A novel next-generation FVIIIa mimetic, Mim8, has a favorable safety profile and displays potent pharmacodynamic effects: Results from safety studies in cynomolgus monkeys.

BACKGROUND: Mim8 is a novel, next-generation factor VIIIa mimetic in development for subcutaneous prophylactic treatment of patients with hemophilia A with and without inhibitors. In vitro and in vivo models indicate that Mim8 has a distinct hemostatic potential. OBJECTIVES: To test the nonclinical safety and pharmacodynamics of Mim8. METHODS: The Mim8 nonclinical safety program in cynomolgus monkeys consisted of three studies of 4-26 weeks in duration with Mim8 doses ranging from 0.3-60 mg/kg/week intravenously or subcutaneously. After sacrifice, macroscopic and microscopic pathological examinations were performed. RESULTS: Mim8 was well tolerated with no noteworthy clinical observations. No signs of excessive coagulation or pathological macroscopic or microscopic findings were observed at doses 0.3-3 mg/kg/week subcutaneous. Thrombosis-related findings were detected during histopathological examination in a small proportion of animals (16%) receiving doses ranging 6-20 mg/kg/week. Dose-dependent increases in factor X (FX) and factor IX (FIX) concentrations were observed. Shortening of activated partial thromboplastin time (APTT) and increased thrombin generation under ex vivo hemophilia A-like conditions were observed at all Mim8 dose levels. CONCLUSIONS: Thrombosis-related findings observed at doses above 6 mg/kg/week Mim8 may have been exaggerated pharmacological reactions to a procoagulant compound in normocoagulant animals. Increases in FX and FIX concentrations could be because of a half-life prolongation due to binding to Mim8, but were limited at clinically relevant exposure levels. Subcutaneous administration of up to 3 mg/kg/week (several fold greater than expected clinical exposure) for 26 weeks resulted in relevant pharmacodynamic effects, observed in thrombin generation and APTT, with no signs of thrombi or excessive coagulation activation.

OSCAR-collagen signaling in monocytes plays a proinflammatory role and may contribute to the pathogenesis of rheumatoid arthritis.

Osteoclast-associated receptor (OSCAR) is an activating receptor expressed by human myeloid cells. Collagen type I (ColI) and collagen type II (ColII) serve as ligands for OSCAR. OSCAR-collagen interaction stimulates RANK-dependent osteoclastogenesis. We have recently reported that OSCAR promotes functional maturation of monocyte-derived dendritic cells. OSCAR is upregulated on monocytes from rheumatoid arthritis (RA) patients with active disease, and these monocytes show an increased proosteoclastogenic potential. In the current study, we have addressed a functional role for an OSCAR-collagen interaction on monocytes. We show that OSCAR-ColII signaling promoted the survival of monocytes. Moreover, ColII stimulated the release of proinflammatory cytokines by monocytes from healthy donors, which could be completely blocked by an anti-OSCAR monoclonal antibody. Mononuclear cells from the synovial fluid of RA patients plated on ColII secreted TNF-α and IL-8 in an OSCAR-dependent manner. Global RNA profiling showed that components of multiple signaling pathways relevant to RA pathogenesis are regulated at the transcriptional level by OSCAR in monocytes. Thus, OSCAR can play a proinflammatory role in monocyte-derived cells and may contribute crucially on multiple levels to RA pathogenesis.

Overexpression of interleukin-1beta induces gastric inflammation and cancer and mobilizes myeloid-derived suppressor cells in mice.

Polymorphisms of interleukin-1beta (IL-1beta) are associated with an increased risk of solid malignancies. Here, we show that stomach-specific expression of human IL-1beta in transgenic mice leads to spontaneous gastric inflammation and cancer that correlate with early recruitment of myeloid-derived suppressor cells (MDSCs) to the stomach. IL-1beta activates MDSCs in vitro and in vivo through an IL-1RI/NF-kappaB pathway. IL-1beta transgenic mice deficient in T and B lymphocytes develop gastric dysplasia accompanied by a marked increase in MDSCs in the stomach. Antagonism of IL-1 receptor signaling inhibits the development of gastric preneoplasia and suppresses MDSC mobilization. These results demonstrate that pathologic elevation of a single proinflammatory cytokine may be sufficient to induce neoplasia and provide a direct link between IL-1beta, MDSCs, and carcinogenesis.

CD11b+/Gr-1+ immature myeloid cells mediate suppression of T cells in mice bearing tumors of IL-1beta-secreting cells.

Tumor cells secreting IL-1beta are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1beta-secreting cells restored immune reactivity and hematological alterations within 7-10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1beta-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1beta, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness.

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1alpha and IL-1beta), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1alpha (pIL-1alpha), mature IL-1beta (mIL-1beta), and mIL-1beta fused to a signal sequence (ssIL-1beta), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1alpha, which expresses IL-1alpha on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-gamma production. Cells transfected with secretable IL-1beta (mIL-1beta and ssIL-1beta) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1beta transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1beta tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1beta transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1beta transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-gamma and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1alpha tumor sites, whereas in mIL-1beta and ssIL-1beta tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1alpha and IL-1beta in malignant processes; IL-1alpha reduces tumorigenicity by inducing antitumor immunity, whereas IL-1beta promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

Characterization of CC-chemokine receptor 7 expression on murine T cells in lymphoid tissues.

Expression of the lymph node homing and CC-chemokine receptor 7 (CCR7), with L-selectin (CD62L), has been shown to divide human memory T cells into two functionally distinct subsets. We generated a polyclonal antibody against murine CCR7 and used this antibody to study CCR7 expression on murine T-cell subsets. Using flow cytometric staining of T cells for visualisation expression of CCR7 in association with CD62L and CD44, a major population of CD4 or CD8 T cells expressing CCR7 were found to be CD62Lhigh CD44low, which would suggest a naïve cell phenotype. By analogy with human studies, memory cells could be subdivided into CCR7high CD62Lhigh CD44high (central memory) and CCR7low CD62Llow CD44high (effector memory). The proportions of these populations were different in lymph node, blood and spleen. Functional, short-term in vitro polyclonal stimulation of blood, spleen and lymph node cells from naive mice demonstrated that CCR7high CD4 T cells produced predominantly interleukin (IL)-2, whereas CCR7low CD4 T cells produced both IL-2 and interferon-gamma (IFN-gamma). However, in contrast to previously published reports, the CCR7high CD8 T-cell subpopulation produced both IFN-gamma and IL-2. Analysis of effector T cells, induced by immunization in vivo, showed that a proportion of activated naïve CD4 T cells down-regulated CCR7 only after multiple cell divisions, and this coincided with the down-regulation of CD62L and production of IL-4 and IFN-gamma. Finally, analysis of effector T cells during the phase of maximal clonal expansion of secondary immune responses in vivo indicated that the vast majority of both IL-2- and IFN-gamma-producing cells are CCR7low, while few cytokine-expressing CCR7high T cells were detected. Our results support the hypothesis, developed from studies with human cells, that CCR7 may separate functionally different murine memory T-cell subpopulations, but indicate additional complexity in that CCR7high CD8 T cells also may produce IFN-gamma.