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Abstract Background TLR7 is a key player in the antiviral immunity. TLR7 signaling activates antigen-presenting cells including DCs and macrophages. This activation results in the adaptive immunity including T cells and B cells. Therefore, TLR7 is an important molecule of the immune system. Based on these observations, TLR7 agonists considered to become a therapy weaponize the immune system against cancer. Radiation therapy (RT) is one of the standard cancer therapies and is reported to modulate the tumor immune response. In this study, we aimed to investigate the anti-tumor activity in combination of TLR7 agonist, DSP-0509, with RT and underlying mechanism. Result We showed that anti-tumor activity is enhanced by combining RT with the TLR7 agonist DSP-0509 in the CT26, LM8, and 4T1 inoculated mice models. We found that once- weekly (q1w) dosing of DSP-0509 rather than biweekly (q2w) dosing is needed to achieve superior anti-tumor activities in CT26 model. Spleen cells from the mice in RT/DSP-0509 combination treatment group showed increased tumor lytic activity, inversely correlated with tumor volume, as measured by the chromium-release cytotoxicity assay. We also found the level of cytotoxic T lymphocytes (CTLs) increased in the spleens of completely cured mice. When the mice completely cured by combination therapy were re-challenged with CT26 cells, all mice rejected CT26 cells but accepted Renca cells. This rejection was not observed with CD8 depletion. Furthermore, levels of splenic effector memory CD8 T cells were increased in the combination therapy group. To explore the factors responsible for complete cure by combination therapy, we analyzed peripheral blood leukocytes (PBLs) mRNA from completely cured mice. We found that Havcr2low, Cd274low, Cd80high, and Il6low were a predictive signature for the complete response to combination therapy. An analysis of tumor-derived mRNA showed that combination of RT and DSP-0509 strongly increased the expression of anti-tumor effector molecules including Gzmb and Il12. Conclusion These data suggest that TLR7 agonist, DSP-0509, can be a promising concomitant when used in combination with RT by upregulating CTLs activity and gene expression of effector molecules. This combination can be an expecting new radio-immunotherapeutic strategy in clinical trials.

Toll-like receptor 7 (TLR7) acts as a crucial component of the innate immune system. Upon TLR7 binding to its ligand, myeloid cells, including dendritic cells (DCs) and macrophages, are activated and play vital roles in initiating adaptive immunity. Consequently, TLR7 agonists have been employed in cancer immunotherapy. We have synthesized DSP-0509, a systemic injectable TLR7 agonist, and in this investigation, we examined the effects of DSP-0509 on tumor-infiltrating lymphocytes (TILs) utilizing single-cell RNA sequencing (scRNA-seq) in a mouse model bearing tumors. Our results demonstrated that DSP-0509 induced an expansion of immune cell populations, such as Natural Killer (NK) cells, CD4+ T cells, and CD4+ regulatory T cells (Tregs). Subsequently, we combined an Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitor with DSP-0509 to enhance the antitumor efficacy by reducing Tregs, as DSP-0509 led to an increase in Treg presence within tumors. Our findings demonstrated that this combination therapy effectively reduced Treg infiltration within the tumor, leading to enhanced antitumor activity. To further prevent CD8+ T cell exhaustion, we combined DSP-0509 with an anti-PD-1 antibody and assessed the alterations in TILs using scRNA-seq. Our results indicated that the combination treatment significantly increased the cluster of CD8+ T cells expressing Gzmb, Prf1, Ctla4, and Icos, when compared to the administration of DSP-0509 alone. Additionally, we observed a marked rise in the M1-like macrophage cluster in the combination treatment group compared to the group receiving only DSP-0509. To validate the potential of modulating myeloid cells within the tumor to enhance antitumor efficacy, we combined DSP-0509 with an inhibitor targeting the receptor tyrosine kinase AXL. In bone marrow derived macrophages (BMDMs), the AXL inhibitor further amplified DSP-0509-stimulated TNFα secretion while reducing IL-10 secretion. As a final step, we evaluated the antitumor activity by combining DSP-0509 and the AXL inhibitor in an in vivo tumor model, which demonstrated increased efficacy. In summary, our study elucidated the effects of DSP-0509 on immune activity within the tumor microenvironment. These findings provided valuable insights that pave the way for the development of novel combination immunotherapy strategies.

