IntroductionThe aim was to determine the effect of the Bruton tyrosine kinase (Btk)-selective inhibitor PCI-32765, currently in Phase I/II studies in lymphoma trials, in arthritis and immune-complex (IC) based animal models and describe the underlying cellular mechanisms.MethodsPCI-32765 was administered in a series of murine IC disease models including collagen-induced arthritis (CIA), collagen antibody-induced arthritis (CAIA), reversed passive anaphylactic reaction (RPA), and passive cutaneous anaphylaxis (PCA). Clinical and pathologic features characteristic of each model were examined following treatment. PCI-32765 was then examined in assays using immune cells relevant to the pathogenesis of arthritis, and where Btk is thought to play a functional role. These included proliferation and calcium mobilization in B cells, cytokine and chemokine production in monocytes/macrophages, degranulation of mast cells and its subsequent cytokine/chemokine production.ResultsPCI-32765 dose-dependently and potently reversed arthritic inflammation in a therapeutic CIA model with an ED50 of 2.6 mg/kg/day. PCI-32765 also prevented clinical arthritis in CAIA models. In both models, infiltration of monocytes and macrophages into the synovium was completely inhibited and importantly, the bone and cartilage integrity of the joints were preserved. PCI-32765 reduced inflammation in the Arthus and PCA assays. In vitro, PCI-32765 inhibited BCR-activated primary B cell proliferation (IC50 = 8 nM). Following FcγR stimulation, PCI-32765 inhibited TNFα, IL-1β and IL-6 production in primary monocytes (IC50 = 2.6, 0.5, 3.9 nM, respectively). Following FcεRI stimulation of cultured human mast cells, PCI-32765 inhibited release of histamine, PGD2, TNF-α, IL-8 and MCP-1.ConclusionsPCI-32765 is efficacious in CIA, and in IC models that do not depend upon autoantibody production from B cells. Thus PCI-32765 targets not only B lymphocytes but also monocytes, macrophages and mast cells, which are important Btk-expressing effector cells in arthritis.
Key Points MCL cells are mobilized into the peripheral blood of patients treated with the BTK inhibitor ibrutinib. Ibrutinib dose-dependently inhibits BCR- and chemokine-mediated adhesion and migration of MCL cells.
954 PCI-32765 is an orally administered, highly potent and specific inhibitor of Bruton tyrosine kinase (BTK) in clinical development for the treatment of B cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often have marked but transient increases of circulating CLL lymphocytes following treatment with PCI-32765, as has been seen with other inhibitors of the B cell receptor (BCR) pathway. In the course of the Phase I study of PCI-32765, we have noted similar effects among treated patients with other types of non-Hodgkin lymphoma (NHL) including mantle cell lymphoma (MCL). We here characterize the patterns and phenotypes of cells mobilized among patients with MCL, and further investigate the mechanism of this effect. Nine patients with MCL treated in the previously reported Phase I study (Advani et al, ASCO, 2010) had baseline absolute lymphocyte counts (ALC) of 1.04 ± 0.42 (x 109/L, Mean ± SD) and had maximal increases during the first 28 day cycle of 12 to 794% (188% increase ± 250, Mean, SD). The ALCs of four patients who were treated on a dosing schedule that included a 1 week drug holiday within each cycle were noted to show intra-cyclic increases of ALC from day 1 to day 15 of each cycle, and decreases following each week off of treatment, for up to 9 cycles (Fig. 1). Patients receiving continuous dosing exhibited gradually decreasing ALCs following the first cycle. The cyclically increasing B lymphocytes were confirmed to be CD5+ (and often also CD45lo), and thus likely to represent circulating, mobilized lymphoma cells. Patient, D005, who attained a complete response, had an easily identifiable CD19+CD45lo subpopulation of 0.47 ×109 cells/L at baseline. This subpopulation increased to 15.2 × 109/L at day 8 of the first cycle, but then decreased markedly as the patient responded clinically. One patient who failed to respond had, by contrast, few if any detectable mobilized cells. Peripheral blood CD19+CD5+ cells from MCL patients treated with PCI-32765 after 8 days were found to have reduced levels of CXC chemokine receptor 4 (CXCR4) levels, whereas pretreatment malignant cells were CXCR4hi. This likely reflects the differences in MCL surface membrane phenotype in solid tissues compared to peripheral blood. Mechanistically, we found that PCI-32765 inhibited BCR- and CXCL12-mediated adhesion and chemotaxis of MCL cell lines in vitro (EC50 = 10–100 nM), and dose-dependently inhibited BCR, stromal cell and CXCL12 stimulations of pBtk, pPLCg and pErk in MCL cells. Importantly, PCI-32765 dose-dependently inhibited the pseudoemperipoleisis of MCL in the presence of stromal cells.Figure 1:Lymphocytic peripheral mobilization of Mantle Cell Lymphoma patients treated with PCI-32765Figure 1:. Lymphocytic peripheral mobilization of Mantle Cell Lymphoma patients treated with PCI-32765 Conclusion: Lymphocyte mobilization into the peripheral blood is notable from MCL in response to treatment with PCI-32765. The majority of these cells are marked with a phenotype (CD19+CD5+ CXCR4lo) which is consistent with malignant cells from secondary lymphoid organs. This effect is likely to be related to PCI-32765 inhibition of BTK activation which results in inhibition of MCL cell chemotaxis, adherence and pseudo-emperipoleisis. We propose that Btk is essential for the homing of MCL cells into secondary lymphoid organs, and that its inhibition results in peripheral blood compartment shift. Disclosures: Chang: Pharmacyclics Inc: Employment. Francesco:Pharmacyclics: Employment, Equity Ownership. Magadala:Pharmacyclics: Employment. Huang:Pharmacyclics: Employment. Spaargaren:Pharmacyclics: Research Funding. Buggy:Pharmacyclics, Inc.: Employment. Elias:Pharmacyclics Inc: Consultancy.
To identify novel protein therapeutics we have utilized an integrated screening system in which a collection of full-length human cDNA clones, encoding virtually all secreted proteins and receptors, was assembled. Soluble secreted proteins were expressed in a high-throughput format using human cells as the expression host and tested in high-throughput cell-based assays. Through this approach, a novel cytokine, FPT025, was identified in a human monocyte proliferation assay. FPT025 has no apparent sequence homology to known cytokines or any other genes and is expressed in human spleen, skin, brain, and other tissues. The purified recombinant FPT025 protein stimulated human primary monocyte proliferation and/or survival. FPT025 protein specifically bound to human primary monocytes and activated ERK1/2 phosphorylation in primary monocytes as well as in a human monocytic cell line, THP-1. FPT025 promoted formation of the myeloid lineage colonies, CFU-M, in a human bone marrow colony formation assay and enhanced proliferation of cells with myeloid cell surface markers from human monocytes. To identify the receptor of FPT025, a collection of extracellular domains (ECD) of transmembrane proteins was screened for their ability to block FPT025 activation of monocyte proliferation. In this screen, we found that FPT025 binds to the M-CSF receptor (M-CSFR) with high affinity. Furthermore, we showed that the binding of FPT025 to M-CSFR was specific and could be competed by M-CSF. The soluble ECD of M-CSFR inhibited both the binding of FPT025 to M-CSFR and the activity of FPT025 in monocyte proliferation. Therefore, FPT025 functions as a novel ligand of the M-CSF receptor and participates in the regulation of myeloid lineage differentiation, proliferation, and survival.
IL-34 is a newly identified cytokine that binds to the CSF-1 receptor (CSF-1R), promotes monocyte viability, and stimulates macrophage colony formation (Lin et al., 2008). Here we demonstrate that human IL-34 induced tyrosine phosphorylation of the CSF-1R in cells of a mouse MacCsf1r−/−.huCSF-1R macrophage line. MacCsf1r−/−.huCSF-1R cells were obtained by retroviral transduction of the human CSF-1R into cells of the MacCsf1r−/− macrophage line (Yu et al., 2008) derived from bone marrow macrophages isolated from a CSF-1R-deficient mouse. In addition, IL-34 stimulated the downstream signaling of ERK1/2 and the IL-34-induced phosphorylation of ERK1/2 was blocked by a CSF-1R-specific inhibitor. IL-34, in synergy with RANK ligand, promoted formation of tartrate resistant acid phosphatase (TRAP)-positive and multinucleated osteoclasts from monocytes in dose-dependent manner. The activity of IL-34 on osteoclast differentiation was inhibited by the soluble CSF-1R extracellular domain. Therefore, IL-34 functions as a new ligand of CSF-1R and participates in the regulation of osteoclast development.
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