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1.
PLoS One ; 19(5): e0298864, 2024.
Article in English | MEDLINE | ID: mdl-38753630

ABSTRACT

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-ß1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.


Subject(s)
Fibrosis , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Renal Insufficiency, Chronic , Animals , Humans , Male , Rabbits , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Disease Models, Animal , Fibrosis/drug therapy , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/immunology , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Macaca fascicularis , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Transglutaminases/antagonists & inhibitors , Transglutaminases/metabolism
2.
MAbs ; 13(1): 1859049, 2021.
Article in English | MEDLINE | ID: mdl-33487120

ABSTRACT

Bispecific antibodies can uniquely influence cellular responses, but selecting target combinations for optimal functional activity remains challenging. Here we describe a high-throughput, combinatorial, phenotypic screening approach using a new bispecific antibody target discovery format, allowing screening of hundreds of target combinations. Simple in vitro mixing of Fab-fusion proteins from a diverse library enables the generation of thousands of screen-ready bispecific antibodies for high-throughput, biologically relevant assays. We identified an obligate bispecific co-targeting CD79a/b and CD22 as a potent inhibitor of human B cell activation from a short-term flow cytometry signaling assay. A long-term, high-content imaging assay identified anti-integrin bispecific inhibitors of human cell matrix accumulation targeting integrins ß1 and ß6 or αV and ß1. In all cases, functional activity was conserved from the bispecific screening format to a therapeutically relevant format. We also introduce a broader type of mechanistic screen whereby functional modulation of different cell subsets in peripheral blood mononuclear cells was evaluated simultaneously. We identified bispecific antibodies capable of activating different T cell subsets of potential interest for applications in oncology or infectious disease, as well as bispecifics abrogating T cell activity of potential interest to autoimmune or inflammatory disease. The bispecific target pair discovery technology described herein offers access to new target biology and unique bispecific therapeutic opportunities in diverse disease indications.


Subject(s)
Antibodies, Bispecific/immunology , CD79 Antigens/immunology , High-Throughput Screening Assays/methods , Immunoglobulin Fab Fragments/immunology , Sialic Acid Binding Ig-like Lectin 2/immunology , Animals , Antibodies, Bispecific/isolation & purification , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Cytokines/immunology , Cytokines/metabolism , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism
3.
Nephron ; 142(4): 328-350, 2019.
Article in English | MEDLINE | ID: mdl-31048591

ABSTRACT

BACKGROUND: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. OBJECTIVE: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. METHOD: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. RESULTS: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor ß neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. CONCLUSION: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.


Subject(s)
Extracellular Matrix/metabolism , Fibrosis/metabolism , Integrins/metabolism , Kidney Diseases/metabolism , Cells, Cultured , Humans , In Vitro Techniques
4.
Sci Rep ; 7(1): 18111, 2017 12 21.
Article in English | MEDLINE | ID: mdl-29269854

ABSTRACT

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

5.
Biol Open ; 6(10): 1423-1433, 2017 Oct 15.
Article in English | MEDLINE | ID: mdl-29032370

ABSTRACT

Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM) in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-ß) blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

6.
Sci Rep ; 7(1): 9963, 2017 08 30.
Article in English | MEDLINE | ID: mdl-28855577

ABSTRACT

Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays: TGFß1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFß antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation: the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.


Subject(s)
Extracellular Matrix/chemistry , Fibrosis/pathology , Kidney Diseases/pathology , Microscopy, Fluorescence/methods , Proteins/analysis , Automation, Laboratory/methods , Cells, Cultured , Epithelial Cells/pathology , Humans , Models, Biological , Staining and Labeling/methods
7.
J Pharmacol Exp Ther ; 361(3): 429-440, 2017 06.
Article in English | MEDLINE | ID: mdl-28442583

ABSTRACT

Phosphoinositide 3-kinases (PI3K) are key signaling enzymes regulating cellular survival, development, and function. Expression of the PI3Kδ isoform is largely restricted to leukocytes and it plays a key role in immune cell development and function. Seletalisib is a novel small-molecule inhibitor of PI3Kδ that was evaluated in biochemical assays, cellular assays of adaptive and innate immunity, and an in vivo rat model of inflammation. Our findings show that seletalisib is a potent, ATP-competitive, and selective PI3Kδ inhibitor able to block protein kinase B (AKT) phosphorylation following activation of the B-cell receptor in a B-cell line. Moreover, seletalisib inhibited N-formyl peptide-stimulated but not phorbol myristate acetate-stimulated superoxide release from human neutrophils, consistent with a PI3Kδ-specific activity. No indications of cytotoxicity were observed in peripheral blood mononuclear cells (PBMCs) or other cell types treated with seletalisib. Findings from cellular assays of adaptive immunity demonstrated that seletalisib blocks human T-cell production of several cytokines from activated T-cells. Additionally, seletalisib inhibited B-cell proliferation and cytokine release. In human whole blood assays, seletalisib inhibited CD69 expression upon B-cell activation and anti-IgE-mediated basophil degranulation. Seletalisib showed dose-dependent inhibition in an in vivo rat model of anti-CD3-antibody-induced interleukin 2 release. Collectively, these data characterize seletalisib as a selective PI3Kδ inhibitor and potential therapeutic candidate for the treatment of B-cell malignancies and autoimmune diseases driven by dysregulated proinflammatory cytokine secretion.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Phosphoinositide-3 Kinase Inhibitors , Pyridines/chemistry , Pyridines/pharmacology , Quinolines/chemistry , Quinolines/pharmacology , Animals , Cells, Cultured , Class I Phosphatidylinositol 3-Kinases , Dose-Response Relationship, Drug , Humans , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/enzymology , Male , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Inbred Lew , Rats, Wistar
8.
Blood ; 115(11): 2203-13, 2010 Mar 18.
Article in English | MEDLINE | ID: mdl-20081091

ABSTRACT

We have previously described critical and nonredundant roles for the phosphoinositide 3-kinase p110delta during the activation and differentiation of naive T cells, and p110delta inhibitors are currently being developed for clinical use. However, to effectively treat established inflammatory or autoimmune diseases, it is important to be able to inhibit previously activated or memory T cells. In this study, using the isoform-selective inhibitor IC87114, we show that sustained p110delta activity is required for interferon-gamma production. Moreover, acute inhibition of p110delta inhibits cytokine production and reduces hypersensitivity responses in mice. Whether p110delta played a similar role in human T cells was unknown. Here we show that IC87114 potently blocked T-cell receptor-induced phosphoinositide 3-kinase signaling by both naive and effector/memory human T cells. Importantly, IC87114 reduced cytokine production by memory T cells from healthy and allergic donors and from inflammatory arthritis patients. These studies establish that previously activated memory T cells are at least as sensitive to p110delta inhibition as naive T cells and show that mouse models accurately predict p110delta function in human T cells. There is therefore a strong rationale for p110delta inhibitors to be considered for therapeutic use in T-cell-mediated autoimmune and inflammatory diseases.


Subject(s)
CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , Cytokines/biosynthesis , Immunity/immunology , Phosphatidylinositol 3-Kinases/metabolism , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Arthritis/enzymology , Arthritis/immunology , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/drug effects , