Ontology highlight
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PROVIDER: PRJNA1125163 | ENA |
REPOSITORIES: ENA
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Project description:Background: Granzyme B (GrB) is a key effector molecule, delivered by cytotoxic T lymphocytes and natural killer cells during immune surveillance to induce cell death. Fusion proteins and immunoconjugates represent an innovative therapeutic approach to specifically deliver a deadly payload to target cells. Epithelial membrane protein-2 (EMP2) is highly expressed in invasive breast cancer, including triple negative breast cancer, and represents an attractive therapeutic target. Methods: We designed a novel fusion protein (GrB-Fc-KS49) composed of an active GrB fused to an anti-EMP2 single chain antibody tethered through the IgG heavy chain (Fc) domain. The protein was expressed by HEK-293E cells. We assessed the construct’s GrB enzymatic activity, anti-EMP2 binding affinity, cytotoxicity against a panel of breast cancer cells, pharmacokinetics, toxicity profile, and in vivo efficacy. Results: GrB-Fc-KS49 exhibited comparable GrB enzymatic activity to commercial GrB, as well as high affinity to an EMP2 peptide, with the dissociation constant in the picomolar range. The fusion protein rapidly internalized into EMP2+ breast cancer cells and showed in vitro cytotoxicity to cell lines expressing surface EMP2, with IC50 values below 100 nM for most positive lines. Ex vivo stability at an elevated temperature (37°C) indicated a half-life exceeding 96h while in vivo pharmacokinetics indicated a bi-exponential plasma clearance, with a moderate initial clearance (t1/2α = 18.4 h) and a much slower terminal clearance rate (t1/2β = 73.1 h). No significant differences between the vehicle control and the GrB-Fc-KS49 was detectable in a Chem16 toxicity panel. In vivo efficacy against a TNBC syngeneic mouse model EMT6 mouse model resulted in a reduction in cell proliferation compared to controls (p=0.02). Treatment using a second syngeneic model (4T1/FLuc) confirmed these results and resulted in a dramatic reduction in cell proliferation (p = 0.001) and increase in cell death by TUNEL (p=0.02) compared to controls. In addition to a significant impact on cell proliferation (p=0.005), GrB-Fc-KS49 treatment also resulted in a dramatic increase of tumor infiltrating CD45+ cells (p=0.002), and redistribution of tumor-associated macrophages (TAMs) which was confirmed by transcriptomic analysis of tumors post treatment. Conclusions: GrB-Fc-KS49 showed high specificity to EMP2 and cytotoxicity towards EMP2 positive cells. In vivo, GrB-Fc-KS49 reduced tumor burden by direct targeting as well as increasing recruitment of immune cells into the tumor microenvironment, suggesting that GrB-Fc-KS49 is a promising therapeutic for further development against breast cancer.
2024-06-22 | GSE270203 | GEO
Project description:GrB-Fc-KS49, a human immuno-oncology therapeutic agent to EMP2 using a human fusion protein containing granzyme b, reshapes the tumor microenvironment by altering immune cell recruitment in breast cancer
| PRJNA1125402 | ENA
Project description:Pathologic retinal neovascularization is a potentially blinding consequence seen in many common diseases including diabetic retinopathy, retinopathy of prematurity, and retinal vascular occlusive diseases, among others. The use of therapeutics targeting pro-angiogenesis factors such as vascular endothelial growth factor (VEGF) has proven to be highly effective, however considerable side effects exist and serial anti-VEGF treatment has been shown to decrease effectiveness over time. Characterization of additional regulators of neovascularization is needed to further understand neovascular disease and identify possible new therapeutic targets. This study investigates epithelial membrane protein 2 (EMP2) and its role as a possible modulator of angiogenesis in human retinal pigment epithelium (RPE) under hypoxia. EMP2 is highly expressed in human RPE and RPE cell lines. Adult retinal pigment epithelial cell line-19 (ARPE-19) cells were genetically modified to either overexpress EMP2 (OE) or knock down EMP2 (KD) and expression at the RNA and protein level was evaluated using RNA sequencing and western blot respectively. Protein expression was evaluated under both normoxic conditions and conditions of hypoxic stress with 0.5% O2. EMP2 expression was found to positively correlate with expression of the pro-angiogenesis factors hypoxia inducible factor 1-alpha (HIF-1a) and VEGF for both RNA and protein. EMP2 mediated changes in ARPE-19 cells was also found to alter the secretion of a paracrine factor(s) in conditioned media that can regulate human umbilical vein endothelial cells (HUVEC) endothelial cell migration and capillary tube formation in in vitro functional angiogenesis assays. This study identifies EMP2 as a potentially important mediator of angiogenesis in the human RPE, a tissue involved in abnormal retinal neovascularization in a number of diseases. EMP2 levels positively correlate with those of the potent pro-angiogenesis mediators HIF-1a and VEGF, however the mechanism of this relationship remains to be clarified. This study supports further investigation of EMP2 as a promising novel target for therapeutic treatment of pathologic neovascularization in the retina.
