Helena Eriksson

Investigating the use of Landsat thematic mapper data for estimation of forest leaf area index in southern Sweden

The study aims at investigating the use of Landsat thematic mapper (TM) for mapping leaf area index (LAI) in coniferous and deciduous forests in southern Sweden. LAI has been estimated in the field with optical measurements, allometric equations, and litter-trap data, and empirical relationships between LAI estimates and satellite-measured reflectances have been analysed. Several common vegetation indices and multiple regressions where estimated LAI is predicted as a function of various spectral bands are tested. The results indicate significant relationships between Landsat TM reflectances and parameters related to LAI, and the relationships are improved when separating coniferous and deciduous stands. The best relationships occur between Landsat TM data and the product of effective LAI as estimated with the LAI-2000 instrument and a needle clumping factor (LG), which explains about 80% of the variation in coniferous stands and about 50% of the variation in deciduous stands. The best single bands in coniferous stands are the middle-infrared bands (TM5 and TM7), and the best vegetation index is the moisture stress index (TM5/TM4). The best single band in deciduous stands is TM4, and the best vegetation index is the simple ratio (SR).

Tasquinimod modulates suppressive myeloid cells and enhances cancer immunotherapies in murine models

Shen, Sundstedt, and colleagues show in murine models that tasquinimod enhanced the antitumor effects of SurVaxM tumor vaccine for prostate cancer and of 5T4Fab-SEA tumor-targeted superantigen for melanoma by inhibiting the accumulation and function of tumor-infiltrating suppressive myeloid cells. A major barrier for cancer immunotherapy is the presence of suppressive cell populations in patients with cancer, such as myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM), which contribute to the immunosuppressive microenvironment that promotes tumor growth and metastasis. Tasquinimod is a novel antitumor agent that is currently at an advanced stage of clinical development for treatment of castration-resistant prostate cancer. A target of tasquinimod is the inflammatory protein S100A9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. Here, we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: a tumor vaccine (SurVaxM) for prostate cancer and a tumor-targeted superantigen (TTS) for melanoma. In the combination strategies, tasquinimod inhibited distinct MDSC populations and TAMs of the M2-polarized phenotype (CD206+). CD11b+ myeloid cells isolated from tumors of treated mice expressed lower levels of arginase-1 and higher levels of inducible nitric oxide synthase (iNOS), and were less immunosuppressive ex vivo, which translated into a significantly reduced tumor-promoting capacity in vivo when these cells were coinjected with tumor cells. Tumor-specific CD8+ T cells were increased markedly in the circulation and in tumors. Furthermore, T-cell effector functions, including cell-mediated cytotoxicity and IFNγ production, were potentiated. Taken together, these data suggest that pharmacologic targeting of suppressive myeloid cells by tasquinimod induces therapeutic benefit and provide the rationale for clinical testing of tasquinimod in combination with cancer immunotherapies. Cancer Immunol Res; 3(2); 136–48. ©2014 AACR.

Prophylactic treatment with S100A9 inhibitor paquinimod reduces pathology in experimental collagenase-induced osteoarthritis

Objectives Alarmins S100A8/A9 regulate pathology in experimental osteoarthritis (OA). Paquinimod is an immunomodulatory compound preventing S100A9 binding to TLR-4. We investigated the effect of paquinimod on experimental OA and human OA synovium. Materials and methods Two OA mouse models differing in level of synovial activation were treated prophylactic with paquinimod. Synovial thickening, osteophyte size and cartilage damage were measured histologically, using an arbitrary score, adapted Pritzker OARSI score or imaging software, respectively. Human OA synovia were stimulated with S100A9, with or without paquinimod. Results Paquinimod treatment of collagenase-induced OA (CIOA) resulted in significantly reduced synovial thickening (57%), osteophyte size at the medial femur (66%) and cruciate ligaments (67%) and cartilage damage at the medial tibia (47%) and femur (75%; n=7, untreated n=6). In contrast, paquinimod did not reduce osteophyte size and reduced cartilage damage at one location only in destabilised medial meniscus, an OA model with considerably lower synovial activation compared with CIOA. In human OA synovium, paquinimod blocked proinflammatory (interleukin (IL)-6, IL-8, tumour necrosis factor-α) and catabolic (matrix metalloproteinases 1 and 3) factors induced by S100A9 (n=5). Conclusions Prophylactic treatment of paquinimod reduces synovial activation, osteophyte formation and cartilage damage in experimental OA with high synovial activation (CIOA) and ameliorates pathological effects of S100A9 in OA synovium ex vivo.

Paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis.

BACKGROUND Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein. OBJECTIVE In this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model. METHODS Seven weeks old female B6.Cg-Fbn1(Tsk)/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test. RESULTS The results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFβ-response in the skin and an abrogation of the increased auto-antibody production in this SSc model. CONCLUSIONS Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

Monotherapeutically nonactive CTLA-4 blockade results in greatly enhanced antitumor effects when combined with tumor-targeted superantigens in a B16 melanoma model.

Immunotherapy aiming to block immune suppression with anti-cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) monoclonal antibodies is a recently clinically established strategy to enhance immune driven antitumor activities. To be successful, this approach depends on the existence of a suppressed immune response against the tumor that can be released by the treatment or alternatively needs to be combined with an immune-enhancing therapy. A tumor-targeted superantigen (TTS) fusion protein utilizes the strong T-cell activating property of bacterial superantigens in concert with the tumor cell binding capacity in antitumor Fab-fragments. Our purpose was to investigate the feasibility of combining anti-CTLA-4 with TTS therapy against the poorly immunogenic B16 mouse melanoma tumor transfected with the human tumor-associated antigen EpCAM recognized by the C215 monoclonal antibody. B16-EpCAM tumors growing in the lung were completely insensitive to anti-CTLA-4 monotherapy. C215Fab-SEA treatment of the B16-EpCAM tumors induced strong infiltration and targeting of both CD4+ and CD8+ T cells. In parallel, Foxp3+CTLA-4high regulatory T cells accumulated in the tumors. Combining activation with C215Fab-SEA and anti-CTLA-4 showed greatly enhanced antitumor effects and prolonged long-term survival. In parallel, when the expansion of regulatory T cells was inhibited, the number of specific effector T cells was enhanced and the cytotoxic T-lymphocyte activity was significantly improved. Collectively, these data emphasize the potential of combining cancer treatment using anti-CTLA-4 monoclonal antibodies with T-cell activation and directed killing by TTS therapy.

FRI0496 An Open-Label Study to Evaluate Biomarkers and Safety in Systemic Sclerosis (SSC) Patients Treated with Paquinimod (ABR-215757)

Background Paquinimod (ABR-215757) is an oral small molecule compound that belongs to the quinoline-3-carboxamide derivatives, a class of structurally related compounds with immunomodulatory properties. Paquinimod has shown beneficial effects in several autoimmune/inflammatory disease models, including experimental models of systemic sclerosis (SSc). Paquinimod binds the S100A9 protein and disrupts its binding to the pro-inflammatory receptors RAGE and TLR4 (1). Mechanistic studies have shown that paquinimod interferes with accumulation of myeloid cells during inflammation (2, 3). Previous clinical experience consists of two phase I dose escalation safety studies and one exploratory study in SLE patients (4). Objectives The aims of the present study in SSc patients were to evaluate safety and changes in disease related biomarkers. Methods Nine patients with a disease duration of 2.0±2.0 (mean ± SD) years and diffuse SSc were treated with paquinimod at 3.0 mg/day for eight weeks in an international, open label, multi-centre study (ClinicalTrials.gov Identifier: NCT01487551). The primary endpoint was changes in disease related biomarkers. Skin biopsies were assessed histologically for α-SMA positive myofibroblast counts. Expression of a panel of extracellular matrix genes and pro-fibrotic genes were analysed by real-time PCR. Secondary end-points included adverse events (AE), extent of skin fibrosis (mRSS) and Quality of Life (QoL) assessments. Results Paquinimod was well tolerated and all patients completed the eight week treatment period. Except for one SAE (peripheral ischemia, considered severe and unrelated to study medication) only mild or moderate AEs, most commonly arthralgia (n=3) and headache (n=3), were reported. Baseline mRSS was 28±10 (mean ± SD) points and as expected, no change in mRSS and QoL measures were observed in this short-term clinical trial. A small but significant reduction of the number of myofibroblasts in the skin was observed after paquinimod treatment compared to baseline (8%, p=0.023). Down-regulation of a number of pro-fibrotic genes such as CCR2, PAI-1, TIMP3, CAV1, CTGF and FN1 was evident after eight weeks of treatment. While no consistent effects on extracellular matrix genes were observed, the expression of several type I IFN-regulated genes also declined in the majority of the patients. The effects on biomarkers are in line with preclinical data in Tsk-1 mice and support that paquinimod could be effective by targeting the innate immune system in SSc. Conclusions Paquinimod was well tolerated and effects on biomarkers relevant for SSc were observed during treatment. References Björk et al., PLoS Biol. 2009;7(4):e97. Deronic et al., International Immunopharmacology 2014; 18:290-297. Helmersson et al., Am J Pathol. 2013;182(5):1671-80. Bengtsson et al., Arthritis Rheum 2012; 64:1579-88. Disclosure of Interest R. Hesselstrand Consultant for: Active Biotech., J. Distler: None declared, G. Riemekasten: None declared, M. Törngren Shareholder of: Active Biotech., Employee of: Active Biotech., H. Nyhlén Shareholder of: Active Biotech., Employee of: Active Biotech., M. Stenström Shareholder of: Active Biotech., Employee of: Active Biotech., F. Andersson Employee of: Active Biotech., H. Eriksson Shareholder of: Active Biotech., Employee of: Active Biotech., B. Sparre Employee of: Active Biotech., H. Tuvesson Shareholder of: Active Biotech., Employee of: Active Biotech., O. Distler Consultant for: Active Biotech. DOI 10.1136/annrheumdis-2014-eular.4207

