CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.

No abstract

Poxvirus-based active immunotherapies mediate anti-tumor efficacy by triggering broad and durable Th1 dominated T cell responses against the tumor. While monotherapy significantly delays tumor growth, it often does not lead to complete tumor regression. It was hypothesized that the induced robust infiltration of IFNγ-producing T cells into the tumor could provoke an adaptive immune evasive response by the tumor through the upregulation of PD-L1 expression. In therapeutic CT26-HER-2 tumor models, MVA-BN-HER2 poxvirus immunotherapy resulted in significant tumor growth delay accompanied by a robust, tumor-infiltrating T cell response that was characterized by low to mid-levels of PD-1 expression on T cells. As hypothesized, this response was countered by significantly increased PD-L1 expression on the tumor and, unexpectedly, also on infiltrating T cells. Synergistic benefit of anti-tumor therapy was observed when MVA-BN-HER2 immunotherapy was combined with PD-1 immune checkpoint blockade. Interestingly, PD-1 blockade stimulated a second immune checkpoint molecule, LAG-3, to be expressed on T cells. Combining MVA-BN-HER2 immunotherapy with dual PD-1 plus LAG-3 blockade resulted in comprehensive tumor regression in all mice treated with the triple combination therapy. Subsequent rejection of tumors lacking the HER-2 antigen by treatment-responsive mice without further therapy six months after the original challenge demonstrated long lasting memory and suggested that effective T cell immunity to novel, non-targeted tumor antigens (antigen spread) had occurred. These data support the clinical investigation of this triple therapy regimen, especially in cancer patients harboring PD-L1neg/low tumors unlikely to benefit from immune checkpoint blockade alone.

The dramatic clinical benefit of immune checkpoint blockade for a fraction of cancer patients suggests the potential for further clinical benefit in a broader cancer patient population by combining immune checkpoint inhibitors with active immunotherapies. The anti-tumor efficacy of MVA-BN-HER2 poxvirus-based active immunotherapy alone or in combination with CTLA-4 checkpoint blockade was investigated in a therapeutic CT26-HER-2 lung metastasis mouse model. MVA-BN-HER2 immunotherapy significantly improved the median overall survival compared to untreated controls or CTLA-4 blockade alone (p < 0.001). Robust synergistic efficacy was achieved with the combination therapy (p < 0.01). Improved survival following MVA-BN-HER2 administration was accompanied by increased tumor infiltration by HER-2-specific cytotoxic T lymphocytes (CTL). These tumor-specific CTL had characteristics similar to antiviral CTL, including strong expression of activation markers and co-expression of IFNγ and TNFα. Combination with CTLA-4 blockade significantly increased the magnitude of HER-2-specific T cell responses, with a higher proportion co-expressing TNFα and/or IL-2 with IFNγ. Furthermore, in mice treated with MVA-BN-HER2 (alone or in combination with CTLA-4 blockade), the inducible T cell co-stimulator (ICOS) protein was expressed predominantly on CD4 and CD8 effector T cells but not on regulatory T cells (Treg). In contrast, mice left untreated or treated solely with CTLA-4 blockade harbored elevated ICOS+ Treg, a phenotype associated with highly suppressive activity. In conclusion, poxvirus-based active immunotherapy induced robust tumor infiltration by highly efficient effector T cells. Combination with CTLA-4 immune checkpoint blockade amplified this response resulting in synergistically improved efficacy. These hypothesis-generating data may help elucidate evidence of enhanced clinical benefit from combining CTLA-4 blockade with poxvirus-based active immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-016-1816-7) contains supplementary material, which is available to authorized users.

The extracellular ATP/adenosine axis in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), otherwise known as CD39, is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by Ecto-5ʹ-nucleotidase/CD73 to immunosuppressive adenosine. Directly inhibiting the enzymatic function of CD39 via an antibody has the potential to unleash an immune-mediated anti-tumor response via two mechanisms: 1) increasing the availability of immunostimulatory extracellular ATP released by damaged and/or dying cells, and 2) reducing the generation and accumulation of suppressive adenosine within the TME. Tizona Therapeutics has engineered a novel first-in-class fully human anti-CD39 antibody, TTX-030, that directly inhibits CD39 ATPase enzymatic function with sub-nanomolar potency. Further characterization of the mechanism of inhibition by TTX-030 using CD39 + human melanoma cell line SK-MEL-28 revealed an uncompetitive allosteric mechanism (α < 1). The uncompetitive mechanism of action enables TTX-030 to inhibit CD39 at the elevated ATP concentrations reported in the TME. Maximal inhibition of cellular CD39 ATPase velocity was 85%, which compares favorably to results reported for antibody inhibitors to other enzyme targets. The allosteric mechanism of TTX-030 was confirmed via mapping the epitope to a region of CD39 distant from its active site, which suggests possible models for how potent inhibition is achieved. In summary, TTX-030 is a potent allosteric inhibitor of CD39 ATPase activity that is currently being evaluated in clinical trials for cancer therapy.