2020-06-03 | GSE151610 | GEO
Project description:We constructed AAV-vectors for systemic expression of a soluble RSPO1 protein in ApcMin/+ mice. We found that the RSPO1-Fc fusion protein suppresses the Wnt/ß-catenin signaling activity in intestinal adenomas and in adenoma-derived intestinal organoids ex vivo, but not in normal intestinal epithelial cells. In the Apc mutant cells, the RSPO1-Fc fusion protein activated the TGFß/SMAD signaling pathway to suppress several Wnt target genes and adenoma growth, which effect was rescued suppressed by the TGFß receptor kinase inhibitor SB-431542. Simultaneously, RSPO1-Fc induced proliferation of the normal intestinal stem cells, giving them a growth advantage over the mutant cells, which enabled the intestinal epithelium to eventually outgrow the adenoma cells. Prolonged systemic expression of AAV-RSPO1-Fc decreased significantly the number of the intestinal adenomas and improved the overall survival of ApcMin/+ mice. Thus RSPO1-Fc provides the normal intestinal epithelial cells a growth advantage when compared to the adenoma cells, which eventually leads to the extrusion of the adenomatous tissue. An attractive idea now is to exploit such differential response of normal vs. cancer cells in cancer therapy.
2020-03-01 | GSE146138 | GEO
Project description:We constructed AAV-vectors for systemic expression of a soluble RSPO1 protein in ApcMin/+ mice. We found that the RSPO1-Fc fusion protein suppresses the Wnt/ß-catenin signaling activity in intestinal adenomas and in adenoma-derived intestinal organoids ex vivo, but not in normal intestinal epithelial cells. In the Apc mutant cells, the RSPO1-Fc fusion protein activated the TGFß/SMAD signaling pathway to suppress several Wnt target genes and adenoma growth, which effect was rescued suppressed by the TGFß receptor kinase inhibitor SB-431542. Simultaneously, RSPO1-Fc induced proliferation of the normal intestinal stem cells, giving them a growth advantage over the mutant cells, which enabled the intestinal epithelium to eventually outgrow the adenoma cells. Prolonged systemic expression of AAV-RSPO1-Fc decreased significantly the number of the intestinal adenomas and improved the overall survival of ApcMin/+ mice. Thus RSPO1-Fc provides the normal intestinal epithelial cells a growth advantage when compared to the adenoma cells, which eventually leads to the extrusion of the adenomatous tissue. An attractive idea now is to exploit such differential response of normal vs. cancer cells in cancer therapy.