FRI0516 Paquinimod (ABR-215757), an Immunomodulatory Compound, Reduces Fibrosis in the Tight Skin-1 (TSK-1) Model for Systemic Sclerosis

Background Paquinimod (ABR-215757), a new oral small molecular drug, belongs to the quinoline-3-carboxamide derivatives, a class of structurally related compounds (1). It targets the S100A9 protein and disrupts its binding to the pro-inflammatory receptors; Receptor for Advanced Glycation Endproducts (RAGE) and Toll-like Receptor 4, (TLR4) (2). Both RAGE and TLR4 are involved in the pathogenesis of many autoimmune and inflammatory diseases and paquinimod has shown convincing beneficial effects in several autoimmune/inflammatory disease models. Preclinical studies have shown that paquinimod affects the infiltration of myeloid cells into sites of inflammation (3). Paquinimod is in development for treatment of Systemic Sclerosis (SSc). SSc is a rare chronic autoimmune connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs. Effects on several biomarkers relevant for SSc have been observed in an exploratory clinical trial of paquinimod (ClinicalTrials.gov Identifier: NCT01487551) in patients with SSc. Objectives The purpose of this study was to evaluate the effects of paquinimod on skin fibrosis in the tight skin 1 (Tsk-1) mouse model, a common model for SSc. Methods Tsk-1 mice were treated for 8 weeks, starting at the age of 7 weeks, with paquinimod ad lib. at 5 and 25 mg/kg/day or with vehicle only. The hypodermal thickness was analyzed in skin sections stained with haematoxylin and eosin. The collagen content in skin was also determined by hydroxyproline assay. Furthermore, the number of α-smooth muscle actin (αSMA) positive myofibroblasts, in the skin sections were determined by immunohistochemical staining. Total IgG levels in serum were measured by ELISA and gene expression in skin biopsies was analyzed by real-time PCR. Results Treatment of Tsk-1 mice with paquinimod resulted in a significant (p<0.05) reduction of 24±6% (mean ± SEM) of the hypodermal thickness, a 22±6% (mean ± SEM) reduction of the hydroxyproline content, a 19±4% (mean ± SEM) decrease in the accumulation of myofibroblasts, and a 59±6% (mean ± SEM) reduction in total levels of serum IgG compared to vehicle treated mice. Paquinimod treated mice also had decreased gene expression of profibrotic biomarkers, such as Col1a2, Ccr2, Ctgf, Il-13 and Fn1 in the skin compared to vehicle treated mice. Conclusions Our results show that paquinimod reduces development of skin fibrosis in the Tsk-1 model measured as hypodermal thickness, hydroxyproline content and number of myofibroblasts in the skin. Paquinimod treatment also resulted in a reduction in the expression of profibrotic genes in the skin. References Jönsson et al. J Med Chem. 2004; 47:2075-88. Björk et al. PLoS Biol. 2009 Apr 28; 7(4):e97. Deronic et al. Int Immunopharmacol. 2014; 18:290-297. Disclosure of Interest M. Stenström Shareholder of: Active Biotech., Employee of: Active Biotech., H. Carlsson Nyhlén Shareholder of: Active Biotech., Employee of: Active Biotech., M. Nilsson Shareholder of: Active Biotech., Employee of: Active Biotech., H. Eriksson Shareholder of: Active Biotech., Employee of: Active Biotech., M. Törngren Shareholder of: Active Biotech., Employee of: Active Biotech., J. Distler: None declared, O. Distler Consultant for: Active Biotech., B. Sparre Employee of: Active Biotech., H. Tuvesson Shareholder of: Active Biotech., Employee of: Active Biotech. DOI 10.1136/annrheumdis-2014-eular.2200

