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  1. Article ; Online: Engineering a natural ligand-based CAR: directed evolution of the stress-receptor NKp30.

    Butler, Savannah E / Brog, Rachel A / Chang, Cheryl H / Sentman, Charles L / Huang, Yina H / Ackerman, Margaret E

    Cancer immunology, immunotherapy : CII

    2021  Volume 71, Issue 1, Page(s) 165–176

    Abstract: B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen ... ...

    Abstract B7H6, a stress-induced ligand which binds to the NK cell receptor NKp30, has recently emerged as a promising candidate for immunotherapy due to its tumor-specific expression on a broad array of human tumors. NKp30 can function as a chimeric antigen receptor (CAR) extracellular domain but exhibits weak binding with a fast on and off rate to B7H6 compared to the TZ47 anti-B7H6 single-chain variable fragment (scFv). Here, directed evolution using yeast display was employed to isolate novel NKp30 variants that bind to B7H6 with higher affinity compared to the native receptor but retain its fast association and dissociation profile. Two variants, CC3 and CC5, were selected for further characterization and were expressed as soluble Fc-fusion proteins and CARs containing CD28 and CD3ς intracellular domains. We observed that Fc-fusion protein forms of NKp30 and its variants were better able to bind tumor cells expressing low levels of B7H6 than TZ47, and that the novel variants generally exhibited improved in vitro tumor cell killing relative to NKp30. Interestingly, CAR T cells expressing the engineered variants produced unique cytokine signatures in response to multiple tumor types expressing B7H6 compared to both NKp30 and TZ47. These findings suggest that natural CAR receptors can be fine-tuned to produce more desirable signaling outputs while maintaining evolutionary advantages in ligand recognition relative to scFvs.
    MeSH term(s) Animals ; B7 Antigens/chemistry ; CD28 Antigens/chemistry ; CD3 Complex/chemistry ; Cell Line, Tumor ; Cell Separation ; Cytokines/metabolism ; Flow Cytometry ; Gene Expression Profiling ; Gene Library ; Genetic Variation ; HEK293 Cells ; Humans ; Immunotherapy ; Kinetics ; Ligands ; Mice ; Mutation ; Natural Cytotoxicity Triggering Receptor 3/chemistry ; Protein Conformation ; Receptors, Chimeric Antigen/chemistry ; Single-Chain Antibodies/chemistry
    Chemical Substances B7 Antigens ; CD28 Antigens ; CD3 Complex ; CD3 antigen, zeta chain ; Cytokines ; Ligands ; NCR3LG1 protein, human ; Natural Cytotoxicity Triggering Receptor 3 ; Receptors, Chimeric Antigen ; Single-Chain Antibodies
    Language English
    Publishing date 2021-05-27
    Publishing country Germany
    Document type Journal Article
    ZDB-ID 195342-4
    ISSN 1432-0851 ; 0340-7004
    ISSN (online) 1432-0851
    ISSN 0340-7004
    DOI 10.1007/s00262-021-02971-y
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1237: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
    Jg. 1995 - 2021: Lesesall (1.OG)
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  2. Article ; Online: Engineering a potent inhibitor of matriptase from the natural hepatocyte growth factor activator inhibitor type-1 (HAI-1) protein.

    Mitchell, Aaron C / Kannan, Deepti / Hunter, Sean A / Parra Sperberg, R Andres / Chang, Cheryl H / Cochran, Jennifer R

    The Journal of biological chemistry

    2018  Volume 293, Issue 14, Page(s) 4969–4980

    Abstract: Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, ... ...

    Abstract Dysregulated matriptase activity has been established as a key contributor to cancer progression through its activation of growth factors, including the hepatocyte growth factor (HGF). Despite its critical role and prevalence in many human cancers, limitations to developing an effective matriptase inhibitor include weak binding affinity, poor selectivity, and short circulating half-life. We applied rational and combinatorial approaches to engineer a potent inhibitor based on the hepatocyte growth factor activator inhibitor type-1 (HAI-1), a natural matriptase inhibitor. The first Kunitz domain (KD1) of HAI-1 has been well established as a minimal matriptase-binding and inhibition domain, whereas the second Kunitz domain (KD2) is inactive and involved in negative regulation. Here, we replaced the inactive KD2 domain of HAI-1 with an engineered chimeric variant of KD2/KD1 domains and fused the resulting construct to an antibody Fc domain to increase valency and circulating serum half-life. The final protein variant contains four stoichiometric binding sites that we showed were needed to effectively inhibit matriptase with a
    MeSH term(s) Amino Acid Sequence ; Animals ; Cell Line, Tumor ; Dogs ; Humans ; Madin Darby Canine Kidney Cells ; Protein Domains ; Protein Engineering/methods ; Proteinase Inhibitory Proteins, Secretory/chemistry ; Proteinase Inhibitory Proteins, Secretory/genetics ; Proteinase Inhibitory Proteins, Secretory/metabolism ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/pharmacology ; Serine Endopeptidases/metabolism ; Serine Proteinase Inhibitors/chemistry ; Serine Proteinase Inhibitors/pharmacology
    Chemical Substances Proteinase Inhibitory Proteins, Secretory ; Recombinant Fusion Proteins ; SPINT1 protein, human ; Serine Proteinase Inhibitors ; Serine Endopeptidases (EC 3.4.21.-) ; matriptase (EC 3.4.21.-)
    Language English
    Publishing date 2018-01-31
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 2997-x
    ISSN 1083-351X ; 0021-9258
    ISSN (online) 1083-351X
    ISSN 0021-9258
    DOI 10.1074/jbc.M117.815142
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Uc I Zs.108: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular
    ab Jg. 2022: Lesesaal (EG)

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  3. Article ; Online: Development of a Protease Biosensor Based on a Dimerization-Dependent Red Fluorescent Protein.