2020-02-29 | GSE146099 | GEO
Project description:Methicillin-resistant Staphylococcus aureus (MRSA) is a major hospital- and community-acquired pathogen, but the mechanisms underlying host-defense to MRSA remain poorly understood. Here, we investigated the role of IL-21 in this process. When administered intra-tracheally into wild-type mice, IL-21 induced granzymes and augmented clearance of pulmonary MRSA but not when neutrophils were depleted or a granzyme B inhibitor was added. Correspondingly, IL-21 induced MRSA killing by human peripheral blood neutrophils. Unexpectedly, however, basal MRSA clearance was enhanced when IL-21 signaling was blocked, both in Il21r KO mice and in wild-type mice injected with IL-21R-Fc fusion-protein. This correlated with increased type I interferon and an IFN-related gene signature, and indeed anti-IFNAR1 treatment diminished MRSA clearance in these animals. Moreover, we found that IFNβ induced granzyme B and promoted MRSA clearance in a granzyme B-dependent fashion. These results reveal an interplay between IL-21 and type-I IFN in the innate immune response to MRSA.
2019-04-22 | GSE129093 | GEO
Project description:Regulatory T (Treg) cells play an important role in the induction and maintenance of peripheral tolerance. Treg cells also suppress a variety of other immune responses, including anti-tumor and alloimmune responses. We have previously reported that tumor-activated Treg cells express granzyme B and that granzyme B is important for Treg cell-mediated suppression of anti-tumor immune responses (GSE13409). Here, we report that allogeneic mismatch also induces the expression of granzyme B. Granzyme B-deficient mice challenged with fully mismatched allogeneic P815 mastocytoma cells have markedly improved survival compared to WT and other granzyme- or perforin-deficient mice, suggesting an immunoregulatory role for granzyme B in this setting. Treg cells harvested from the tumor environment of P815-challenged mice express granzyme B. Treg cells also express granzyme B in vitro during mixed lymphocyte reactions and in vivo in a mouse model of graft-versus-host disease (GVHD). However, in contrast to findings from our previously published tumor model, granzyme B is not required for the suppression of effector T cell (Teff) proliferation in in vitro Treg suppression assays stimulated by either Concanavalin A or allogeneic antigen presenting cells. Additionally, in an ex vivo assay, sort-purified in vivo-activated CD4+Foxp3+ Treg cells from mice with active GVHD -- under conditions known to induce granzyme B expression in Treg cells -- suppressed Teff cell proliferation in a granzyme B-independent manner. Adoptive transfer of naive granzyme B-deficient CD4+CD25+ Treg cells into a mouse model of GVHD rescued hosts from lethatlity equivalently to naive wild-type Treg cells. Serum analysis of GVHD-associated cytokine production in these recipients also demonstrated that Treg cells suppressed production of IL-2, IL-4, IL-5, GM-CSF, and IFN-gamma in a granzyme B-independent manner. In order to determine whether the context in which Treg cells are activated alters the intrinsic properties of Treg cells, we used Foxp3 reporter mice to obtain gene expression profiles of CD4+Foxp3+ Treg cells purifed from naive resting spleens, spleens from mice with acute GVHD, and from ascites fluid of mice challenged intraperitoneally with allogeneic P815 tumor cells. Unsupervised analyses revealed distinct activation signatures of Treg cells among the 3 experimental groups. Taken together, these findings demonstrate that granzyme B is not required for Treg cell-mediated suppression of GVHD, which is in contrast to what we have previously reported for Treg cell function in the setting of tumor challenge. Cell intrinsic differences could partially account for these differential phenotypes. These data also suggest the therapeutic potential of targeting specific Treg cell suppressive functions in order to segregate GVHD and graft-versus-tumor effector functions. Experiment Overall Design: Six replicates of Naive CD4+Foxp3+ Treg cells were purified from resting spleens, five replicates of allogeneic tumor-activated Treg cells and three samples of GVHD-activated Treg cells. Experiment Overall Design: Naive reps 1-3 are controls for the GVHD-activated samples. Experiment Overall Design: Naive reps 4-6 are controls for the Allogeneic tumor-activated samples.