Extracellular S100A9 protein in bone marrow supports multiple myeloma survival by stimulating angiogenesis and cytokine secretion

In mouse multiple myeloma, treatment with the small molecule ABR-238901 interfered with calcium-binding protein S100A9, affected cytokine secretion and angiogenesis, and reduced tumor load. Results improved further when ABR-238901 was delivered in combination with the proteasome inhibitor bortezomib. Dysregulated expression of S100 protein family members is associated with cancer proliferation, invasion, angiogenesis, and inflammation. S100A9 induces myeloid-derived suppressor cell (MDSC) accumulation and activity. MDSCs, immunosuppressive cells that contribute to tumor immune escape, are the main producers of S100A9. In this study, we evaluated the role of extracellular S100A9 and the therapeutic relevance of S100A9 inhibition in multiple myeloma (MM), using the immunocompetent murine 5T33MM model. We demonstrated the presence of S100A9 and its receptor TLR4 in both monocytic and granulocytic MDSCs in human and mouse samples. We showed that S100A9 acted as a chemoattractant for MM cells and induced MDSCs to express and secrete inflammatory and pro-myeloma cytokines, including TNFα, IL6, and IL10. Blocking S100A9 interactions in vivo with the small molecule ABR-238901 did not directly affect MDSC accumulation but did reduce IL6 and IL10 cytokine expression by MDSC. ABR-238901 treatment in vivo reduced angiogenesis but had only minor effects on tumor load as single agent (6% reduction). However, ABR-238901 treatment in combination with bortezomib resulted in an increased reduction in tumor load compared with single treatments (50% relative reduction compared with bortezomib alone). Our data suggest that extracellular S100A9 promotes MM and that inhibition of S100A9 may have therapeutic benefit. Cancer Immunol Res; 5(10); 839–46. ©2017 AACR.

Abstract B17: Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects.

