Effect of quinoline-3-carboxamides on myeloid cells in inflammation and cancer

The quinoline-3-carboxamides (Q compounds) are a family of small molecules that ameliorate various types of murine inflammatory autoimmune disease. One such compound has also demonstrated anti-tumor effects in murine tumor models. Several Q compounds have reached clinical trials and one compound, laquinimod, is currently in phase III clinical trial for relapsing-remitting multiple sclerosis. The Q compounds bind to S100A9, which can act as a damage-associated molecular pattern by binding to its receptors TLR4 and RAGE. S100A9 has also been associated with the development of cancer as S100A9 promotes the accumulation and migration of an immunosuppressive myeloid cell population during tumor growth. The aim of this thesis was to investigate the effects of the Q compounds during inflammation and cancer, with particular focus on myeloid cells, in order to better understand the mode of action of these compounds. In the first paper presented, the effect of the Q compound paquinimod was evaluated on myeloid cell accumulation during acute peritonitis. Here, paquinimod specifically reduced the accumulation of inflammatory monocytes and eosinophils in the peritoneum and omentum. These observations were confirmed in subcutaneous matrigel plugs, suggesting that paquinimod may affect the influx of these cells to inflammatory sites. In the second paper, the same cell populations were studied in a mouse mammary carcinoma tumor. Short-term treatment with tasquinimod similarly reduced the recruitment of inflammatory monocytes to tumors. The anti-tumor effect of this compound mainly operated during the first few days of tumor growth and a comparable anti-tumor effect was observed when monocytes were depleted using an anti-Gr1 antibody. In addition, long-term tasquinimod treatment reduced the tumor-induced myeloid cell expansion in the spleen and normalized the changes in the composition of myeloerythroid progenitors in this compartment. In the third paper, the effect of paquinimod was evaluated in a mouse model of spontaneously developed inflammation and fibrosis. Paquinimod reduced the established inflammation in these mice and, remarkably, the fibrosis also regressed. This was associated with a decreased number of M2-polarized macrophages and also NKT cells, which have been shown to be integral for the development of the phenotype in these mice. Paquinimod also dramatically reduced the production of several Th2-associated cytokines by the NKT cells. In summary, the work in this thesis has identified monocytes as one cellular target of the Q compounds. As these cells have important roles in promoting development of inflammatory disease and cancer, these observations may explain the broad effects of the Q compounds observed in various disease models

The anti-tumor effect of the quinoline-3-carboxamide tasquinimod : Blockade of recruitment of CD11b+ Ly6Chi cells to tumor tissue reduces tumor growth

Background: Previous work has demonstrated immunomodulatory, anti-tumor, anti-metastatic and anti-angiogenic effects of the small molecule quinoline-3-carboxamide tasquinimod in pre-clinical cancer models. To better understand the anti-tumor effects of tasquinimod in transplantable tumor models, we have evaluated the impact of the compound both on recruitment of myeloid cells to tumor tissue and on tumor-induced myeloid cell expansion as these cells are known to promote tumor development. Methods: Mice bearing subcutaneous 4 T1 mammary carcinoma tumors were treated with tasquinimod in the drinking water. A BrdU-based flow cytometry assay was utilized to assess the impact of short-term tasquinimod treatment on myeloid cell recruitment to tumors. Additionally, long-term treatment was performed to study the anti-tumor effect of tasquinimod as well as its effects on splenic myeloid cells and their progenitors. Myeloid cell populations were also immune-depleted by in vivo antibody treatment. Results: Short-term tasquinimod treatment did not influence the proliferation of splenic Ly6Chi and Ly6Ghi cells, but instead reduced the influx of Ly6Chi cells to the tumor. Treatment with tasquinimod for various periods of time after tumor inoculation revealed that the anti-tumor effect of this compound mainly operated during the first few days of tumor growth. Similar to tasquinimod treatment, antibody-mediated depletion of Ly6Chi cells within that same time frame, caused reduced tumor growth, thereby confirming a significant role for these cells in tumor development. Additionally, long-term tasquinimod treatment reduced the splenomegaly and expansion of splenic myeloid cells during a later phase of tumor development. In this phase, tasquinimod normalized the tumor-induced alterations in myeloerythroid progenitor cells in the spleen but had only limited impact on the same populations in the bone marrow. Conclusions: Our results indicate that tasquinimod treatment reduces tumor growth by operating early after tumor inoculation and that this effect is at least partially caused by reduced recruitment of Ly6Chi cells to tumor tissue. Long-term treatment also reduces the number of splenic myeloid cells and myeloerythroid progenitors, but these effects did not influence established rapidly growing tumors

Amelioration of Experimental Autoimmune Encephalomyelitis by the Quinoline-3-Carboxamide Paquinimod (ABR-215757): Reduced Priming of Proinflammatory Effector CD4(+) T Cells.

