A major barrier for cancer immunotherapy is the presence of suppressive cell populations in cancer patients, such as myeloid-derived suppressor cells MDSC
Active biotech byter vd 3 tumor-associated macrophages TAMwhich 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 SA9, which has been demonstrated to affect the accumulation and function of tumor-suppressive myeloid cells. "Active biotech byter vd 3," we report that tasquinimod provided a significant enhancement to the antitumor effects of two different immunotherapeutics in mouse models of cancer: 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.
Immunotherapies have gained momentum in cancer therapeutics following the recent approvals of drugs for the treatment of prostate cancer and melanoma. Sipuleucel-T dendritic cell DC vaccine is now available for treatment of patients with asymptomatic or minimally symptomatic, metastatic, castration-resistant prostate cancer [ 1 ].
Clinical observations have indicated that melanoma is an immunogenic tumor [ 2 ], and extended survival data have led to the approval of immune checkpoint inhibitor ipilimumab for the treatment of metastatic melanoma [ 3 ].
However, despite these clinical advances, immunotherapies for these diseases and solid tumors in general, benefit only a subset of patients, as intrinsic or acquired tumor immune tolerance remains a major hurdle. A significant barrier in vaccine therapy is the presence of immunosuppressive soluble and cellular components including myeloid-derived suppressor cells MDSC [ 4 ] and tumor-associated macrophages TAM [ 5 ], which are Active biotech byter vd 3 by tumor- and stroma-secreted inflammatory mediators [ 6 - 8 ].
Tasquinimod, a quinolinecarboxyamide analog, is in clinical development for treatment of prostate cancer and other solid tumors. In a placebo-controlled, phase II randomized trial, tasquinimod doubled the median progression-free survival PFS period and prolonged survival of patients with metastatic, castration-resistant prostate cancer [ 1415 ]. Tasquinimod has been shown to inhibit prostate cancer growth and metastasis in animal models [ 16 - 18 ]. However, in an orthotopic, metastatic prostate cancer model, tasquinimod reduced the metastatic rate without affecting microvessel density in the primary tumor [ 18 ].
Therefore, mechanisms other than impairing angiogenesis may play an important role in the antitumor and anti-metastasis activities of tasquinimod. SA9 interacts with pro-inflammatory receptors Toll-like receptor 4 TLR4 and receptor of advanced glycation end products RAGEand this interaction is inhibited by the specific binding of tasquinimod to SA9 [ 22 - 23 ]. These receptors are expressed on the surface of multiple myeloid-cell populations, including MDSCs, macrophages, DCs, as well as endothelial cells.
Functionally, SA9 regulates the accumulation of MDSCs and inhibits DC differentiation [ 24 ] [ 25 ], which may lead to suppression of immune responses and tumor progression. Therefore, by targeting SA9, tasquinimod has immunomodulatory activity and the potential to regulate multiple myeloid populations.
In this study, we tested the effect of tasquinimod "Active biotech byter vd 3" immunosuppressive myeloid-cell populations and investigated its immunomodulatory activity. We conducted preclinical studies of tasquinimod in combination with two different immunotherapeutic approaches in mouse models of prostate cancer and melanoma.
Our results suggest that treatment with tasquinimod affects the tumor microenvironment by modulating suppressive myeloid-cell populations, leading to augmented immune responses and enhanced antitumor effects of immunotherapies. The tumor size was measured by a caliper twice a week. At the of the 3—4 week experiment, tumors and spleens were collected and analyzed.
Bh5T4 cells were cultured as described above, counted, re-suspended and maintained in iced-cold matrigel BD Biosciences, San Jose, CA at a concentration of 0. Tumor cells were implanted s. Experiments were terminated between day 16 and day Cell pellets were treated with ACK lysing buffer Biosource. Single-cell suspensions were prepared from tumors with mouse tumor dissociation kit Miltenyi Biotech.
Alternatively, the tumors were cut into small pieces and incubated in 0. The following fluorochrome-labeled antibodies were used: Brefeldin A Sigma was added to the cultures to block protein secretion.
