab) fragments by panning against either epitope peptide-BSA conjugates or live HeLa cells. RFab fragments retrieved from peptide-BSA panning showed no reactivity to HeLa cells. Their variable-region sequences were different from HBJ127. However, rFab fragments retrieved from HeLa cell panning showed reactivity to CD98 by indirect immunofluorescence and immunoprecipitation. Moreover, they were structurally almost identical to HBJ127. Although the immunogenicity of epitope peptides may be insufficient for induction of expected antitumor activity in vivo, we used antibody phage display to show that IgG antibodies almost identical to HBJ127 were an undetectable population in epitope peptide-induced immune sera.

Figure 1

Reactivity of antisera against peptide–BSA conjugates. Serum (#1S–#4S) from peptide (#1–#4)–KLH immunized mice was examined against four peptide–BSA conjugates. Reactivity is shown relative to the reactivity against the corresponding peptide–BSA.

Figure 2

Reactivity of cell panning (CP) clones against live HeLa cells by indirect immunofluorescence. The recombinant Fab (a) CP1‐5, (b) CP1‐10, (c) and CP3‐10 were examined. (d) The monoclonal antibody HBJ127 was used as a positive control.

Figure 3

Reactivity of epitope peptide panning (EP) clones against peptide–BSA conjugates by direct enzyme‐linked immunosorbent assay. Recombinant Fab, EP3‐1, EP3‐6, and EP3‐31 were examined against four peptide (#1–#4)–BSA conjugates.

Figure 4

Reactivity of cell panning (CP) and epitope peptide panning (EP) clones against HeLa lysates by capture sandwich enzyme‐linked immunosorbent assay. HeLa lysates captured by purified recombinant Fab (CP and EP clones) were detected by biotinylated anti‐CD98 monoclonal antibody 1‐10. Murine leukemia P388 cell lysates were used as a negative control. HBJ127 was used as a positive control, and SER4 directed against HER2/neu was used as a negative control.

Figure 5

Immunoprecipitation of cell panning (CP) clone‐reactive antigen from HeLa cell lysates. Recombinant Fab, CP1‐5 (lane 2), CP1‐10 (lane 3), and CP3‐10 (lane 4) were examined. The monoclonal antibody HBJ127 (lane 1) was used as a positive control. The recombinant Fab EP3‐1 (lane 5) was used as a negative control. The arrows indicate the molecular weights of intact CD98 (125 kDa) and CD98 heavy chain (80 kDa).

Figure 6

Effects of cell panning (CP) clones on in vitro growth of HeLa cells. Cells (1 × 10⁴) were cultured with recombinant Fab (CP1‐5, CP1‐10, CP3‐10) plus antimouse IgG F(ab′)2 or with monoclonal antibody (HBJ127) for 72 h at 37°C. The cell growth activity was evaluated by alamar blue assay.

Figure 7

Deduced amino acid sequences of cell panning (CP) and epitope peptide panning (EP) clones compared with HBJ127 and the closest known germline. (a) Variable heavy domain (VH) sequences and (b) variable light domain (VL) sequences.