TLR7 is an innate immune receptor that recognizes single-stranded RNAs, and its activation leads to anti-tumor immune effects. Although it is the only approved TLR7 agonist in cancer therapy, imiquimod is allowed to be administered with topical formulation. Thus, systemic administrative TLR7 agonist is expected in terms of expanding applicable cancer types. Here, we demonstrated the identification and characterization of DSP-0509 as a novel small-molecule TLR7 agonist. DSP-0509 is designed to have unique physicochemical features that could be administered systemically with a short half-life. DSP-0509 activated bone marrow-derived dendritic cells (BMDCs) and induced inflammatory cytokines including type I interferons. In the LM8 tumor-bearing mouse model, DSP-0509 reduced tumor growth not only in subcutaneous primary lesions but also in lung metastatic lesions. DSP-0509 inhibited tumor growth in several syngeneic tumor-bearing mouse models. We found that the CD8+ T cell infiltration of tumor before treatment tended to be positively correlated with anti-tumor efficacy in several mouse tumor models. The combination of DSP-0509 with anti-PD-1 antibody significantly enhanced the tumor growth inhibition compared to each monotherapy in CT26 model mice. In addition, the effector memory T cells were expanded in both the peripheral blood and tumor, and rejection of tumor re-challenge occurred in the combination group. Moreover, synergistic anti-tumor efficacy and effector memory T cell upregulation were also observed for the combination with anti-CTLA-4 antibody. The analysis of the tumor-immune microenvironment by using the nCounter assay revealed that the combination of DSP-0509 with anti-PD-1 antibody enhanced infiltration by multiple immune cells including cytotoxic T cells. In addition, the T cell function pathway and antigen presentation pathway were activated in the combination group. We confirmed that DSP-0509 enhanced the anti-tumor immune effects of anti-PD-1 antibody by inducing type I interferons via activation of dendritic cells and even CTLs. In conclusion, we expect that DSP-0509, a new TLR7 agonist that synergistically induces anti-tumor effector memory T cells with immune checkpoint blockers (ICBs) and can be administered systemically, will be used in the treatment of multiple cancers.

Forkhead box P3 (FOXP3) is a “master regulator” of regulatory T cells (Tregs), which are a subset of T cells that can suppress the antigen-specific immune reaction and that play important roles in host tolerance and immune homeostasis. It is well known that FOXP3 forms complexes with several proteins and can be regulated by various post-translational modifications (PTMs) such as acetylation, phosphorylation, ubiquitination, and methylation. As a consequence, the PTMs change the stability of FOXP3 and its capability to regulate gene expression, and eventually affect Treg activity. Although FOXP3 per se is not an ideal drug target, deacetylases, acetyltransferases, kinases, and other enzymes that regulate the PTMs of FOXP3 are potential targets to modulate FOXP3 and Treg activity. However, FOXP3 is not the only substrate for most of these enzymes; thus, unwanted “on target/off FOXP3” side effects must be considered when inhibitors to these enzymes are used. In this review, we summarize recent progress in the study of FOXP3 cofactors and PTM proteins, and potential clinical applications in autoimmunity and cancer immunity. Keywords: Treg, Forkhead box P3 (FOXP3), Post-translational modification, Autoimmune, Cancer

Arginine methylation is a post-translational modification that regulates many biological processes. However, the role of arginine methylation in immune cells is not well studied. Here we report an essential role of protein arginine methyltransferase 5 (PRMT5) in T cell homeostasis and activation-induced expansion. Using T cell-specific PRMT5 conditional knockout mice, we found that PRMT5 is required for natural killer T (NKT) cell but not for conventional or regulatory T (Treg) cell development after the double positive (DP) stage in the thymus. In contrast, PRMT5 was required for optimal peripheral T cell maintenance, for the transition of naïve T cells to effector/memory phenotype, and for early T cell development before the DP stage in a cell-intrinsic manner. Accordingly, PRMT5-deleted T cells showed impaired IL-7-mediated survival and TCR-induced proliferation in vitro. The latter was more pronounced and attributed to reduced responsiveness to IL-2. Acute deletion of PRMT5 revealed that not only naïve but also effector/memory T cells were impaired in TCR-induced proliferation in a development-independent manner. Reduced expression of common γ chain (γc), a shared receptor component for several cytokines including IL-7 and IL-2, on PRMT5-deleted T cells may be in part responsible for the defect. We further showed that PRMT5 was partially required for homeostatic T cell survival but absolutely required for lymphopenic T cell expansion in vivo. Thus, we propose that PRMT5 is required for T cell survival and proliferation by maintaining cytokine signaling, especially during proliferation. The inhibition of PRMT5 may provide a novel strategy for the treatment of diseases where uncontrolled T cell activation has a role, such as autoimmunity.

