Ken Takashima

Adjuvant for vaccine immunotherapy of cancer – focusing on Toll‐like receptor 2 and 3 agonists for safely enhancing antitumor immunity

Immune‐enhancing adjuvants usually targets antigen (Ag)‐presenting cells to tune up cellular and humoral immunity. CD141+ dendritic cells (DC) represent the professional Ag‐presenting cells in humans. In response to microbial pattern molecules, these DCs upgrade the maturation stage sufficient to improve cross‐presentation of exogenous Ag, and upregulation of MHC and costimulators, allowing CD4/CD8 T cells to proliferate and liberating cytokines/chemokines that support lymphocyte attraction and survival. These DCs also facilitate natural killer‐mediated cell damage. Toll‐like receptors (TLRs) and their signaling pathways in DCs play a pivotal role in DC maturation. Therefore, providing adjuvants in addition to Ag is indispensable for successful vaccine immunotherapy for cancer, which has been approved in comparison with antimicrobial vaccines. Mouse CD8α+ DCs express TLR7 and TLR9 in addition to the TLR2 family (TLR1, 2, and 6) and TLR3, whereas human CD141+ DCs exclusively express the TLR2 family and TLR3. Although human and mouse plasmacytoid DCs commonly express TLR7/9 to respond to their agonists, the results on mouse adjuvant studies using TLR7/9 agonists cannot be simply extrapolated to human adjuvant immunotherapy. In contrast, TLR2 and TLR3 are similarly expressed in both human and mouse Ag‐presenting DCs. Bacillus Calmette–Guerin peptidoglycan and polyinosinic–polycytidylic acid are representative agonists for TLR2 and TLR3, respectively, although they additionally stimulate cytoplasmic sensors: their functional specificities may not be limited to the relevant TLRs. These adjuvants have been posted up to a certain achievement in immunotherapy in some cancers. We herein summarize the history and perspectives of TLR2 and TLR3 agonists in vaccine‐adjuvant immunotherapy for cancer.

Vaccine immunotherapy with ARNAX induces tumor-specific memory T cells and durable anti-tumor immunity in mouse models

Immunological checkpoint blockade therapies benefit a limited population of cancer patients. We have previously shown that vaccine immunotherapy with Toll‐like receptor (TLR)3‐adjuvant and tumor antigen overcomes anti‐programmed death ligand‐1 (PD‐L1) resistance in mouse tumor models. In the present study, 4 different ovalbumin (OVA)‐expressing tumor cell lines were implanted into syngeneic mice and subjected to anti‐tumor immunotherapy using ARNAX and whole OVA protein. ARNAX is a TLR3‐specific agonist that does not activate the mitochondrial antiviral‐signaling protein (MAVS) pathway, and thus does not induce systemic inflammation. Dendritic cell priming and proliferative CTL were induced by ARNAX + OVA, but complete remission was achieved only in a PD‐L1‐low cell line of EG7. Addition of anti‐PD‐L1 antibody to the ARNAX + OVA therapy brought complete remission to another PD‐L1‐high subline of EG7. Tumor shrinkage but not remission was observed in MO5 in that regimen. We analyzed tumor cells and tumor‐infiltrating immune cells to identify factors associated with successful ARNAX vaccine therapy. Tumors that responded to ARNAX therapy expressed high levels of MHC class I and low levels of PD‐L1. The tumor‐infiltrating immune cells in ARNAX‐susceptible tumors contained fewer immunosuppressive myeloid cells with low PD‐L1 expression. Combination with anti‐PD‐L1 antibody functioned not only within tumor sites but also within lymphoid tissues, augmenting the therapeutic efficacy of the ARNAX vaccine. Notably, ARNAX therapy induced memory CD8+ T cells and rejection of reimplanted tumors. Thus, ARNAX vaccine + anti‐PD‐L1 therapy enabled permanent remission against some tumors that stably present antigens.

