Function of CSF1 and IL34 in Macrophage Homeostasis, Inflammation, and Cancer.

Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model.

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.

Lead optimization of a 4-aminopyridine benzamide scaffold to identify potent, selective, and orally bioavailable TYK2 inhibitors.

Herein we report our lead optimization effort to identify potent, selective, and orally bioavailable TYK2 inhibitors, starting with lead molecule 3. We used structure-based design to discover 2,6-dichloro-4-cyanophenyl and (1R,2R)-2-fluorocyclopropylamide modifications, each of which exhibited improved TYK2 potency and JAK1 and JAK2 selectivity relative to 3. Further optimization eventually led to compound 37 that showed good TYK2 enzyme and interleukin-12 (IL-12) cell potency, as well as acceptable cellular JAK1 and JAK2 selectivity and excellent oral exposure in mice. When tested in a mouse IL-12 PK/PD model, compound 37 showed statistically significant knockdown of cytokine interferon-γ (IFNγ), suggesting that selective inhibition of TYK2 kinase activity might be sufficient to block the IL-12 pathway in vivo.

Evaluation of heterophilic antibody blocking agents in reducing false positive interference in immunoassays for IL-17AA, IL-17FF, and IL-17AF.

IL-17AA, IL-17FF, and IL-17AF are proinflammatory cytokines that have been implicated in the pathogenesis of autoimmune diseases such as rheumatoid arthritis (RA). In order to measure the levels of these cytokines in synovial fluid and serum samples from RA patients, immunoassays specific for IL-17AA, FF, and AF were developed. Although these assays could tolerate up to 50% pooled normal human serum, false positive reactivity was problematic in patient samples suggesting interference from heterophilic antibodies. We therefore evaluated the ability of several commercially available heterophilic antibody blocking agents to reduce false positive reactivity by testing them against samples that were confirmed as false positives in the IL-17AA, FF, and AF assays. Several of the blockers performed well, including HBR-1, HBR-9, HBR-11, HBR-Plus, Serum Cytokine Assay Diluent, and IIR. We chose to move forward using IIR blocker for sample analysis and verified that IIR had no effect on the assay standard curves and did not affect IL-17 quantitation in plasma from ex vivo stimulated human whole blood. IL-17FF and IL-17AF were below the limits of quantitation of the assays (12.3 and 10.5pg/ml, respectively) in synovial fluid and serum samples from patients with RA and osteoarthritis (OA). For the more sensitive IL-17AA assay (1.6pg/ml limit of quantitation), low levels of IL-17AA were measurable in 48% of RA synovial fluid samples (mean, 7.9pg/ml; median, <1.6pg/ml; range, <1.6-29.7pg/ml; n=23) but not in synovial fluid from patients with OA (n=33). For serum samples, however, IL-17AA was below the limit of detection for both RA and OA patients. When these same serum samples were analyzed in the absence of a heterophilic antibody blocker, false positive reactivity yielded apparent mean IL-17AA levels of 43.3pg/ml (28% positive; n=50) and 14.8pg/ml (12% positive; n=50) for RA and OA patients, respectively, results that could potentially be interpreted as consistent with disease biology. These studies demonstrate the importance of ensuring that HAb interference is well controlled, particularly when measuring low concentrations of cytokines in samples from patients with autoimmune disease.

Antibodies specific for a segment of human membrane IgE deplete IgE-producing B cells in humanized mice.

IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.