Breast cancer affects one out of eight women in their lifetime. Many factors contribute to the development of breast cancer, such as hereditary mutations and lifetime exposure to environmental factors, including estrogen. In addition, overweight and obesity, especially with increased waist circumference, are known to be associated with breast cancer risk. This review will summarize our understanding of the effect of diet on breast cancer incidence and progression. Since some inflammatory cytokines that are changed by a high-fat diet are known to promote the growth of breast cancer cells, these cytokines may serve as biomarkers to monitor the dietary influence for women at high risk of breast cancer and as future therapeutic targets for breast cancer treatment.

Regulatory T cells (Tregs) are engaged in maintaining immune homeostasis and preventing autoimmunity. Treg cells include thymic Treg cells and peripheral Treg cells, both of which can suppress the immune response via multiple distinct mechanisms. The differentiation, proliferation, suppressive function and survival of Treg cells are affected by distinct energy metabolic programs. Tissue-resident Treg cells hold unique features in comparison with the lymphoid organ Treg cells. Foxp3 transcription factor is a lineage master regulator for Treg cell development and suppressive activity. Accumulating evidence indicates that the activity of Foxp3 protein is modulated by various post-translational modifications (PTMs), including phosphorylation, O-GlcNAcylation, acetylation, ubiquitylation and methylation. These modifications affect multiple aspects of Foxp3 function. In this review, we define features of Treg cells and roles of Foxp3 in Treg biology, and summarize current research in PTMs of Foxp3 protein involved in modulating Treg function. This review also attempts to define Foxp3 dimer modifications relevant to mediating Foxp3 activity and Treg suppression. Understanding Foxp3 protein features and modulation mechanisms may help in the design of rational therapies for immune diseases and cancer.

Regulatory T cells (Tregs) are a subpopulation of T cells that are specialized in suppressing immune responses. Here we show that the arginine methyl transferase protein PRMT5 can complex with FOXP3 transcription factors in Tregs. Mice with conditional knock out (cKO) of PRMT5 expression in Tregs develop severe scurfy-like autoimmunity. In these PRMT5 cKO mice, the spleen has reduced numbers of Tregs, but normal numbers of Tregs are found in the peripheral lymph nodes. These peripheral Tregs that lack PRMT5, however, display a limited suppressive function. Mass spectrometric analysis showed that FOXP3 can be di-methylated at positions R27, R51, and R146. A point mutation of Arginine (R) 51 to Lysine (K) led to defective suppressive functions in human CD4 T cells. Pharmacological inhibition of PRMT5 by DS-437 also reduced human Treg functions and inhibited the methylation of FOXP3. In addition, DS-437 significantly enhanced the anti-tumor effects of anti-erbB2/neu monoclonal antibody targeted therapy in Balb/c mice bearing CT26Her2 tumors by inhibiting Treg function and induction of tumor immunity. Controlling PRMT5 activity is a promising strategy for cancer therapy in situations where host immunity against tumors is attenuated in a FOXP3 dependent manner.

Despite substantial clinical progress with targeted therapies, current antibody-based approaches have limited efficacy at controlling HER2/neu-positive breast cancers, especially in the absence of chemotherapies. Previously, we showed that the combination of IFNγ and anti-HER2/neu antibody synergistically reduces tumor growth in an in vivo implanted mammary tumor model. Here, we report a recombinant approach to produce an anti-HER2/neu scFv and IFNγ fusion protein using an engineered effector domain (EED) scaffold. The new molecule induces in vitro apoptosis in an IFNγ receptor-dependent manner. At a very low dose in the in vivo xenografted tumor models, the new EED-IFNγ fusion protein demonstrates superior activity over the anti-HER2/neu antibody and is even active on tumors that are resistant to anti-HER2/neu antibody therapy. Examination of tumor infiltrated macrophages and lymphocytes reveals that the fusion protein can induce changes in tumor microenvironment to support immune reactivity against tumors. Our studies have defined a targeted immunotherapy approach for the treatment of cancers.

Reversion of the malignant phenotype of erbB2-transformed cells can be driven by anti-erbB2/neu monoclonal antibodies (mAbs), which disrupt the receptor’s kinase activity. We examined the biologic effects of IFN-γ alone or after anti-erbB2/neu mAb treatment of erbB2-positive cells. IFN-γ had no effect on its own. Treatment of the tumors with anti-erbB2/neu mAbs followed by IFN-γ led to dramatic inhibition of tumor growth in vitro and in vivo with minimal mAb dosing. Sequential therapy enhanced the effects of chemotherapy. Moreover, IFN-γ with mAb treatment of mice with IFNγR knockdown tumors did not demonstrate marked synergistic eradication effects, indicating an unexpected role of IFN-γ on the tumor itself. Additionally, mAb and IFN-γ treatment also induced immune host responses that enhanced tumor eradication. Biochemical analyses identified loss of Snail expression in tumor cells, reflecting diminution of tumor-stem-cell-like properties as a consequence of altered activity of GSK3-β and KLF molecules.

The human FOXP3 molecule is an oligomeric transcriptional factor able to mediate activities that characterize T regulatory cells, a class of lymphocytes central to the regulation of immune responses. The activity of FOXP3 is regulated at the posttranslational level, in part by two histone acetyltransferases (HATs): TIP60 and p300. TIP60 and p300 work cooperatively to regulate FOXP3 activity. Initially, p300 and TIP60 interactions lead to the activation of TIP60 and facilitate acetylation of K327 of TIP60, which functions as a molecular switch to allow TIP60 to change binding partners. Subsequently, p300 is released from this complex, and TIP60 interacts with and acetylates FOXP3. Maximal induction of FOXP3 activities is observed when both p300 and TIP60 are able to undergo cooperative interactions. Conditional knockout of TIP60 in Treg cells significantly decreases the Treg population in the peripheral immune organs, leading to a scurfy-like fatal autoimmune disease.

In recent years, casting molds made by sand-type additive manufacturing (AM) technology are increasingly being used to build prototypes and small-lot production casting products. The development of this technology aiming at Mass-production applications in the future is progressing through the advancement of AM technologies and casting technologies. The application to Mass production is expected to increase the potentials of the whole casting, by realizing more complicated internal structure, reducing product thickness and weight by improving the cavity precision, etc. One method which can meet such needs is the current AM technique using furan sand molds. It is performed on large molding machines and is based on the organic self-hardening process utilizing mainly furan binder. This 3D AM sand mold using furan binder (hereafter referred to as "furan AM sand mold") contains sulfurous acid gas generated by the thermal decomposition of the catalyst used or organic gases generated by the thermal decomposition of the cured binder during pouring. In some cases, it is necessary to take measures such as removal of the gases from the molds. Other issues also need to be improved in terms of the working environment and casting quality. Examples of practically applied 3D AM methods using inorganic binders are; ink jetting water to plaster or cement, or ink jetting water to coated sand (laminated sand coated with water glass). However, various challenges are met with these methods, such as lack of high-speed large 3D printers and mold collapsibility is difficult after pouring. Under these circumstances, we attempted to fill a furan AM sand mold with an inorganic binder consisting of phosphate and sulfate, and then sinter the sand mold in an atmosphere of 850°C so that the furan AM sand mold changes into inorganic mold. With this mechanism, strong bonded layers are formed by the formation of polyphosphoric acid by the high molecularization of phosphate and the fusion of sulfate, while the thermal decomposition of the cured furan binder is progressing. As a result, this transforms the sand mold into a mold composed of only the inorganic binder. The resultant mold has been found to be sufficiently practical with almost no gas generation even under the condition of 1000°C. As for the problem of mold breakage, when water is added to the mold, the sulfate that forms the adhesion layer dissolves in the water, and this action allows the mold to be collapsed easily. These results confirm that this mold has a balanced combination of good heat resistance, reduced harmful gases, and good mold collapsibility. [ABSTRACT FROM AUTHOR]

TLR7 is an innate immune receptor that recognizes single-stranded RNAs, and its activation leads to anti-tumor immune effects. Although it is the only approved TLR7 agonist in cancer therapy, imiquimod is allowed to be administered with topical formulation. Thus, systemic administrative TLR7 agonist is expected in terms of expanding applicable cancer types. Here, we demonstrated the identification and characterization of DSP-0509 as a novel small-molecule TLR7 agonist. DSP-0509 is designed to have unique physicochemical features that could be administered systemically with a short half-life. DSP-0509 activated bone marrow-derived dendritic cells (BMDCs) and induced inflammatory cytokines including type I interferons. In the LM8 tumor-bearing mouse model, DSP-0509 reduced tumor growth not only in subcutaneous primary lesions but also in lung metastatic lesions. DSP-0509 inhibited tumor growth in several syngeneic tumor-bearing mouse models. We found that the CD8+ T cell infiltration of tumor before treatment tended to be positively correlated with anti-tumor efficacy in several mouse tumor models. The combination of DSP-0509 with anti-PD-1 antibody significantly enhanced the tumor growth inhibition compared to each monotherapy in CT26 model mice. In addition, the effector memory T cells were expanded in both the peripheral blood and tumor, and rejection of tumor re-challenge occurred in the combination group. Moreover, synergistic anti-tumor efficacy and effector memory T cell upregulation were also observed for the combination with anti-CTLA-4 antibody. The analysis of the tumor-immune microenvironment by using the nCounter assay revealed that the combination of DSP-0509 with anti-PD-1 antibody enhanced infiltration by multiple immune cells including cytotoxic T cells. In addition, the T cell function pathway and antigen presentation pathway were activated in the combination group. We confirmed that DSP-0509 enhanced the anti-tumor immune effects of anti-PD-1 antibody by inducing type I interferons via activation of dendritic cells and even CTLs. In conclusion, we expect that DSP-0509, a new TLR7 agonist that synergistically induces anti-tumor effector memory T cells with immune checkpoint blockers (ICBs) and can be administered systemically, will be used in the treatment of multiple cancers. [ABSTRACT FROM AUTHOR]

Regulation of the extent of immune responses is a requirement to maintain self-tolerance and limit inflammatory processes. CD4+CD25+Foxp3+ regulatory T (Treg) cells play a role in regulation. The Foxp3 transcription factor is considered a dominant regulator for Treg cell development and function. Foxp3 function itself is directly regulated by multiple posttranslational modifications that occur in response to various external stimuli. The Foxp3 protein is a component of several dynamic macromolecular regulatory complexes. The complexes change constituents over time and through different signals to regulate the development and function of regulatory T cells. Here we identified a mechanism regulating Foxp3 level and activity that operates through discrete phosphorylation. The Pim-2 kinase can phosphorylate Foxp3, leading to decreased suppressive functions of Treg cells. The amino-terminal domain of Foxp3 is modified at several sites by Pim-2 kinase. This modification leads to altered expression of proteins related to Treg cell functions and increased Treg cell lineage stability. Treg cell suppressive function can be up-regulated by either pharmacologically inhibiting Pim-2 kinase activity or by genetically knocking out Pim-2 in rodent Treg cells. Deficiency of Pim-2 activity increases murine host resistance to dextran sodium sulfate-induced colitis in vivo, and a Pim-2 small molecule kinase inhibitor also modified Treg cell functions. Our studies define a pathway for limiting the regulation of Foxp3 function because the Pim-2 kinase represents a potential therapeutic target for modulating the Treg cell suppressive activities in controlling immune responses. [ABSTRACT FROM AUTHOR]

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here, we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a "Grababody" that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the use of this platform recognizes p185her2/neu and includes an IgG binding domain. The recombinant scFv Grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185her2/neu. The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved antitumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications. [ABSTRACT FROM AUTHOR]