pAc/Emm55 increases anti-melanoma immunity via TLR2 signaling in dendritic cells and improves checkpoint blockade efficacy.

BACKGROUND: pAc/Emm55 (IFx-Hu2.0) is a plasmid DNA therapy encoding a Streptococcus pyogenes-derived bacterial gene. It shows anti-tumor activity in preclinical models, but the mechanisms of anti-melanoma immunity and additive effects with immune checkpoint blockade remain unclear. METHODS: B16 melanoma tumor-bearing mice were treated intratumorally with pAc/Emm55 alone and/or intraperitoneally with PD-1, CTLA-4, or LAG-3 blockade and monitored for tumor growth. Antibodies against Emm55 and melanoma cells (B16 or YUMM) were quantified by flow cytometry. Bone marrow-derived dendritic cells (BMDCs) from WT, MyD88 KO, TLR2 KO, or TLR7 KO mice were assessed for antigen uptake, OVA cross-presentation, and OT-I priming after pulsing with cell lysates from Emm55- or empty-transfected melanoma cells. Tumor immune infiltration by CD8 and CD11c cells were evaluated by immunohistochemistry. RESULTS: IFx-Hu2.0 significantly reduced tumor growth, correlating with increased intratumoral CD8+ T cell infiltration and enhanced systemic antibody responses against melanoma cells. TLR deficiency did not alter OVA uptake or basal BMDC cross-presentation. However, Emm55-enhanced OT-I priming and IFN-γ production required TLR2 and MyD88, but not TLR7, supporting a TLR2-MyD88-dependent mechanism. Emm55 monotherapy and anti-PD-1 each reduced tumor burden. The combination further improved inhibition. Adding anti-CTLA-4 or anti-LAG-3 to anti-PD-1 did not provide additional meaningful benefit. CONCLUSIONS: Emm55 facilitates the TLR2-MyD88 pathway in DCs and subsequent IFN-γ production by OT-I CD8 T cells. Combining intratumoral IFx-Hu2.0 with PD-1 blockade improves tumor control, supporting clinical evaluation of this strategy as also suggested by the first-in-human clinical trial of IFx-Hu2.0.