The TLR3/TICAM-1 signal constitutively controls spontaneous polyposis through suppression of c-Myc in Apc Min/+ mice

BackgroundIntestinal tumorigenesis is promoted by myeloid differentiation primary response gene 88 (MyD88) activation in response to the components of microbiota in ApcMin/+ mice. Microbiota also contains double-stranded RNA (dsRNA), a ligand for TLR3, which activates the toll-like receptor adaptor molecule 1 (TICAM-1, also known as TRIF) pathway.MethodsWe established ApcMin/+Ticam1−/− mice and their survival was compared to survival of ApcMin/+Myd88−/− and wild-type (WT) mice. The properties of polyps were investigated using immunofluorescence staining and RT-PCR analysis.ResultsWe demonstrate that TICAM-1 is essential for suppression of polyp formation in ApcMin/+ mice. TICAM-1 knockout resulted in shorter survival of mice compared to WT mice or mice with knockout of MyD88 in the ApcMin/+ background. Polyps were more frequently formed in the distal intestine of ApcMin/+Ticam1−/− mice than in ApcMin/+ mice. Infiltration of immune cells such as CD11b+ and CD8α+ cells into the polyps was detected histologically. CD11b and CD8α mRNAs were increased in polyps of ApcMin/+Ticam1−/− mice compared to ApcMin/+ mice. Gene expression of inducible nitric oxide synthase (iNOS), interferon (IFN)-γ, CXCL9 and IL-12p40 was increased in polyps of ApcMin/+Ticam1−/− mice. mRNA and protein expression of c-Myc, a critical transcription factor for inflammation-associated polyposis, were increased in polyps of ApcMin/+Ticam1−/− mice. A Lactobacillus strain producing dsRNA was detected in feces of ApcMin/+ mice.ConclusionThese results imply that the TLR3/TICAM-1 pathway inhibits polyposis through suppression of c-Myc expression and supports long survival in ApcMin/+ mice.

Adjuvant immunotherapy for cancer: both dendritic cell-priming and check-point inhibitor blockade are required for immunotherapy

The immune system eliminates advanced cancer when treated with programmed cell death protein-1 (PD-1) or its ligand (PD-L1) blockade, but PD-1 therapy is effective in only ∼20% of patients with solid cancer. The PD-1 antibody mainly acts on the effector phase of cytotoxic T lymphocytes (CTLs) in tumors but induces no activation of the priming phase of antigen-presenting dendritic cells (DCs). It is reasonable that both DC-priming and PD-1/L1 blocking are mandatory for efficient CTL-mediated tumor cytolysis. For DC-priming, a therapeutic vaccine containing Toll-like receptor (TLR) agonists, namely a priming adjuvant, is a good candidate; however, a means for DC-targeting by TLR adjuvant therapy remains to be developed. TLR adjuvants usually harbor cytokine toxicity, which is a substantial barrier against drug approval. Here, we discuss the functional properties of current TLR adjuvants for cancer immunotherapy and introduce a TLR3-specific adjuvant (ARNAX) that barely induces cytokinemia in mouse models.

Toll-like receptor 3 signal augments radiation-induced tumor growth retardation in a murine model

Radiotherapy induces anti‐tumor immunity by induction of tumor antigens and damage‐associated molecular patterns (DAMP). DNA, a representative DAMP in radiotherapy, activates the stimulator of interferon genes (STING) pathway which enhances the immune response. However, the immune response does not always parallel the inflammation associated with radiotherapy. This lack of correspondence may, in part, explain the radiation‐resistance of tumors. Additive immunotherapy is expected to revive tumor‐specific CTL facilitating radiation‐resistant tumor shrinkage. Herein pre‐administration of the double‐stranded RNA, polyinosinic‐polycytidylic acid (polyI:C), in conjunction with radiotherapy, was shown to foster tumor suppression in mice bearing radioresistant, ovalbumin‐expressing Lewis lung carcinoma (LLC). Extrinsic injection of tumor antigen was not required for tumor suppression. No STING‐ and CTL‐response was induced by radiation in the implant tumor. PolyI:C was more effective for induction of tumor growth retardation at 1 day before radiation than at post‐treatment. PolyI:C targeted Toll‐like receptor 3 with minimal effect on the mitochondrial antiviral‐signaling protein pathway. Likewise, the STING pathway barely contributed to LLC tumor suppression. PolyI:C primed antigen‐presenting dendritic cells in draining lymph nodes to induce proliferation of antigen‐specific CTL. By combination therapy, CTL efficiently infiltrated into tumors with upregulation of relevant chemokine transcripts. Batf3‐positive DC and CD8+ T cells were essential for therapeutic efficacy. Furthermore, polyI:C was shown to stimulate tumor‐associated macrophages and release tumor necrosis factor alpha, which acted on tumor cells and increased sensitivity to radiation. Hence, polyI:C treatment prior to radiotherapy potentially induces tumor suppression by boosting CTL‐dependent and macrophage‐mediated anti‐tumor responses. Eventually, polyI:C and radiotherapy in combination would be a promising therapeutic strategy for radiation‐resistant tumors.

