Specificity of mimotope-induced anti-high molecular weight-melanoma associated antigen (HMW-MAA) antibodies does not ensure biological activity

Abstract

Vaccines based on peptide mimics (mimotopes) of conformational tumor antigen epitopes have been investigated for a variety of human tumors including breast cancer, tumors expressing the carcinoembryonic antigen, B cell lymphoma, neuroblastoma, and melanoma. In our previous work, we designed a vaccine based on a mimotope of the high molecular weight-melanoma associated antigen (HMW-MAA) that elicited HMW-MAA-specific antibodies (Abs) with anti-tumor activity in vitro and in vivo. In this study, we aimed to identify mimotopes of additional distinct HMW-MAA epitopes, since they could be used to construct a polymimotope melanoma vaccine. For this purpose, random peptide phage libraries were screened with the anti-HMW-MAA monoclonal antibodies (mAbs) VT80.12 and VF1-TP43 yielding one peptide ligand for each mAb. Both peptides inhibited the binding of the corresponding mAb to the HMW-MAA. Furthermore, when coupled to the carrier protein keyhole limpet hemocyanin (KLH), both HMW-MAA mimotopes elicited peptide-specific Abs in rabbits or BALB/c mice, but only the mimotope isolated with the mAb VT80.12 elicited HMW-MAA-specific Abs and only in mice. However, the latter Abs had no detectable effect on HMW-MAA expressing human melanoma cells in vitro. These results describe limitations related to the phage display technique and emphasize the need to characterize the functional properties of the mAb utilized to isolate mimotopes of the corresponding epitopes.

Figure 1. Inhibition of mAb binding to HMW-MAA by synthetic peptides.

Microtiter plates were coated with mAb TP61.5 and incubated with microsomal preparation of 518A2 melanoma cells to catch HMW-MAA. Biotinylated mAbs were preincubated with increasing concentrations of synthetic peptides, followed by incubation with HMW-MAA. (A) Biotinylated mAb VT80.12 was preincubated with peptide 15/3/6 (•) or an irrelevant control peptide (▴). (B) Biotinylated mAb VF1-TP43 was preincubated with peptide 43.12p3 (▪) as well as the control peptide (▴). Binding of biotinylated mAbs was measured using an AP-conjugated streptavidin. Percentage of inhibition was calculated as follows: 100−(OD (inhibited)/OD (uninhibited)×100).

Figure 2. Antigen- and HMW-MAA-specific immune response of immunized rabbits.

Left panel: Microtiter plates were coated with 15/3/6-BSA conjugate, KLH or BSA and incubated with increasing concentrations of purified rabbit IgG from immunizations with 15/3/6-KLH conjugate or KLH to detect peptide- and KLH-specific Abs. Right panel: Microtiter plates were coated with mAb TP61.5 and incubated with microsomal preparations of the melanoma cell lines 518A2 or M14. Purified rabbit IgG were analyzed by administration of increasing Ab concentrations.

Figure 3. HMW-MAA-specific Ab response determined by FACS and IHC.

(A) The human melanoma cell lines 518A2 and M14 were each incubated with 1 µg mAb VT80.12 (as positive control to detect HMW-MAA) or 100 µg purified rabbit IgG from immunizations with 15/3/6-KLH conjugate or KLH. Bound IgG were detected with FITC-labeled goat anti-mouse IgG or FITC-labeled goat anti-rabbit IgG. Histogram overlays show unstained (white) vs. stained cells (grey). (B) IHC on 518A2 tumor tissues of C.B.17 SCID/SCID mice . HMW-MAA staining was done with PBS buffer (negative control), mAb VT80.12 (positive control), and biotinylated IgG purified from rabbits immunized with 15/3/6-KLH conjugate (anti-15/3/6) or KLH (anti-KLH). Bound Abs were detected with StrepAB/HRP and visualized by DAB-chromogen solution and subsequent hematoxylin counterstaining.

Figure 4. Antigen-specific immune response of immunized BALB/c mice.

Serum samples from day 0 (preimmune serum; white bars), day 22 (grey bars), and day 41 (black bars) were tested in ELISA. The mean value of the individual responses of each group is shown and standard deviations are indicated by error bars. Peptide specificity of sera from mice either immunized with 15/3/6- or 43.12p3-KLH conjugate was assessed using the respective peptide-BSA conjugates. Sera from KLH immunized mice were tested for specificity to KLH.

Figure 5. HMW-MAA-specific immune response of immunized BALB/c mice.

Human melanoma cell lines 518A2 and M14 were either incubated with 1 µg mAb VT80.12 (as positive control) or a 1∶10 dilution of pooled mouse sera of each group. Bound IgG were detected with FITC-labeled goat anti-mouse IgG. Histogram overlays show unstained (white) and stained cells (grey).

Figure 6. Inhibition of melanoma cell proliferation in vitro.

[³H]thymidine proliferation assay demonstrating effects of purified mouse IgG on HMW-MAA expressing melanoma cells. 518A2 or M14 cells were incubated with increasing concentrations of purified IgG from mice immunized with 15/3/6-KLH conjugate or KLH for 72 h and pulsed with [³H]thymidine. Data are presented as percentage of inhibition of proliferation compared to untreated cells. Percentage of inhibition was calculated as follows: 100−(cpm (treated)/cpm (untreated)×100). Values represent the mean of three independent experiments.