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Comparative Study
. 2025 Dec;45(1):1-16.
doi: 10.1080/01652176.2025.2473717. Epub 2025 Mar 10.

Clinical evaluation of HuDo-CSPG4 DNA electroporation as adjuvant treatment for canine oral malignant melanoma: comparison of two vaccination protocols

Affiliations
Comparative Study

Clinical evaluation of HuDo-CSPG4 DNA electroporation as adjuvant treatment for canine oral malignant melanoma: comparison of two vaccination protocols

Mariateresa Camerino et al. Vet Q. 2025 Dec.

Abstract

Canine oral malignant melanoma (OMM) is an aggressive, spontaneously occurring tumor carrying a poor to guarded prognosis and relatively limited therapeutic strategies. In this landscape, chondroitin sulfate proteoglycan (CSPG)4 represents a promising immunotherapeutic target. The objective of this bi-center prospective study was to examine the clinical outcome of OMM-bearing dogs treated with surgery and adjuvant electroporation using a DNA vaccine (HuDo-CSPG4) encoding both human (Hu) and canine (Do) portions of CSPG4 through two different vaccination protocols. Dogs with stage I-III surgically resected CSPG4-positive OMM underwent HuDo-CSPG4 plasmid electroporation starting at the 3rd-4th post-operative week; electrovaccination was repeated after 2 weeks. In protocol 1, electrovaccination was then delivered monthly while in protocol 2, electrovaccination was performed monthly four additional times followed by semestral boosters. The survival rates of HuDo-CSPG4-vaccinated dogs were estimated and compared with a control group treated with surgery alone. Significantly longer overall survival times were observed in HuDo-CSPG4 vaccinated dogs as compared with non-vaccinated controls. Dogs receiving protocol 2 showed similar outcomes to those of dogs undergoing protocol 1, despite fewer vaccinations. The comparable humoral response against CSPG4 resulting from the administration of protocol 1 and 2 appears to have similar clinical relevance, highlighting protocol 2 as the optimal vaccination schedule.

Keywords: CSPG4; Canine oral malignant melanoma; DNA electroporation; HuDo-CSPG4; adjuvant immunotherapy.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Immunization protocol 1 and protocol 2.
Figure 2.
Figure 2.
Immunohistochemical staining of canine OMM with an anti-CSPG4 antibody. (A) Moderate expression within tumor cells (score = 5/8). (B-D) high expression of CSPG4 in scattered neoplastic cells (score 8/8, B; score 7/8, C and D). Melanin (stained in brown) is clearly distinguishable (C) from the immunohistochemical reaction. Skeletal muscle used as negative antibody tissue control (E).
Figure 3.
Figure 3.
Kaplan-Meier curves comparing the MST of HuDo-CSPG4 (black line) and control (grey line) groups. Log-rank test, *p = 0.01.
Figure 4.
Figure 4.
Kaplan-Meier curves comparing the MST (A) and DFI (B) of dogs undergoing protocol I (P1, grey line) and protocol 2 (P2, black line). Log Rank test, MST, p = 0.99; DFI, p = 0.18.
Figure 5.
Figure 5.
(A) Kaplan-Meier curves comparing the MST of dogs undergoing protocol I (P1, black line) and control group (ctrl, grey line). Log Rank test, p = 0.99. (B) Kaplan-meier curves comparing the MST of dogs undergoing protocol II (P2, black line) and control group (ctrl, grey line). Log Rank test, *p = 0.001.
Figure 6.
Figure 6.
HuDo-CSPG4 electroporation induces a specific cspg4 antibody response in OMM-affected dogs included in both vaccination protocols. Analysis, by means of ELISA assay, of the presence of IgG against the human D1 (a and C) and canine D2 + D3 (B and D) domains of the CSPG4 protein in the sera of dogs collected before the first (Pre-vax), after the 6th (6-vax) vaccination (a and B) and at three-month intervals thereafter (ctrl 1,2,3; C and D). Results express the optical density (O.D.) at the absorbance measured at 450 nm or the fold change between the O.D. measured in the post-6th vax/Pre-vax. Paired student’s t-test: *p < 0,0238; **p = 0.0046.
Figure 7.
Figure 7.
(A and B) Kaplan-Meier curves correlating the overall survival of responder (R, continuous black lines) versus non-responders (NR, dotted black lines) dogs to the anti-CSPG4 vaccination. “responders” are the dogs which developed a specific IgG response against the do–D2 + D3 domains, with a higher level in the Post-Vax sera as compared to Pre-Vax sera (measured by ELISA, fold change Post-Vax/Pre-vax O.D. at 450 nm >1.1), considering the 6th vaccination (A) or any time points during treatment. (C and D) Kaplan-Meier curves correlating the overall survival of the entire population (C) or of the responder dogs only (D), with a high (continuous black lines) or low (dotted black lines) absolute antibody titer, measured in their post-vaccination sera by ELISA, considering as cut-off the median of the absorbance at 450 nm. Log Rank test, **p = 0.008.

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