2009-09-15 | E-GEOD-16210 | biostudies-arrayexpress
Project description:Epithelial membrane protein-2 (EMP2) is a tetraspan protein predicted to regulateplacental development. Highly expressed in secretory endometrium and trophectoderm cells, previous studies suggest that it may regulate implantation by orchestrating the surface expression of integrins and other membrane proteins. In order to confirm the role of EMP2 in pregnancy, mice lacking EMP2 (Emp2-/- ) were generated. These animals are fertile but have reduced litter sizes. Moreover, their placentas exhibit dysregulation in pathways related to neoangiogenesis, coagulation, and oxidative stress, and have increased fibrin deposition and altered vasculature. Given that these findings often occur due to placental insufficiency resulting in an oxygen-poor environment, expression of hypoxia-inducible factor-1 alpha (HIF-1α) was examined. Emp2-/- animals have reduced HIF-1α expression within cells of trophoblast origin. However, they appear to have a compensatory increase in uterine NK (uNK) cells, demonstrating a unique interplay between uNK cells and trophoblasts modulated through EMP2. To determine if these results translated to human pregnancy, placentas from normal, term deliveries or those complicated by intrauterine growth restriction (IUGR) were stained for EMP2. EMP2 was significantly reduced in both villous and extravillous trophoblast populations in IUGR placentas. Experiments in vitro using human trophoblast cells lines indicate that EMP2 modulates angiogenesis by altering HIF-1a expression. Our results reveal a novel role for EMP2 in regulating trophoblast function and vascular development in conditions of altered oxygen availability in mice and humans and suggest it may be a novel biomarker for placental insufficiency.
2017-02-23 | GSE81940 | GEO
Project description:Although different sarcomas have been modeled in mice upon expression of fusion oncogenes in MSCs, sarcomagenesis has not been successfully modeled in human MSCs (hMSCs). We report that FUS-CHOP, a hallmark fusion gene in mixoid liposarcoma (MLS), has an instructive role in lineage commitment, and its expression in hMSC sequentially immortalized/transformed with up to 5 oncogenic hits (p53 and Rb deficiency, hTERT over-expression, c-myc stabilization and H-RASv12 mutation) drives the formation of serially transplantable MLS. This is the first model of sarcoma based on the expression of a sarcoma-associated fusion protein in hMSC, and allowed us to unravel the differentiation processes and signaling pathways altered in the MLS-initiating cells. This study will contribute to test novel therapeutic approaches, and constitutes a proof-of-concept to employ hMSCs as target cell for modeling other fusion gene-associated human sarcomas. Wild type (MSC-0H) or transformed (MSC-5H) BM-hMSCs were transduced with concentrated viral particles expressing either pRRL-EF1?-PGK-GFP (empty vector; GFP) or pRRL-EF1?-FUS-CHOP-PGK-GFP (FUSCHOP expressing vector; FC) in order to generate MSC-0H-GFP, MSC-0H-FC, MSC-5H-GFP and MSC-5H-FC cell lines. MSC-0H-GFP and MSC-0H-FC cells did not develop tumors, meanwhile MSC-5H-GFP cells gave rise to undifferentiated sarcomas and MSC-5H-FC originated mixoid liposarcoma tumors when inoculated into immunedeficient mice. Several cell lines were derived from tumors developed from MSC-5H-GFP (T-5H-GFP-1 to -3 cell lines) and MSC-5H-FC (T-5H-FC-1 to -3 cell lines) cells. Gene expression analysis was performed using MSC-0H, MSC-5H and T-5H cell lines and lists of differentially expressed genes were created by comparing the gene expression profiles of MSC-0H-FC, MSC-5H-FC and T-5H-FC cell types to the control MSC-0H-GFP cells.
2013-06-18 | E-GEOD-48030 | biostudies-arrayexpress
Project description:Here, we describe a secreted HLA (sHLA) Fc-fusion construct for simple single HLA allele profiling in hypoxic pancreatic ductal adenocarcinoma (PDAC) and cellular senescence. This method streamlines sample preparation, enables temporal control, and provides allele-restricted target identification. Over 30,000 unique HLA-associated peptides were identified across two different HLA alleles and seven cell lines, with ~9,300 peptides newly discovered. The sHLA Fc-fusion capture technology holds potential to expedite immunopeptidomics and advance therapeutic interest in peptide-HLA complexes.
2024-01-26 | PXD045796 | Pride