Anti-tumor efficacy of plasmid encoding emm55 in a murine melanoma model

Abstract

Emm55 is a bacterial gene derived from Streptococcus pyogenes (S. pyogenes) that was cloned into a plasmid DNA vaccine (pAc/emm55). In this study, we investigated the anti-tumor efficacy of pAc/emm55 in a B16 murine melanoma model. Intralesional (IL) injections of pAc/emm55 significantly delayed tumor growth compared to the pAc/Empty group. There was a significant increase in the CD8+ T cells infiltrating into the tumors after pAc/emm55 treatment compared to the control group. In addition, we observed that IL injection of pAc/emm55 increased antigen-specific T cell infiltration into tumors. Depletion of CD4+ or CD8+ T cells abrogated the anti-tumor effect of pAc/emm55. Combination treatment of IL injection of pAc/emm55 with anti-PD-1 antibody significantly delayed tumor growth compared to either monotherapy. pAc/emm55 treatment combined with PD-1 blockade enhanced anti-tumor immune response and improved systemic anti-tumor immunity. Together, these strategies may lead to improvements in the treatment of patients with melanoma.

Keywords: Bacterial protein; Intralesional injection; Melanoma; Streptococcus pyogenes; emm55.

Figures

Fig. 1. pAc/emm55 delays tumor growth in B16 melanoma bearing mice.

B16 bearing mice with palpable tumors (approximately 25 mm2) received three doses of IL injection of pAc/emm55 or pAc/Empty vector on a weekly interval. Invivo-jetPEI transfection reagent (VWR) was mixed with pAc/emm55 or pAc/Empty vector to get homogenous complex for IL injection. Tumor growth was measured and monitored 2–3 times a week. (a) end point PCR expression on tumor cells transfected with pAc/emm55 in vitro and (b) qRT-PCR on one representative tumor per group; (c) Tumor weight * p<0.05 Students t-test. (d) Tumor growth curves, CGGC permutation test. (e) Survival curves, log rank test. Data represents mean + SEM, N=12 per group.

Fig. 2. IL injection of pAc/emm55 enhances T cell infiltration in B16 tumors.

(a) B16 melanoma tumors were harvested and the numbers of CD3+ CD8+ T cells and CD4+ T cells was measured within the tumor by flow cytometry. The cell number per mg of tumor was compared (p < 0.05, unpaired T test). (b) Representative images of Immunohistochemistry analysis for T cell infiltration within tumors. Bar graphs represent the positive staining of T cell intensities in the membrane of each cell. We observed trends in the numbers of infiltrating CD3+ and CD8+ cells with CD4+ cells reaching statistical significance p = 0.049. * p<0.05

Fig. 3

Systemic immune responses elicited by the pAc/emm55 vector in B16 bearing mice is T cell dependent. C57BL/6 mice were injected with (a) 3 weekly doses of pAc/Empty or pAc/emm55 plasmid IL. CGGC permutation test p=0.02. Mice were pretreated with (b) anti-CD4 or (c) anti-CD8 antibody (300 μg/200μL/intraperitoneal) starting on day −3 and continuing twice a week until the endpoint. Control mice received isotype control. CD4 or CD8 T cells abrogates the activity of the emm55 vector. The p values utilizing the student t-test is presented for the last two time points on the growth curve. (d) Combined graph with all controls represented. N = 6 per group.

Fig. 4. Systemic immune responses elicited by pAc/emm55 vector in B16 bearing mice are antigen specific.

C57BL/6 were injected subcutaneously with M05 cells and mice received intralesional injection of pAc/empty or pAc/emm55 vector on days 7 and 14. In addition, the mice received 5*106 OT-1 T cells (CD45.1+) systemically. After 48 hours, tumors were harvested and tetramer specific CD8+ T cells were measured by flow cytometry. The bar graphs represent the percent of (a) CD8+ and (b) CD8+ OVA tetramer+ positive cells (n=5 in each group). (c) Representative flow plots comparing both treatment groups illustrating the increase in tetramer recognition with emm55 plasmid therapy. * p<0.05

Fig. 5. IFNγ production increases upon the addition of pAc/emm55 vector and anti-PD-1 therapy.

Murine melanoma B16 cells were injected subcutaneously on day 0. pEmpty vector or pEmm55 vector was injected on days 7, 14, and 21. Starting on day 8, mice received intraperitoneal injection of either isotype control antibody or anti-PD-1 blocking antibody (250 μg/200μL/IP) twice weekly (a). (b) Splenocytes collected from mice treated with emm55 vector ± anti-PD-1 therapy were co-cultured with B16 melanoma cells and culture supernatants were collected after 24 hours. Levels of IFNγ were measured (p < 0.01 utilizing unpaired T test with Bonferroni correction). (c) Tumor sizes for these B16 tumors at day 25 were also measured and demonstrated an increased response with pEmm55/anti-PD-1 compared to anti-PD-1 therapy alone (p < 0.05; unpaired T test) (d) Tumor growth curves from each treatment group. (N=12 for treatment groups, N=9 for control pEmpty group). * p<0.05

Fig. 6. Expression of emm55 enhances the ability of DC to stimulate T cells.

M05 tumor cells were not transfected (NT) or transfected with pAc/empty or pAc/emm55 plasmid. (a) Tumor cells or (b) DC pulsed with tumor cell lysates were co-cultured alone or with OT-I T cells for 24 hours and supernatants were collected. Controls included OT-I T cells alone (CM) or co-cultured with OVA-SIINFEKL peptide (SIINFEKL). IFN-gamma was measured by ELISA. (c) Lysates of tumor cells transfected with pEmpty or pEmm55 were pulsed onto wild-type or MyD88 k/o DC and co-cultured with OT-I T cells. Controls included OT-I T cells alone or co-cultured with DC pulsed with OVA-SIINFEKL peptide. IFN-gamma was measured in supernatants after a 24-hour co-culture.