High-molecular-weight melanoma-associated antigen mimotope immunizations induce antibodies recognizing melanoma cells

Abstract

Size and posttranslational modifications are obstacles in the recombinant expression of high-molecular-weight melanoma-associated antigen (HMW-MAA). Creating a tumor antigen mimic via the phage display technology may be a means to overcome this problem for vaccine design. In this study, we aimed to generate an immunogenic epitope mimic of HMW-MAA. Therefore we screened a linear 9mer phage display peptide library, using the anti-HMW-MAA monoclonal antibody (mAb) 225.28S. This antibody mediates antibody-dependent cellular cytotoxicity (ADCC) and has already been used for anti-idiotype therapy trials. Fifteen peptides were selected by mAb 225.28S in the biopanning procedure. They share a consensus sequence, but show only partial homology to the amino acid sequence of the HMW-MAA core protein, indicating mimicry with a conformational epitope. One mimotope was chosen to be fused to albumin binding protein (ABP) as an immunogenic carrier. Immunoassays with 225.28S indicated that the mimotope fusion protein was folded correctly. Subsequently, the fusion protein was tested for immunogenicity in BALB/c mice. The induced anti-mimotope antibodies recognized HMW-MAA of 518A2 human melanoma cells, whereas sera of mice immunized with the carrier ABP alone showed no reactivity. These anti-mimotope antibodies were capable of inducing specific lysis of 518A2 melanoma cells in ADCC assays with murine effector cells. In conclusion, the presented data indicate that mimotopes fused to an immunogenic carrier are suitable tools to elicit epitope-specific anti-melanoma immune responses.

Fig. 1

Alignment and sequence of peptides selected by mAb 225.28S. The peptide sequences were subjected to the multiple alignment algorithm of the Vector NTI Suite, which produces a phylogenetic tree indicating the degree of similarities of the peptides to each other. Residues in boxes were found at a specific position four or more times

Fig. 2

Specific recognition of the K11-ABP fusion protein by mAb 225.28S in immunoblot. K11-ABP (lane 1) and ABP (lane 2) fusion proteins were subjected to 12% SDS-PAGE and blotted onto a nitrocellulose membrane. Strips were incubated with A an anti-His tag antibody, B mAb 225.28S, C isotype control, and D buffer. Bound antibodies were detected by a radioactively (¹²⁵I) labeled sheep anti-mouse Ig antibody

Fig. 3

Sera of mice immunized with K11-ABP recognize the HMW-MAA band at 250 kDa in an 518A2 cell lysate immunoblot. a Immunoblot. 518A2 membrane preparations were separated by 6% SDS-PAGE and proteins were transferred to a nitrocellulose membrane. Strips were incubated with controls or pooled sera as indicated (b buffer control, neg isotype control, pos 225.28S, PIS preimmune serum, IS immune serum). b Immunization schedule. BALB/c mice (n=5 per group) were immunized ip with 10 μg of the respective fusion protein at the indicated time points (Imm immunization). Blood samples were taken 1 week after each immunization

Fig. 4

Sera from mimotope-immunized mice recognize HMW-MAA in immunofluorescence staining of 518A2 melanoma cells. Panel A: 225.28S positive control. Panel B: Serum IgG from mice immunized with K11-ABP specifically recognize cell surface HMW-MAA. Serum IgG from mice immunized with ABP alone (panel C) and pre-immune sera (panel D) only show background staining. Column 1: A–D antibody staining (green), membrane staining (red) and nuclear staining (blue). Column 2: FITC channel. Column 3: Texas red channel

Fig. 5

Sera of mice immunized with K11-ABP induce specific lysis of 518A2 melanoma cells in an ADCC assay. 518A2 target cells were labeled with a substance fluorescent at contact with europium. Following incubation with 10 μg mAb/10 μl pooled sera (equaling ~2 μg HMW-MAA-specific antibodies), murine effector cells were added at an effector-to-target ratio of 50:1. Aliquots of supernatants were mixed with europium solution, and fluorescence was measured in a time-resolved fluorometer. Percentage of specific lysis was calculated relative to maximum release, corrected by spontaneous release values. Column heights represent mean ± SD. Effectors: lytic activity of effector cells alone. Other columns: ADCC mediated by mAb/pooled sera as indicated on the x-axis