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
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ABSTRACT:
PROVIDER: PRJNA1125402 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR29457977.fastq.gz | Fastqsanger.gz | |||
| SRR29457977_1.fastq.gz | Fastqsanger.gz | |||
| SRR29457977_2.fastq.gz | Fastqsanger.gz | |||
| SRR29457978_1.fastq.gz | Fastqsanger.gz | |||
| SRR29457978_2.fastq.gz | Fastqsanger.gz |
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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:Previous studies have suggested that overexpression of the oncogenic protein epithelial membrane protein-2 (EMP2) correlates with endometrial carcinoma progression and ultimately poor survival from disease. To understand the role of EMP2 in the etiology of disease, gene analysis was performed to show transcripts that are reciprocally regulated by EMP2 levels. In particular, EMP2 expression correlates with and helps regulate the expression of several cancer stem cell associated markers including aldehyde dehydrogenase 1 (ALDH1).
2018-01-01 | GSE92886 | GEO
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:Genetic investigation in Mendelian skin disorders featuring generalized or localized skin scaling and redness, known as the ichthyoses, has revealed novel pathways relevant to epidermal integrity, barrier function, and desquamation. Here we show that a recurrent de novo missense variant in EMP2 (epithelial membrane protein 2), which encodes a cell surface tetraspan protein in the growth-arrest specific 3 (GAS3)/peripheral myelin protein 22 (PMP22) family, is associated with a previously undescribed Mendelian disorder in the progressive symmetric erythrokeratoderma spectrum. The disorder features severely thickened, red and scaly skin at sites of wound healing or repetitive movement including on the face, genitals, flexural areas, and the palms and soles. EMP2 has previously been shown to directly associate with focal adhesion kinase (FAK), which links cell junction forces to signaling pathways relevant to proliferation, migration, and wound healing. Using single-cell spatial transcriptomics in affected tissue, we discovered ectopic suprabasal activation of signaling pathways downstream of receptor tyrosine kinases including epidermal growth factor receptor (EGFR), which we confirmed with Western blotting in affected cells, supporting a gain-of-function mechanism for mutant EMP2. Remarkably, treatment with erlotinib, an EGFR inhibitor, led to marked clinical improvement underscoring the key role of EMP2 in epidermal differentiation and proliferation.
2025-07-09 | GSE301486 | GEO
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:Bacillus inaquosorum strain:EMP2 Genome sequencing
| PRJNA1134279 | ENA
Project description:Little is know about the role of epithelial membrane protein-2 in breast cancer development or progression. In this study, we tested the hypothesis that EMP2 may regulate the formation or self-renewal of breast cancer stem cells (BCSC) in the tumor microenvironment.
2019-11-30 | GSE134522 | GEO
Project description:Elucidating the true physiological functions of granzymes has been challenging since the origin of the granzyme studied, the use of non-physiological granzyme concentrations and the interspecies use of granzymes, with extrapolation of all these data to orthologous granzymes has led to inconsistencies. This is highly relevant for granzymes A and K where a debate is ongoing concerning their involvement in either cytotoxic or inflammatory processes, or in both. In order to tackle such contradictions, detailed knowledge of the substrate repertoires and substrate specificities of granzymes as well as their cleavage efficiencies may add in unraveling the primary signaling pathways these granzymes are involved in. Therefore, a degradome analysis using N-terminal COFRADIC was performed to unravel the substrate repertoires and subsite determinants for the closely related homologous human tryptases. We furthermore also profiled the uncharacterized mouse granzyme K in a comparative analysis with its human ortholog. Despite the subtle differences observed in the primary specificity profiles of these granzymes, for each granzyme distinguishing substrate subsite features could be elucidated.
2015-09-30 | PXD000570 | Pride
Project description:Cryopreserved synovial fluid mononuclear cells from three patients with seropositive rheumatoid arthritis were thawed and stained for surface antibodies, then fixed and stained for intracellular granzyme K and granzyme B. CD8 T cells with the following expression patterns were then isolated by FACS.
2022-05-08 | GSE202365 | GEO