Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases. Here, we describe how structure-based optimization of compound 2 resulted in the discovery of 34 (MHV370, (S)-N-(4-((5-(1,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methyl)bicyclo[2.2.2]octan-1-yl)morpholine-3-carboxamide). Its in vivo activity allows for further profiling toward clinical trials in patients with autoimmune disorders, and a Phase 2 proof of concept study of MHV370 has been initiated, testing its safety and efficacy in patients with Sjögren's syndrome and mixed connective tissue disease.

Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.

Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.

Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs). It was functionally cross reactive with mouse TLR7 but lacked oral exposure and had only modest potency. Chemical optimization resulted in 2, which showed in vivo efficacy following intraperitoneal administration. Further optimization resulted in 8 which had excellent in vitro activity, exposure and in vivo activity. Additional work to improve physical properties resulted in 15, an advanced lead that had favorable in vitro and exposure properties. It was further demonstrated that activity of the series tracked with binding to the extracellular domain of TLR7 implicating that the target of this series are endosomal TLRs rather than downstream signaling pathways.

Checkpoint inhibition has transformed immunotherapy by alleviating T cell exhaustion in a subset of patients. However, an important component of effective immune targeting to expand the benefit of immune response requires engagement of both innate and adaptive responses. Our understanding of safe and effective engagement of the innate immune system is evolving, with multiple preclinical and clinical agents targeting pathways such the Toll-like Receptors. Here we disclose the structure and preclinical activity of LHC165, a benzonapthyridine TLR7 agonist that is adsorbed to aluminum hydroxide. The interaction between LHC165 and aluminum hydroxide allows for a slow release from the injection site resulting in improved efficacy in mouse models compared with free LHC165. This localization allows for immune activation at the site of the tumor and also results in lower systemic exposure and cytokine induction. Intratumoral studies in syngeneic preclinical studies show single agent activity and a benefit when dosed in combination with checkpoint blockade. LHC165 as a single agent and in combination with PDR001 is currently enrolling patients with advanced malignancies in CLHC165X2101. Citation Format: Jonathan A. Deane, German A. Cortez, Chun Li, Nora Eifler, Shailaja Kasibhatla, Nehal Parikh, Shifeng Pan, Steven Bender. Identification and characterization of LHC165, a TLR7 agonist designed for localized intratumoral therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4128.

Pathogenic infections are important environmental modifiers of autoimmune disease as they can alter the immune system in a way that either promotes or reduces autoimmune responses. We tested the effect of infection with vesicular stomatitis virus (VSV) on the FcγR2B−/− (R2) mouse strain that spontaneously develops lupus disease. Infecting FcγR2B−/− mice with live VSV reduces autoantibody levels and inflammatory pathology and prolongs survival. We showed that VSV-mediated protection can be transferred in a CD8 cell adoptive transfer experiment. After further testing the transfer of various CD8 T cell subsets, we have identified CD8+CD122+NKG2D+ cells as mediator in VSV-induced Lupus amelioration. RNA Sequencing data for gene expression profiles among various CD8 subsets, indicate that the protective cells express higher level of genes linked to memory phenotype. Our working hypothesis is that the viral infection may induce the expansion of long lived CD8 populations that regulate the antibody response. In support of the idea we initiated to identify the target of the VSV-primed CD8 T cells in vivo. We examined the effect of VSV-primed CD8 T cells on the immune phenotypes of recipient R2 mice at the initial stages, e.g. after 1, 2 or 3 weeks of transfer. Interestingly, we found that Tfh and germinal center B cells are significantly reduced after three weeks of VSV-CD8 transfer. Thus, the protective CD8 T cells from VSV-infected mice might target autoreactive germinal center to regulate the antibody response. How VSV-CD8 T cells might control germinal center & why it takes about 3 weeks for controlling is under investigation.

Pathogenic infections are important environmental modifiers of autoimmune disease as they can alter the immune system in a way that either promotes or reduces autoimmune responses. We tested the effect of infection with vesicular stomatitis virus (VSV) on the FcγR2B−/− (R2) mouse strain that spontaneously develops lupus disease. Infecting FcγR2B−/− mice with live VSV reduces autoantibody levels and inflammatory pathology, and prolongs survival. We showed that VSV-mediated protection can be transferred in a CD8 cell adoptive transfer experiment. Legend screening for surface markers on CD8 T cells indicated that CD8 T cells from VSV infected mice express high level of markers linked to exhausted and regulatory phenotypes. After testing the transfer of various CD8 T cell subsets, we have identified CD8+CD122+NKG2D+ cells as mediator in VSV-induced Lupus amelioration. Preliminary analysis of RNA Sequencing data for gene expression profiles among various CD8 subsets, indicate that the protective cells express higher level of genes linked to memory phenotype. Our working hypothesis is that the viral infection may induce the expansion of long lived CD8 populations that regulate the humoral response.