Brefeldin A Sigma was added to the cultures during the last 5 hours of culture to block protein secretion. Cells were harvested and stained for surface markers, then fixed and stained for intracellular Granzyme B eBioscience and analyzed by flow cytometry. Cells were then harvested and the incorporated 3 H-thymidine was detected Active biotech byter vd 3 scintillation counting.
After a 5-hour incubation, all cells in culture were harvested and PI staining was performed to detect dead cells. Cell cytotoxicity was analyzed by calculating percentage of dead cells with Dio label compared to the whole cell population with Dio label.
Representative photos were taken and the density of CDpositive cells fluorescence was measured with Leica QWin image analysis system. Sections were de-paraffinized and rehydrated through graded alcohol washes. Sections were further incubated in hydrogen peroxide to reduce endogenous activity. Then tissue section were blocked with 2. Section were dehydrated and mounted with cytoseal 60 Thermo Scientific.
Corresponding isotype negative controls were used for evaluation of specific staining. Stained sections were analyzed under bright field using the Zeiss Axio microscope. The number of positive cells was determined in a blinded fashion by analyzing four random 20x fields per tissue and quantified using Image J software.
The primers used for target genes were: The assay was performed with an ultra sensitive assay for nitric oxide synthase from Oxford Biomedical Research Cat: Briefly, lysates from isolated CD11b cells were first incubated with substrates and cofactors.
Then the mixtures were incubated with nitrate reductase to transform nitrate to nitrite, and mixed with coloring reagent to quantify total end-product concentration. These reactions were performed in a well plate and absorbance was read at nm. The difference in tumor weight between treatment groups was statistically evaluated by non-parametric Mann-Whitney U test.
Results from previous studies in experimental tumor models indicated that immunomodulatory effects of tasquinimod may contribute to its antitumor activity [ 23 To investigate the potential immunomodulatory activities of tasquinimod, we tested this agent in combination with a survivin peptide vaccine SurVaxM in survivin-expressing CR Myc-CaP prostate cancer model and "Active biotech byter vd 3" a tumor-targeted superantigen in a B16 melanoma model.
Survivin is an intracellular TAA expressed in several solid tumors, including prostate cancer [ 29 ]. The combination treatment also significantly inhibited tumor growth compared to that of single treatment groups tasquinimod vs. Mice were inoculated s. When the tumors reached an average size of 25 mm 2mice were treated with vehicle, survivin, tasquinimod or the combination of survivin and tasquinimod.
Left panel shows tumor growth curves by serial caliper measurements. Right panel shows tumor weights at the endpoint. Right panel shows end of treatment tumor weights. The experiments were repeated at least twice. Results from one representative experiment are shown. Error bars indicate s. In parallel, we tested tasquinimod in combination with a Active biotech byter vd 3 immunotherapy approach, tumor-targeted superantigens TTS in a transplantable B16 melanoma model.
TTS immunotherapy activates and directs T lymphocytes to attack tumor cells by means of fusion proteins between bacterial superantigens, such as staphylococcal enterotoxin A SEAand Fab-fragments of tumor-reactive monoclonal antibodies mAb [ 32 ].
Tasquinimod treatment began the day after tumor-cell inoculation and 5T4Fab-SEA was administrated on days
Active biotech byter vd 3 the combination of tasquinimod with two different immunotherapeutic strategies resulted in a significant enhancement of antitumor effects. Splenocytes were isolated from differentially treated mice, stimulated and then stained for cell-surface markers and intracellular proteins.
Propidium iodide was added at the end of incubation to detect tumor cell death. FACS analysis of tumor-infiltrating T cells performed at the endpoint of the experiment depicted in Fig. Consistent with enhanced antitumor activity observed following combination treatment, splenocytes from these mice displayed significantly improved tumor-cell killing capacity as compared to those from other treatment groups Fig.
Thus, these results suggest that the combination therapy does not enhance CTL activity per se but rather inhibits T cell-suppressing factor s in the cultured splenocytes. Tumor-infiltrating cells were analyzed at different days day to follow the kinetics of specific T-cell expansion. Tasquinimod has been reported to display anti-angiogenic activity in prostate cancer models [ 1934 ]. To determine whether the anti-angiogenic effect of tasquinimod was involved in enhancing the antitumor effects of immunotherapy, we assessed the microvasculature density CD31 expression in the harvested tumor tissue by either immunofluorescence or immunohistochemistry analysis in the two therapeutic strategies, respectively.
