Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling.

Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown in vitro to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an in vivo human functional readout. AZ617 elicited a strong human TNFα and IL-6 response in vivo that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release in vitro. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to ex vivo stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an in vivo model system.

NRF2 activator A-1396076 ameliorates inflammation in autoimmune disease models by inhibiting antigen dependent T cell activation.

Nuclear factor (erythroid-derived 2) like 2 (NRF2) is a nuclear transcription factor activated in response to oxidative stress that induces a gene program that dampens inflammation and can limit cell damage that perpetuates the inflammatory response. We have identified A-1396076, a potent and selective NRF2 activator with demonstrated KEAP1 binding and modulation of cellular NRF2 mediated effects. In vivo administration of A-1396076 inhibits inflammation across several rodent models of autoimmunity when administered at or before the time of antigen challenge while also inducing NRF2 modulated gene transcription in the liver of the animals. It was not effective when administered after the time of antigen challenge or in a T cell independent model of arthritis induced by passive transfer of anti-collagen antibodies. A-1396076 inhibited antigen dependent T cell activation as measured by IFN-γ production in an ex vivo re-stimulation assay and following anti-CD3 challenge of MOG-sensitized mice. A-1396076 reduced costimulatory molecule expression on dendritic cells in the lungs of OVA LPS challenged mice suggesting that the mechanism of T cell inhibition was mediated at least partially by interfering with antigen presentation. These data suggest that NRF2 activation may be an effective strategy to dampen inflammation for treatment of autoimmune disease.

Treatment with a CD40 Antagonist Antibody Reverses Severe Proteinuria and Loss of Saliva Production and Restores Glomerular Morphology in Murine Systemic Lupus Erythematosus.

CD40 is a costimulatory receptor on APCs that is critical for the induction and maintenance of humoral and cell-mediated immunity. Accordingly, CD40 and its ligand, CD40L, have long been considered targets for the treatment of autoimmune diseases. We developed a rat/mouse chimeric anti-mouse CD40 antagonist mAb, 201A3, and evaluated its ability to alleviate murine lupus. Treatment of NZB/W-F1 mice with 201A3 after the onset of severe proteinuria rapidly reversed established severe proteinuria and nephritis and largely restored normal glomerular and tubular morphology. This coincided with a normalization of the expression of genes associated with proteinuria and injury by kidney parenchymal cells. Anti-CD40 treatment also prevented and reversed loss of saliva production and sialadenitis. These effects on kidney and salivary gland function were confirmed using mice of a second strain, MRL/Mp-lpr/lpr, and extended to alleviating joint inflammation. Immunologically, anti-CD40 treatment disrupted multiple processes that contribute to the pathogenesis of systemic lupus erythematosus (SLE), including autoreactive B cell activation, T effector cell function in target tissues, and type I IFN production. This ability to disrupt disease-critical immunological mechanisms, to reverse glomerular and tubular injury at the cellular and gene expression levels, and to confer exceptional therapeutic efficacy suggests that CD40 is a central disease pathway in murine SLE. Thus, a CD40 antagonist Ab could be an effective therapeutic in the treatment of SLE.

ABBV-105, a selective and irreversible inhibitor of Bruton's tyrosine kinase, is efficacious in multiple preclinical models of inflammation.

OBJECTIVES: Bruton's tyrosine kinase (BTK) is a non-receptor tyrosine kinase required for intracellular signaling downstream of multiple immunoreceptors. We evaluated ABBV-105, a covalent BTK inhibitor, using in vitro and in vivo assays to determine potency, selectivity, and efficacy to validate the therapeutic potential of ABBV-105 in inflammatory disease. METHODS: ABBV-105 potency and selectivity were evaluated in enzymatic and cellular assays. The impact of ABBV-105 on B cell function in vivo was assessed using mechanistic models of antibody production. Efficacy of ABBV-105 in chronic inflammatory disease was evaluated in animal models of arthritis and lupus. Measurement of BTK occupancy was employed as a target engagement biomarker. RESULTS: ABBV-105 irreversibly inhibits BTK, demonstrating superior kinome selectivity and is potent in B cell receptor, Fc receptor, and TLR-9-dependent cellular assays. Oral administration resulted in rapid clearance in plasma, but maintenance of BTK splenic occupancy. ABBV-105 inhibited antibody responses to thymus-independent and thymus-dependent antigens, paw swelling and bone destruction in rat collagen induced arthritis, and reduced disease in an IFNα-accelerated lupus nephritis model. BTK occupancy in disease models correlated with in vivo efficacy. CONCLUSION: ABBV-105, a selective BTK inhibitor, demonstrates compelling efficacy in pre-clinical mechanistic models of antibody production and in models of rheumatoid arthritis and lupus.