Subunit vaccines are typically poorly immunogenic when administered alone, and require adjuvants for robust responses. Triggering TLRs to boost antigen-specific adaptive immunity is an attractive approach to increase the potency and quality of vaccines. However, recent reports suggest that alterations in TLR expression are associated with the pathogenesis of inflammatory and autoimmune diseases. To compare genetic studies with adjuvant studies, we examined whether stimulation through a TLR agonist induces or increases the autoimmune phenotype of healthy or autoimmune mice. C57BL/6, MRL/lpr, and Fc gamma r2b-deficient mice were dosed i.p. with Poly I:C every other day for 3 weeks, and monitored for signs of autoimmunity over 3 months. A separate group of mice was vaccinated three times i.m. with rPA anthrax antigen with or without Poly I:C with 2 weeks between doses. Immunized groups exhibited robust responses to vaccine and C57BL/6 and MRL/lpr mice showed a statistically significant increase in anti-rPA IgG responses in the presence of Poly I:C. Interestingly, Fc gamma r2b-/- mice showed increases with the base rPA vaccine, which was not significantly increased when Poly I:C was used as an adjuvant. In the chronically dosed groups, we also observed subtle alterations in levels of total antibody and some autoantibodies. However, there were no statistically significant differences in autoimmune syndrome, as measured by proteinurea, kidney pathology, weight loss, and mortality, with Poly I:C administration in chronic or vaccination mode. Taken together, these results suggest that administration of TLR3 agonists in chronic or vaccination mode does not induce or exacerbate models of systemic lupus erythematosus.

Toll-like receptor 7 (Tlr7) has been linked to systemic lupus disease incidence in humans and mice, but how TLR7 potentiates autoimmunity is unclear. We used a Tlr7 transgenic (tg) mouse model to investigate the cellular and molecular events required to induce spontaneous autoimmunity through increased TLR7 activity. We determined that Tlr7 exerts B-cell-intrinsic effects in promoting spontaneous germinal center (GC) and plasmablast B-cell development, and that these B-cell subsets are dependent on T-cell-derived signals through CD40L and SLAM-associated protein (SAP), but not IL-17. Antigen specificity also factored into TLR7-induced disease, as both a restricted T cell receptor (TCR) specificity and MHC haplotype H2(k/k) protected Tlr7tg mice from spontaneous lymphocyte activation and autoantibody production. Inflammatory myeloid cell expansion and autoimmunity did not develop in Tlr7tgIgH(-/-) mice, suggesting either that spontaneous TLR7 activation does not occur in dendritic cells, or, if it does occur, cannot drive these events in the absence of B-cell aid. These data indicate that autoimmune disease in Tlr7tg mice is contingent upon B cells receiving stimulation both through innate pathways and T-cell-derived signals and suggest a codependent relationship between B cells and T cells in the development of autoimmunity.

Type I IFNs (IFN-I) are normally produced during antiviral responses, yet high levels of chronic IFN-I expression correlate with autoimmune disease. A variety of viral sensors generate IFN-I in their response, but other than TLRs, it is not fully known which pathways are directly involved in the development of spontaneous immune pathologies. To further explore the link between IFN-I induced by viral pathways and autoimmunity, we generated a new transgenic mouse line containing multiple copies of Ifih1, a gene encoding the cytoplasmic dsRNA sensor MDA5 with proven linkage to diabetes and lupus. We show that MDA5 overexpression led to a chronic IFN-I state characterized by resistance to a lethal viral infection through rapid clearance of virus in the absence of a CD8(+) or Ab response. Spontaneous MDA5 activation was not sufficient to initiate autoimmune or inflammatory pathology by itself, even though every immune cell population had signs of IFN activation. When combined with the lupus-susceptible background of the FcγR2B deficiency, MDA5 overexpression did accelerate the production of switched autoantibodies, the incidence of glomerulonephritis, and early lethality. Thus, MDA5 transgenic mice provide evidence that chronic elevated levels of IFN-I are not sufficient to initiate autoimmunity or inflammation although they might exacerbate an ongoing autoimmune pathology.

Abstract MDA5 is a pattern recognition receptor responsible for detecting double stranded viral RNA in the cytoplasm. Ligation of MDA5 leads to the induction of inflammatory cytokines and type one interferons (IFN-I) that help coordinate the adaptive and innate immune response against the virus. Here, we describe the phenotype of mice overexpressing MDA5 under its endogenous promoter. MDA5 transgenic (MDA5 Tg) mice have normal numbers of B and T cells, yet expanded numbers of CD11b+ cells expressing Ly6C. Splenocytes isolated from MDA5 Tg display an interferon gene expression signature. MDA5 Tg CD8+ T cells have increased surface expression of CD44, CD69, Ly6C and Ly6A/E, indicative of an activated phenotype. In vitro, CD8+ T cells from MDA5 tg hyperrespond to stimulation. Surprisingly, MDA5 tg mice fail to mount a CD8+ T cell-driven antigen-specific immune response against Vesicular stomatitis virus (VSV). MDA5 tg mice also lacked VSV neutralizing antibodies 16 days after infection with the virus. Although MDA5 tg mice failed to mount a full immune response against the virus, they were resistant to a lethal dose of VSV. This result suggests that increased viral resistance of MDA5 Tg is independent of antibody-neutralization and CD8+ T cell-mediated clearance, and might be due to high expression levels of interferon inducible inhibitors of virus replication. MDA5 tg mice could serve as a model for diseases involving type one interferon and CD8+ T cells, such as Type One Diabetes.

We have investigated whether infection by pathogens affects the development of autoimmune diseases such as lupus. We tested the effect of infection with vesicular stomatitis virus (VSV) on two mouse strains that spontaneously develop lupus disease. Infection of VSV in lupus prone FcγR2B-/- (R2) mice significantly reduced autoantibody titers and inflammatory cytokines and chemokines in serum, reduced glomerulonephritis, and improved survival. In contrast, VSV infection in R2yaa mice, which develop a more aggressive lupus disease, showed slightly reduced auto-antibody titers in the serum without any major changes in serum cytokines and chemokines, kidney glomreulonephritis, or survival. Characterization of immune cell phenotypes in VSV infected R2 mice showed that B cell development in the bone marrow was not affected whereas germinal center, plasma, and IgG producing mature B cell numbers were reduced. Additionally, VSV infected R2 mice had lower number of follicular helper T cells and CD11c+ cells in the spleen. Compared to uninfected mice, microarray analysis of naïve resting B cells from VSV infected R2 splenocytes showed up-regulation of several candidate genes including a non receptor type protein tyrosine phosphatase, PTPN22 that has been shown to be associated with Lupus and other autoimmune diseases. Studies of the functional implications of Ptpn22 overexpression in VSV-infected R2 B cells are in progress.

Abstract Nucleic-acid sensing molecules provide a first line of defense against microbial infections, but recent studies have shown that their inappropriate activation could promote autoimmunity. MDA-5 senses virally-derived RNA in the cytoplasm, which leads to type one interferon (IFN) production, recently shown to be associated with systemic lupus erythematosus. Here, we have generated mice with multiple copies of Ifih1, the gene that encodes MDA-5, and crossed them to the lupus-prone FcgammaRIIB-/- mouse. Mice with enhanced MDA-5 expression and FcgammaRIIB deficiency have accelerated autoimmunity with splenomegaly and anti-nuclear antibodies in their serum compared to FcgammaRIIB-deficient mice alone. They also display elevated levels of splenic plasma and germinal center B cells, indicating possible early autoreactive B cell formation. Bone marrow and splenic macrophage/monocytes from MDA5 transgenic animals also display an activated phenotype, characterized by elevated Ly6A/E, PDCA-1 and CD80 expression. B cells from MDA-5-transgenic animals also express elevated levels of type one IFN signature genes. These results suggest that increased expression of MDA-5 could exacerbate lupus, possibly through the early production of type one IFN.

Both genetic and environmental factors, including infection by pathogens, affect the development of autoimmune diseases such as lupus. We are investigating the effect of viral infections on the lupus model of FcγR2B-/- mice. We infected FcγR2B-/- mice a single time at 2 months of age with vesicular stomatitis virus (VSV) and observed the development of autoimmune disease for the following 6 months. Infection with VSV significantly reduced autoantibody titers in serum and improved survival of FcγR2B-/- mice. B cell development in the bone marrow was not affected in VSV infected mice whereas memory B cells, and germinal center, plasma, and IgG producing mature B cell numbers were reduced. VSV infected FcγR2B-/- mice had lower number of follicular helper T cells and CD11c+ cells in the spleen. Increased survival in these mice correlated with a reduction of inflammatory cytokines and chemokines in the serum. The fact that a single round of infection with VSV confers long time protection from lupus in this mouse model indicates that the suppressor mechanism underlying the VSV effect on autoimmunity could be an excellent candidate for therapies seeking long term efficacy.

Phosphoinositide kinase (PI3K) is activated by various receptors on lymphocytes and regulates development, activation, and tolerance. Genetic ablation of PI3K function in T cells leads to the appearance of autoimmune disorders. In B cells, loss of the class IA regulatory subunit p85 alpha causes a partial defect in B cell development and proliferation, whereas loss of p85 beta alone causes no apparent changes in B cell function. Here we investigate further the consequences of p85 beta deletion in B cells, in the presence or absence of p85 alpha. We demonstrate that p85 beta partially compensates for loss of p85 alpha in B cell development and peripheral survival, with greater defects observed when both isoforms are absent. BCR-mediated AKT phosphorylation is partially reduced in p85 alpha-deficient B cells and further diminished with concomitant loss of p85 beta. Unexpectedly, loss of p85 beta results in increased BCR-mediated proliferation and ERK phosphorylation. These results indicate that the p85 beta regulatory isoform has partially overlapping functions with p85 alpha in B cells as well as a unique role in opposing BCR responses.