The results showed that tasquinimod treatment reduced microvasculature density in B16 tumors Fig. In summary, these results suggest Active biotech byter vd 3 the immunomodulatory effects of tasquinimod may be dissociated from its anti-angiogenic activity; and in the Bh5T4 tumor model, the tasquinimod-induced inhibition of tumor blood vessel formation, may account at least in part for its antitumor effect in this model.
SA9 is an inflammatory protein that affects the accumulation of immunosuppressive myeloid cells, including MDSCs [ 24 ] [ 25 ]. Tasquinimod binds to SA9, inhibiting its downstream signaling, and thus has the potential to affect myeloid cells. To investigate the mechanism of immune-promoting activity of tasquinimod in combination with immunotherapy, we analyzed the peripheral and tumor-infiltrating myeloid-cell populations.
In the CR Myc-CaP tumor model, blood samples were taken from differentially treated mice after two weeks of treatments and subjected to immunofluorescence staining and FACS analysis. In addition, tumors were harvested from differentially treated mice and processed into suspension. Interestingly, tasquinimod significantly reduced the number of tumor-infiltrating MDSCs when given as a single agent or in combination with the vaccine Fig.
FACS plots and quantifications are depicted throughout. A comparable picture was also seen in the spleen Active biotech byter vd 3 Fig. Thus, the decrease in microvasculature density by tasquinimod in the B16 model could be the consequence of reducing pro-angiogenic monocytic cells within the tumors. Tumor-associated macrophages are important components of the immunosuppressive TME.
When infiltrating the tumor tissue, these cells adapt to the environment and differentiate into TAMs by losing Gr1 marker expression and gaining an even more immunosuppressive M2 macrophage phenotype [ 36 ] [ 37 ] [ 38 ]. Therefore, we assessed the effect of tasquinimod on TAMs. Similarly, analysis of macrophages in Bh5T4 tumors also revealed a strong reduction of this subpopulation in tasquinimod-treated mice Fig.
In addition to MDSCs and TAMs, we also investigated whether tasquinimod treatment affects immune-promoting activities of other myeloid and lymphoid cells. Tasquinimod did not impair T-cell expansion upon activation either in T cells isolated from differentially treated mice Supplementary Fig.
Tregs represent an immunosuppressive lymphocyte population whose accumulation can be regulated by MDSCs. DC differentiation has been shown to be regulated by the SA9 protein [ 24 ]. Although tasquinimod slightly reduced the number of DCs in the spleen Supplementary Fig. These data suggest that immunosuppressive myeloid cells, such as MDSCs and TAMs but not other myeloid or lymphoid populations are the potential cellular targets of tasquinimod and they may be responsible for the immune-promoting activity of tasquinimod in combination with immunotherapies.
Taken together, these results suggest that tasquinimod modulates not only the infiltration but also the suppressive capacity of tumor-infiltrating myeloid-cell populations. This observation led us to investigate the expression of two mechanistically relevant genes, Arg1 arginase-1 and iNOS induced nitric oxide synthase in the tumor-infiltrating myeloid cells Fig.
It has been reported that Arg1 gene expression can be regulated by TLR4 pathway [ 39 ], which is target receptor for SA9. Li Shen. 1Genitourinary Program, Roswell Park Cancer Institute, Buffalo NY. A phase III clinical trial to test the effect of tasquinimod in the same patient at Roswell Park Cancer Institute and Active Biotech, respectively. groups ( tasquinimod vs. combination p= ; survivin vs. combination p= ). FBL manufactures Cholecalciferol (Vitamin D3), Penicillin G Acylase, Candida Fermenta Biotech Ltd (FBL), incepted under the auspices of Duphar Interfran Ltd (now D3 feed grade for use in animal feed supplements; and activated.
Biotechnology and Bioengineering Symposium Series 15, — Phelan, M. B., Crawford, D. L. and Pometto III, A. L. (). Cloning and expression in biologically active form of the gene for human interferon Ryu, D. D.
Y., Kim, K. 5 ., Che, N. Y. and Pai, H. S. Scott, C. D., Strandberg, G. W. and Lewis, S. N. ( ).
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