    Mitchell, Aaron C / Alford, Spencer C / Hunter, Sean A / Kannan, Deepti / Parra Sperberg, R Andres / Chang, Cheryl H / Cochran, Jennifer R

    ACS chemical biology

    2017  Volume 13, Issue 1, Page(s) 66–72

    Abstract: Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address ... ...

    Abstract Dysregulated activity of the protease matriptase is a key contributor to aggressive tumor growth, cancer metastasis, and osteoarthritis. Methods for the detection and quantification of matriptase activity and inhibition would be useful tools. To address this need, we developed a matriptase-sensitive protein biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) reporter system. In this platform, two adjoining protein domains, connected by a protease-labile linker, produce fluorescence when assembled and are nonfluorescent when the linker is cleaved by matriptase. A panel of ddRFP-based matriptase biosensor designs was created that contained different linker lengths between the protein domains. These constructs were characterized for linker-specific cleavage, matriptase activity, and matriptase selectivity; a biosensor containing a RSKLRVGGH linker (termed B4) was expressed at high yields and displayed both high catalytic efficiency and matriptase specificity. This biosensor detects matriptase inhibition by soluble and yeast cell surface expressed inhibitor domains with up to a 5-fold dynamic range and also detects matriptase activity expressed by human cancer cell lines. In addition to matriptase, we highlight a strategy that can be used to create effective biosensors for quantifying activity and inhibition of other proteases of interest.
    MeSH term(s) Biosensing Techniques/methods ; Blotting, Western ; Cell Line, Tumor ; Drug Evaluation, Preclinical/instrumentation ; Drug Evaluation, Preclinical/methods ; Escherichia coli/genetics ; Humans ; Luminescent Proteins/chemistry ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Peptide Hydrolases/analysis ; Peptide Hydrolases/metabolism ; Protein Multimerization ; Serine Endopeptidases/analysis ; Serine Endopeptidases/metabolism ; Serine Proteinase Inhibitors/pharmacology ; Structure-Activity Relationship ; Red Fluorescent Protein
    Chemical Substances Luminescent Proteins ; Serine Proteinase Inhibitors ; Peptide Hydrolases (EC 3.4.-) ; Serine Endopeptidases (EC 3.4.21.-) ; matriptase (EC 3.4.21.-)
    Language English
    Publishing date 2017-12-12
    Publishing country United States
    Document type Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ISSN 1554-8937
    ISSN (online) 1554-8937
    DOI 10.1021/acschembio.7b00715
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  4. Article ; Online: A critical role for the mTORC2 pathway in lung fibrosis.

    Chang, Wenteh / Wei, Ke / Ho, Lawrence / Berry, Gerald J / Jacobs, Susan S / Chang, Cheryl H / Rosen, Glenn D

    PloS one

    2014  Volume 9, Issue 8, Page(s) e106155

    Abstract: A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory ... ...

    Abstract A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.
    MeSH term(s) Animals ; Benzoxazoles/pharmacology ; Bleomycin ; Carrier Proteins/antagonists & inhibitors ; Carrier Proteins/genetics ; Carrier Proteins/metabolism ; Coculture Techniques ; Epithelial Cells/cytology ; Epithelial Cells/drug effects ; Epithelial Cells/metabolism ; Extracellular Matrix Proteins/antagonists & inhibitors ; Extracellular Matrix Proteins/genetics ; Extracellular Matrix Proteins/metabolism ; Fibroblasts/drug effects ; Fibroblasts/metabolism ; Fibroblasts/pathology ; Gene Expression Regulation ; Mechanistic Target of Rapamycin Complex 1 ; Mechanistic Target of Rapamycin Complex 2 ; Mice ; Multiprotein Complexes/antagonists & inhibitors ; Multiprotein Complexes/genetics ; Multiprotein Complexes/metabolism ; Protective Agents/pharmacology ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors ; Proto-Oncogene Proteins c-akt/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Pulmonary Fibrosis/chemically induced ; Pulmonary Fibrosis/drug therapy ; Pulmonary Fibrosis/genetics ; Pulmonary Fibrosis/pathology ; Pyrimidines/pharmacology ; Rapamycin-Insensitive Companion of mTOR Protein ; Signal Transduction ; TOR Serine-Threonine Kinases/antagonists & inhibitors ; TOR Serine-Threonine Kinases/genetics ; TOR Serine-Threonine Kinases/metabolism ; Transforming Growth Factor beta/pharmacology
    Chemical Substances Benzoxazoles ; Carrier Proteins ; Extracellular Matrix Proteins ; Multiprotein Complexes ; Protective Agents ; Pyrimidines ; Rapamycin-Insensitive Companion of mTOR Protein ; Transforming Growth Factor beta ; rictor protein, mouse ; Bleomycin (11056-06-7) ; mTOR protein, mouse (EC 2.7.1.1) ; Mechanistic Target of Rapamycin Complex 1 (EC 2.7.11.1) ; Mechanistic Target of Rapamycin Complex 2 (EC 2.7.11.1) ; Proto-Oncogene Proteins c-akt (EC 2.7.11.1) ; TOR Serine-Threonine Kinases (EC 2.7.11.1) ; sapanisertib (JGH0DF1U03)
    Language English
    Publishing date 2014-08-27
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't
    ZDB-ID 2267670-3
    ISSN 1932-6203 ; 1932-6203
    ISSN (online) 1932-6203
    ISSN 1932-6203
    DOI 10.1371/journal.pone.0106155
    Database MEDical Literature Analysis and Retrieval System OnLINE

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