Identification of anti-CD98 antibody mimotopes for inducing antibodies with antitumor activity by mimotope immunization

Abstract

A mimotope is an antibody-epitope-mimicking peptide retrieved from a phage display random peptide library. Immunization with antitumor antibody-derived mimotopes is promising for inducing antitumor immunity in hosts. In this study, we isolated linear and constrained mimotopes from HBJ127, a tumor-suppressing anti-CD98 heavy chain mAb, and determined their abilities for induction of antitumor activity equal to that of the parent antibody. We detected elevated levels of antipeptide responses, but failed to detect reactivity against native CD98-expressing HeLa cells in sera of immunized mice. Phage display panning and selection of mimotope-immunized mouse spleen-derived antibody Fab library showed that HeLa cell-reactive Fabs were successfully retrieved from the library. This finding indicates that native antigen-reactive Fab clones represented an undetectable minor population in mimotope-induced antibody repertoire. Functional and structural analysis of retrieved Fab clones revealed that they were almost identical to the parent antibody. From these results, we confirmed that mimotope immunization was promising for retrieving antitumor antibodies equivalent to the parent antibody, although the co-administration of adjuvant compounds such as T-cell epitope peptides and Toll-like receptor 4 agonist peptides is likely to be necessary for inducing stronger antitumor immunity than mimotope injection alone.

Keywords: CD98; mimotope; monoclonal antibody; phage display; recombinant Fab.

Fig. 1

Reactivity of recombinant Fab clones against live HeLa cells by indirect immunofluorescence. Recombinant Fab (a) A18, (b) B3, and (c) C17 retrieved from peptide–KLH immunized mice were examined. The parental antibody (d) HBJ127 was used as a positive control.

Fig. 2

Reactivity of recombinant Fab (rFab) clones against HeLa lysates by capture sandwich ELISA. HeLa lysates captured by purified rFabs (A18, B3, C17) or parental antibody HBJ127 (positive control) were detected by biotinylated anti-CD98 mAb 1-10. Murine leukemia P388 cell lysates were used as a negative control.

Fig. 3

Effects of recombinant Fab clones on in vitro growth of HeLa cells. Cells (1 × 10⁴) were cultured with recombinant Fabs (A18, B3, C17) plus anti-mouse IgG F(ab')2 or with mAbs (HBJ127 and isotype-matched unrelated antibody [NC]) for 72 h at 37°C. Cell growth activity was evaluated by alamarBlue assay.

Fig. 4

Deduced amino acid sequences of recombinant Fab clones (A18, B3, C17) compared with HBJ127 and the closest known germline. (a) Variable heavy domain (VH) sequences and (b) variable light domain (VL) sequences.