cGAMP Promotes Germinal Center Formation and Production of IgA in Nasal-Associated Lymphoid Tissue

Induction of immunoglobulin (Ig) A in the mucosa of the upper respiratory tract and the nasal cavity protects against influenza virus infection. Cyclic dinucleotides (CDNs) are used as mucosal adjuvants to enhance the immunogenicity of intranasal influenza hemagglutinin (HA) vaccines. The adjuvant activity of 2′3′ cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) on Ig production was investigated in nasal-associated lymphoid tissue (NALT), serum of wild-type C57BL/6J, and stimulator of interferon genes (STING)-deficient mice, which do not recognize cGAMP. Mice were vaccinated intranasally with a HA vaccine with or without the cGAMP adjuvant. IgA and IgG production, T-cell responses, germinal center formation, and cytokine expression in NALT were assayed. cGAMP enhanced IgA and IgG production, and promoted T-cell responses. Intranasal administration of cGAMP activated both NALT and systemic immune cells, induced a favorable cytokine environment for IgA induction, and promoted germinal center formation. The cGAMP effect was STING-dependent. Taken together, cGAMP as an HA vaccine adjuvant promoted a STING-dependent NALT environment suitable for the enhancement of IgA production.

DNAJB1/HSP40 Suppresses Melanoma Differentiation-Associated Gene 5-Mitochondrial Antiviral Signaling Protein Function in Conjunction with HSP70

Melanoma differentiation-associated gene 5 (MDA5) is a pattern recognition receptor that recognizes cytoplasmic viral double-stranded RNA (dsRNA) and initiates rapid innate antiviral responses. MDA5 forms a filament-like multimer along the dsRNA leading to oligomerization, which in turn activates the adaptor protein mitochondrial antiviral signaling protein (MAVS) to provide a signal platform for the induction of type I interferon (IFN) and proinflammatory cytokines. The conformational switch of MDA5 causes antiviral defense, but excessive activation of the MDA5-MAVS pathway may result in autoimmune diseases. The regulatory mechanisms of MDA5 activation remain largely unknown. By yeast 2-hybrid, we identified DNAJB1, a member of the HSP40 (heat shock protein 40) family, as an MDA5-binding protein. HSP40s usually cowork with HSP70s. We found that dsRNA stimulation with physiological conditions upregulated the expression levels of DNAJB1 and HSP70; then the proteins were coupled and translocated into the stress granules, where MDA5 encounters dsRNA. DNAJB1 disrupted MDA5 multimer formation, resulting in the suppression of type I IFN induction. The disruption of endogenous DNAJB1 increased MDA5- and MAVS-mediated IFN promoter activation and rendered cells virus resistant. HSP70 inhibitor also enhanced the IFN-inducing function of MDA5 and MAVS. These results suggest that the DNAJB1-HSP70 complex functions for the natural maintenance of RNA sensing by interacting with MDA5/MAVS.

