Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma

Abstract

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies.

Fig. 1

Vaccination protocol used in 34 dogs with advanced stage, spontaneously arising malignant melanoma

Fig. 2

Detection of canine gp100 mRNA expression in canine melanoma cells. Total RNA samples purified from a human melanoma M21 cell line (positive control; lane A), four canine melanoma cell lines (lanes B, C, D, E), and two osteosarcoma cell lines (human, lane F; canine, lane G) were subjected to reverse transcription-PCR analysis. The expected PCR products for gp100 mRNA (399 bp) were size fractionated onto a 1% agarose gel and stained with ethidium bromide. Sequence analysis of the PCR product confirmed gp100 identification. gp100 transcripts were strongly expressed in melanocytic cell lines (lanes A–E) and weakly expressed in cells of non-melanocytic origin (lanes F and G). PCR amplification of β-actin was used as an internal control

Fig. 3

Detection of canine gp100, MART-1, and tyrosinase mRNA expression in the canine melanoma cell line, 17CM98. Total RNA was purified from several different canine melanoma cell lines and subjected to RT-PCR analysis. The expected PCR products for various melanoma antigens were size fractionated onto a 1% agarose gel and stained with ethidium bromide. Pictured is a representative gel of the canine melanoma cell line 17CM98 that strongly expressed three melanoma-associated antigens: gp100, MART-1, and tyrosinase

Fig. 4

Flow cytometric analysis in canine melanoma cells (17CM98) transfected with hgp100. 17CM98 cells were transfected with hgp100 using the Accell gene gun and transferred to flasks containing growth media. Adherent cells were harvested the next day, permeablized, stained with anti-gp100 HMB45 antibody, and secondary GAM-FITC antibody. A transfection efficiency of 12% hgp100-positive cells was observed in 17CM98 cells transfected with hgp100 (line) when compared with mock-transfected cells (filled peak)

Fig. 5

Kaplan–Meier curves for (a) time to progression (TTP) for dogs achieving tumor control (CR/PR/SD) and (b) overall survival time for dogs experiencing tumor control (CR/PR/SD; n=12) or no response (n=22) after receiving hgp100-ACTV

Fig. 6

PBMC cytotoxic activity against allogeneic tumor cells pre- and post-ATCV treatment. PBMC collected pre-treatment and at the time of the eighth vaccine treatment were co-cultured for 18 h with autologous melanoma cells from the dog’s primary tumor. Following incubation, cytolytic activity was measured using the CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). PBMC cytotoxic activity pre and post hgp100-ATCV treatment. At PBMC to tumor cell ratios of 10:1, 20:1 and 40:1, there was a marked increase in percent cytotoxicity by PBMC post-treatment, compared to pre-treatment