Purpose: The novel anti-tumor agent tasquinimod (ABR-215050) is being developed for treatment of metastatic castration-resistant prostate cancer. Proof of concept has been demonstrated in a Phase II trial by delaying disease progression and increasing overall survival, and enrollment of patients to a global pivotal Phase III trial (10TASQ10) is ongoing. Tasquinimod targets the tumor microenvironment resulting in anti-angiogenesis, inhibition of metastasis and immunomodulation. A target molecule of tasquinimod is the S100A9 protein which has been demonstrated to affect the accumulation and function of CD11b+Gr-1+ regulatory myeloid cell subsets. Given the major contribution of these cells to the immunosuppressive tumor environment, modulation of this cell population could contribute to the anti-tumor effects by tasquinimod and enhance the effects of cancer immunotherapy. In this study, we evaluated the immunomodulatory effects of tasquinimod when combined with tumor-targeted superantigens (TTS); an immunotherapeutic strategy which is highly dependent on activation of effector T cells directed against the tumor. Experimental design: The effect of tasquinimod treatment on CD11b+ myeloid cell populations was analyzed by flow cytometry in cell suspensions from spleens and from B16 tumors growing subcutaneously. In addition, B16-5T4 expressing tumors were treated with tasquinimod (30 mg/kg), the TTS fusion protein 5T4Fab-SEA (25 μg/kg), or the combination. Tumor growth and tumor-directed T cell responses were monitored. Results: The number of CD11b+ myeloid cells significantly increased in tumor bearing mice. Tasquinimod treatment inhibited tumor growth and reduced the CD11b+Ly6G-Ly6Chigh monocytic subpopulation of CD11b+Gr-1+ cells. In addition, less CD206-expressing tumor-associated macrophages were detected. Combining tasquinimod and TTS immunotherapy significantly enhanced the anti-tumor effects compared to the mono therapies. This was accompanied with increased numbers of TTS-activated CD8+ T cells in the tumors over a prolonged period of time. Conclusions: This study demonstrates that tasquinimod modulates the tumor infiltrating myeloid cell subsets and suppresses tumor growth. In line with an effect on the immunosuppressive tumor microenvironment, combining tasquinimod with the immunotherapy TTS gave rise to significantly improved anti-tumor effects. These data suggest that tasquinimod reduces tumor-induced immune suppression and that tasquinimod combined with immunotherapy could represent a novel and promising anti-cancer strategy. Citation Format: Anette Sundstedt, Anders Olsson, Martin Stenstrom, Mona Celander, Marie Torngren, Helena Eriksson, Gunnar Hedlund, David Liberg, Tomas Leanderson. Immunomodulation by tasquinimod: Combination with immunotherapy results in enhanced CD8 T cell responses and improved antitumor effects. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B17.

Abstract B78: Immunomodulation by tasquinimod: Skewing of tumor infiltrating myeloid cell populations.

Purpose: Tasquinimod is in phase III development (10TASQ10) for treatment of metastatic castration-resistant prostate cancer. Tasquinimod has shown proof of concept by delaying disease progression and increasing overall survival in a randomized phase II study. Tasquinimod binds to the S100A9 protein and inhibits its interaction with the RAGE and TLR4 receptors, an interaction postulated to be important for the accumulation and function of tumor infiltrating myeloid cells. Treatment with tasquinimod in preclinical tumor models has been shown to result in modulation of immune responses, inhibition of angiogenesis and inhibition of metastasis. Since myeloid cells can contribute to all of the above mentioned areas, we analyzed the effect of tasquinimod on infiltrating cell populations in MC38 adenocarcinoma tumors. Experimental design: Murine colon carcinoma cells (MC38) were inoculated in matrigel in wt or nude C57/Bl6 mice and tumor growth was monitored in the presence or absence of tasquinimod. Effects on vascularization and cell populations were studied by immunohistochemistry on tumors or flow cytometry on isolated cell suspensions. Results: Treatment with tasquinimod inhibited growth of subcutaneously inoculated MC38 tumors. This effect was not restricted to a changed T cell response since tumor growth was also inhibited in nude mice. Treatment effects were associated with a decrease in tumor neovascularization as evident by CD31 immunostaining. A distinct unvascularized necrotic core can be observed in the treated tumors compared to vehicle treated mice. Untreated MC38 tumors showed a substantial infiltration of primarily myeloid cells where most were Ly6ClowCD206+ (M2) macrophages, a population that has been shown to have pro-angiogenic properties. Treatment with tasquinimod led to a reduction in this CD206+ cell population and a concomitant increase in CCR7+ (M1) macrophages. No change was detected in infiltration of CD4+ or CD8+ T cells, including Foxp3+ cells, in accordance with the lack of T cell dependency of tasquinimod effects in this tumor. Conclusions: S100A9 and RAGE has previously been shown to have roles in infiltration of myeloid cells in MC38 tumors. Treatment with tasquinimod, an S100A9-RAGE/TLR4 inhibitor, changes the distribution of myeloid cell populations in the tumor which includes a reduced number of M2 macrophages in the infiltrate. These data suggest that tasquinimod has an effect on tumor infiltrating myeloid cells which could contribute to T cell independent effects on angiogenesis and inhibition of tumor growth. Citation Format: David Liberg, Anders Olsson, Pascale Plas, Marie Torngren, Catherine George, Martin Stenstrom, Fabien Schmidlin, Helena Eriksson, Tomas Leanderson. Immunomodulation by tasquinimod: Skewing of tumor infiltrating myeloid cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B78.