Specific antibodies elicited by a novel DNA vaccine targeting gastrin-releasing peptide inhibit murine melanoma growth in vivo

Abstract

The elevated expression and receptor binding of gastrin-releasing peptide (GRP) in various types of cancer, especially in malignant melanoma of the skin, suggest that GRP might be a putative target for immunotherapy in neoplastic diseases. We have therefore constructed a novel DNA vaccine coding for six tandem repeats of a fragment of GRP from amino acids 18 to 27 (GRP6) flanked by helper T-cell epitopes for increased immunogenicity, including HSP65, a tetanus toxoid fragment from amino acids 830 to 844 (T), pan-HLA-DR-binding epitope (PADRE) (P), and two repeats of a mycobacterial HSP70 fragment from amino acids 407 to 426 (M). The anti-GRP DNA vaccine (pCR3.1-VS-HSP65-TP-GRP6-M2) was constructed on a backbone of a pCR3.1 plasmid vector with eight 5'-GACGTT-3' CpG motifs and the VEGF183 signal peptide (VS). Intramuscular (IM) injections of anti-GRP vaccine in mice stimulated the production of high titers of specific antibodies against GRP and suppressed the growth of subcutaneous tumors of B16-F10 melanoma cells. Parallel results were obtained in vitro, showing inhibition of B16-F10 cell proliferation by GRP antisera. IM injections of the DNA vaccine also significantly attenuated tumor-induced angiogenesis associated with intradermal tumors of B16-F10 cells. In addition, lung invasion of intravenously injected cells was highly diminished, suggesting potent antimetastatic activity of the DNA vaccine. These findings support the highly immunogenic and potent antitumorigenic activity of specific anti-GRP antibodies elicited by the anti-GRP DNA vaccine.

FIG. 1.

Characterization of GRP-specific IgG from immunized mice. (A) Schematic diagram of pCR3.1-VS-HSP65-TP-GRP6-M2. In this DNA vaccine, the VS cDNA (VS) was placed under the control of promoter pCMV (arrow), followed sequentially by the genes encoding for HSP65 (black box), tetanus toxoid 830-844 (T), PADRE (P), six tandem repeats of human GRP_18-27 (6 X hGRP 18-27; GRP6), and two copies of mycobacterial HSP70_407-426 (mHSP70 407-426; M2). (B) Mice immunized with pCR3.1-VS-HSP65-TP-GRP6-M2 produced the highest titers of anti-GRP antibody than any other groups, especially at 7 weeks (wks) after the initial immunization (P < 0.001). OD₄₅₀, OD at 450 nm; KD, kilodaltons. (C) Specificity of anti-GRP antibodies was verified using immunoblot analysis. Proteins transferred onto nitrocellulose membranes were stained with Ponceau red (a) or incubated with sera from immunized mice (b). Lanes 1, protein markers; lanes 2, rhVEGF121-GRP_18-27 without DTT; lanes 3, rhVEGF121-GRP_18-27 with DTT; lanes 4, rhVEGF121 without DTT; lanes 5, rhVEGF121 with DTT. (D) Anti-GRP antibodies from sera of mice immunized with HSP70-containing plasmids had relative avidity significantly (P < 0.0001) higher than that for the group without, according to modified ELISA.

FIG. 2.

Effects of GRP and the anti-GRP vaccine on the proliferation of tumor cells. (A and B) GRP peptides stimulated the proliferation of cultured B16-F10 cells in a dose-dependent manner (*, P < 0.05; **, P < 0.01) (A), while GRP antisera suppressed their growth (*, P < 0.05), as seen with an MTT assay (B). OD₅₇₀, OD at 570 nm. (C) Immunization scheme. Arrows indicate immunizations (a); the tumors were extracted from mice immunized with saline (b; control, n = 8), pCR3.1-VS-HSP65-TP-M2 (c; control, n = 8), or pCR3.1-VS-HSP65-TP-GRP6-M2 (d; n = 8) 14 days after the tumor cell challenge. (D) Tumor weights from mice immunized with pCR3.1-VS-HSP65-TP-GRP6-M2 were significantly lower than those from the saline group or the vaccine control group. ***, P < 0.001.

FIG. 3.

Effects of the anti-GRP vaccine on the growth and angiogenesis of intradermal tumors. (A) Light microscope picture of B16-F10 tumor cells implanted intradermally in the anterior abdominal wall and the development of new blood vessels. Tumor-associated angiogenesis in mice injected with saline (a and b) and the pCR3.1-VS-HSP65-TP-M2 control vaccine (c and d) appeared to be significantly greater than in mice immunized with the pCR3.1-VS-HSP65-TP-GRP6-M2 anti-GRP vaccine (e and f). Representative images were taken with an objective that was ×10 (a, c, and e) or higher (b, d, and f). (B) The total number of blood vessels was determined within the precise 1-cm² area around each implant site. **, P < 0.01.

FIG. 4.

Effects of the anti-GRP vaccine on lung metastasis of intravenous B16-F10 cells. (A) Representative lungs from mice (n = 8 for each group) immunized with saline (a) or with the pCR3.1-VS-HSP65-TP-M2 control vaccine (b) showed numerous darkly pigmented metastatic melanoma cells compared with mice immunized with the pCR3.1-VS-HSP65-TP-GRP6-M2 anti-GRP vaccine (c). (B and C) Lungs of mice (n = 8 for each group) immunized with the pCR3.1-VS-HSP65-TP-GRP6-M2 anti-GRP vaccine were significantly (P < 0.05) lighter and contained fewer tumor nodules than lungs from the saline group or from the pCR3.1-VS-HSP65-TP-M2 control vaccine group. Values are means ± standard deviations. *, P < 0.05; ***, P < 0.001.