CD20-Mimotope Peptides: A Model to Define the Molecular Basis of Epitope Spreading

Abstract

Antigen-mimicking peptide (mimotope)-based vaccines are one of the most promising forms of active-immunotherapy. The main drawback of this approach is that it induces antibodies that react poorly with the nominal antigen. The aim of this study was to investigate the molecular basis underlying the weak antibody response induced against the naïve protein after peptide vaccination. For this purpose, we analyzed the fine specificity of monoclonal antibodies (mAb) elicited with a 13-mer linear peptide, complementary to theantigen-combining site of the anti-CD20 mAb, Rituximab, in BALB/c mice. Anti-peptide mAb competed with Rituximab for peptide binding. Even so, they recognized a different antigenic motif from the one recognized by Rituximab. This explains their lack of reactivity with membrane (naïve) CD20. These data indicate that even on a short peptide the immunogenic and antigenic motifs may be different. These findings highlight an additional mechanism for epitope spreading and should be taken into account when designing peptides for vaccine purposes.

Keywords: CD20; Rituximab; active immunotherapy; antigenicity; epitope spreading; immunogenicity; mimotope; peptide; vaccine.

Figure 1

(A) Specificity of the reactivity of sera antibodies elicited in a BALB/c mouse immunized with Rituximab-specific peptide Rp5-L. 10-fold serial dilutions of serum were added to wells previously coated with Rp5-L peptide (closed triangle). Following a 4h incubation at R/T, bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG. The Qp-1a peptide was used as a negative control (open triangle). (B) The occurrence of anti-CD20 Ab in serum from mice immunized with Rp5-L. Anti-Rp5-L serum (diluted 1:20) was added to rabbit IgG-treated CD20⁺ B lymphoid Raji cells. Bound antibodies were detected with an appropriate dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Rituximab, the anti-HLA class I mAb TP25.99 and the CD20⁻ human T lymphoid CEM cells were used as controls.

Figure 2

Binding assay to define the differential reactivity of anti-Rp5-L mAb with a panel of Rituximab-specific peptides. Hybridoma-supernatants secreting anti-Rp5-L mAb were added to wells previously coated with Rituximab-specific peptides Rp5-L (black bar), Rp1-L (red bar), Rp10-L (blue bar), pASMLPD (green bar), Rev-pASMLPD (yellow bar), RpCD20-L (grey bar), and Qp-1a (white bar) for 4 hours at R/T. Peptide-antibody binding was detected with HRP-conjugated goat anti-mouse Ig. Rituximab, the anti-HLA class I mAb HC-10, and the peptide Qp-1a were used as controls. The data are representative of two experiments.

Figure 3

(A) Competitive binding assay to define the relative avidity of mAb FE-718 and FE-341 for Rp5-L. Ten-fold serial dilution of mAb FE-718 and FE-341 were mixed with an equal volume of an appropriate dilution of Rituximab and added to wells previously coated with Rp5-L. Following a 2-hour incubation at R/T, Rituximab-bound was detected with HRP-conjugated goat anti-human Ig (Fc portion). The anti-HLA class I mAb HC-10 was used as control. Results are expressed as percentage inhibition of binding compared with binding in the absence of inhibitor. The data are representative of two experiments. The O.D. of uninhibited Rituximab was 2.41±0.23 S.D. (B) Reactivity of mAb FE-718 and FE-341 with CD20⁺ B lymphoid cells. mAb FE-718 and FE-341 were added to rabbit IgG-treated CD20⁺ B lymphoid Raji cells. Bound antibodies were detected with an appropriate dilution of FITC-conjugated goat anti-mouse IgG. Rituximab was used as control.

Figure 4

Reactivity of Rituximab with the mAb FE-341-specific phage clone pc718-3. Supernatants from pc718-3 and pcR1L phage clones were diluted 16-fold and added to wells previously coated with mAb or mouse IgG (mIgG). After a 4h incubation, the antibody-phage clone interaction was detected with HRP-conjugated anti-M13 mAb. The data are representative of two experiments.