Quinoline-3-carboxamide compounds (Q compounds) have demonstrated efficacy in treating autoimmune disease in both humans and mice. However, the mode of action of these compounds is poorly understood. Here, we show that preventive treatment with the Q compound paquinimod (ABR-215757) during the first 5 days after induction of experimental autoimmune encephalomyelitis is sufficient to significantly ameliorate disease symptoms. Parallel cell-depletion experiments demonstrated that Ly6C(hi) inflammatory monocytes play an essential role in this phase. The paquinimod-induced amelioration correlated with reduced priming of antigen-specific CD4(+) T cells and reduced frequency of IFN-γ- and IL-17-producing cells in draining lymph nodes. Importantly, the treatment did not inhibit T-cell division per se. In mice with established experimental autoimmune encephalomyelitis, the numbers of Ly6C(hi) CD115(+) inflammatory monocytes and CD11b(+)CD11c(+) dendritic cells (DCs) were reduced in spleen, but not in bone marrow or draining lymph nodes of treated mice. Inflammatory monocyte-derived DCs and CD4(+) T cells were also reduced in the brain. In contrast, there was no decrease in DC subsets previously shown to be critical for effector CD4(+) T-cell development in lymph nodes. Taken together, these data indicate that preventive treatment with paquinimod ameliorates experimental autoimmune encephalomyelitis by reducing effector T-cell priming and, on prolonged treatment, displays a selective effect by decreasing distinct subpopulations of splenic CD11b(+) myeloid cells

Rationale and clinical development of CD40 agonistic antibodies for cancer immunotherapy

Introduction: CD40 signaling activates dendritic cells leading to improved T cell priming against tumor antigens. CD40 agonism expands the tumor-specific T cell repertoire and has the potential to increase the fraction of patients that respond to established immunotherapies. Areas covered: This article reviews current as well as emerging CD40 agonist therapies with a focus on antibody-based therapies, including next generation bispecific CD40 agonists. The scientific rationale for different design criteria, binding epitopes, and formats are discussed. Expert opinion: The ability of CD40 agonists to activate dendritic cells and enhance antigen cross-presentation to CD8+ T cells provides an opportunity to elevate response rates of cancer immunotherapies. While there are many challenges left to address, including optimal dose regimen, CD40 agonist profile, combination partners and indications, we are confident that CD40 agonists will play an important role in the challenging task of reprogramming the immune system to fight cancer

A1M/α1-microglobulin is proteolytically activated by myeloperoxidase, binds its heme group and inhibits low density lipoprotein oxidation.

α1-microglobulin (A1M) is a 26 kDa plasma and tissue protein with reductase activity and radical- and heme-binding anti-oxidative functions. In addition, exposure of A1M to hemoglobin has been shown to induce proteolytic elimination of a C-terminal tetrapeptide yielding a heme-degrading form, truncated A1M (t-A1M). Myeloperoxidase (MPO), a heme-containing enzyme that catalyzes the production of free radicals and hypochlorite, is released by neutrophils during the inflammatory response to bacterial infections. MPO-induced low density lipoprotein (LDL)-oxidation in blood has been suggested as a causative factor in atherosclerosis. In this study we have hypothesized that A1M interacts with MPO in a similar mode as with hemoglobin, and is a regulator of its activity. The results show that A1M is proteolytically cleaved, with formation of t-A1M, after exposure to MPO, and that t-A1M contains iron and heme-degradation products. The reaction is dependent of pH, time and concentration of substrates and a pH-value around 7 is shown to be optimal for cleavage. Furthermore, A1M inhibits MPO- and hydrogen peroxide-induced oxidation of LDL. The results suggest that A1M may have a role as an inhibitor of the damaging effects of the neutrophil respiratory burst on bystander tissue components

The human anti-CD40 agonist antibody mitazalimab (ADC-1013; JNJ-64457107) activates antigen-presenting cells, improves expansion of antigen-specific T cells, and enhances anti-tumor efficacy of a model cancer vaccine in vivo

Non-responders to checkpoint inhibitors generally have low tumor T cell infiltration and could benefit from immunotherapy that activates dendritic cells, with priming of tumor-reactive T cells as a result. Such therapies may be augmented by providing tumor antigen in the form of cancer vaccines. Our aim was to study the effects of mitazalimab (ADC-1013; JNJ-64457107), a human anti-CD40 agonist IgG1 antibody, on activation of antigen-presenting cells, and how this influences the priming and anti-tumor potential of antigen-specific T cells, in mice transgenic for human CD40. Mitazalimab activated splenic CD11c+ MHCII+ dendritic cells and CD19+ MHCII+ B cells within 6 h, with a return to baseline within 1 week. This was associated with a dose-dependent release of proinflammatory cytokines in the blood, including IP-10, MIP-1α and TNF-α. Mitazalimab administered at different dose regimens with ovalbumin protein showed that repeated dosing expanded ovalbumin peptide (SIINFEKL)-specific CD8+ T cells and increased the frequency of activated ICOS+ T cells and CD44hi CD62L− effector memory T cells in the spleen. Mitazalimab prolonged survival of mice bearing MB49 bladder carcinoma tumors and increased the frequency of activated granzyme B+ CD8+ T cells in the tumor. In the ovalbumin-transfected tumor E.G7-OVA lymphoma, mitazalimab administered with either ovalbumin protein or SIINFEKL peptide prolonged the survival of E.G7-OVA tumor-bearing mice, as prophylactic and therapeutic treatment. Thus, mitazalimab activates antigen-presenting cells, which improves expansion and activation of antigen-specific T cells and enhances the anti-tumor efficacy of a model cancer vaccine

The immunomodulatory quinoline-3-carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis

Quinoline-3-carboxamides (Q substances) are small molecule compounds with anti-inflammatory properties. In this study, we used one of these substances, Paquinimod, to treat a novel model for chronic liver inflammation and liver fibrosis, the NOD-Inflammation Fibrosis (N-IF) mouse. We show that treatment of N-IF mice significantly reduced inflammation and resulted in the regression of fibrosis, even when the treatment was initiated after onset of disease. The reduced disease phenotype was associated with a systemic decrease in the number and reduced activation of disease-promoting transgenic natural killer T (NKT)-II cells and their type 2-cytokine expression profile. Paquinimod treatment also led to a reduction of CD115+ Ly6Chi monocytes and CD11b+ F4/80+ CD206+ macrophages