RIOK3 keeps MDA5 inactive

RIG-I and MDA5 are cytoplasmic RNA sensors that recognize dsRNA patterns and activate MAVS to induce innate antiviral gene program. RNA sensing is regulated by ubiquitination in RIG-I while by phosphorylation in MDA5. Thus, de novo phosphorylation is an essential step for keeping MDA5 inactive. We identified a protein kinase RIO kinase 3 (RIOK3) targeting MDA5, and here discuss its possible relationship to autoimmune diseases. RNA pattern-sensing is a pivotal event in host defense against virus infections, which induces innate immune response, inflammation, and augmentation of lymphocyte functions. These are rooted in RNA sensor-mediated dendritic cell (DC) maturation. TLR3, 7 and 8 in endosome and RIG-I and MDA5 RNA helicases in cytoplasm are involved in RNA sensing in DCs. MDA5 recognizes relatively long double-stranded RNA yielded as a virus replication intermediate, leading to the formation of MDA5 filament required for activating the adaptor MAVS, then inducing IRF3 activation followed by type I IFN production. [1] Poliovirus, EMCV and measles virus are representative virus species recognized by MDA5. Notably, only a little RIG-I and MDA5 exist in resting cells and viral infection markedly up-regulates their mRNA levels in affected cells. Then, the proteins are activated sufficient to recognize cytoplasmic RNA. For RIG-I activation, ubiquitin ligases TRIM25 and Riplet are indispensable, whereas no ligase is responsible for MDA5 activation. Recent report suggested that MDA5 was activated by dephosphorylation by PP1 [2]; if so, phosphorylation of de novo MDA5 is a prerequisite for keeping MDA5 inactive. We identified that RIO kinase 3 (RIOK3) phosphorylates MDA5 to be inactivated (Figure ​(Figure1).1). RIOK3 selectively promotes C-terminal Ser- 828 phosphorylation of MDA5, which blocks MDA5 multimerazation and attenuates MDA5 signaling. [3]. Although another kinase might phosphorylate N-terminal region of MDA5, phosphorylation brings a dysfunctional conformation to MDA5 [2]. Figure 1 Sensing dsRNA by MDA5 Excess activation of MDA5 was recently reported to link the process of autoimmune diseases such as SLE and type I diabetes [4]. Viral infections sometimes trigger autoimmune disorders as reported clinically [5]. However, the mechanism by which autoimmune diseases are exacerbated by MDA5 over-activation remains undetermined. G821S mutation near the Ser- 828 in MDA5 appears to be associated with constitutive activation of MDA5 and closely links to autoimmune triggering [6]. In virus infections or oncogenesis, RNA is released outside the affected cells with exosomes. Thus, the source of MDA5 ligands would be provided through RNA replication or cell destruction. However, how RIOK3 is regulated in cells that take RNA into the cytoplasm is unknown yet. RIOK3 knockout (RIOK3 KO) in culture cells produced more robust type I IFN and inflammatory cytokines than wild-type cells in response to polyI:C or viral infections, which can increase MDA5 levels (Takashima K et al, unpublished data). The results infer that RIOK3 KO surely promotes activation of MDA5 (Figure ​(Figure11). We are aware that the autoimmune disorder involves a number of signal axes in a variety of cells in patients. Regulatory T cells (Treg), B cells producing anti-DNA/RNA Ab, and other lymphocytes are involved in the process of autoimmunity. We notice here that innate immune response to RNA may trigger autoimmune disorder. Recent reports further suggest that Regnase-1 vs Loquin recognize 3′-stem-structured mRNA and regulate cytokine production such as IL-6 and TNF−α, which may suppress autoimmune disorder [7]. RIG-I and MDA5 are upstream of the cytokine producing gene program. Regulatory mechanism of RIG-I mechanistically differs from that of MDA5. What happens for MDA5 activation in oncogenesis, which contrasts to autoimmunity, is also intriguing. What is the role of RIOK3 in the pathogenic process of autoimmunity in the context of MDA5 activation will be the next issue to be analyzed. Since MDA5 is ubiquitously expressed, what cell types are responsible for a trigger of auto-reactive lymphocytes is a coming topic. We find we are in a new gate to the clue for the mechanistic mystery of RNA-dependent induction of autoimmune diseases. We have elucidated the process of MDA5 activation, by which it recognizes cytoplasmic RNA. Excess RNA production via viral infections or tumor growth allows the cells to liberate a large amounts of structured RNA and facilitate autoimmune disorders (Figure ​(Figure1).1). We may find a new strategy to the early diagnosis, prevention and treatment of autoimmune diseases by investigating RIOK3 knockout mice.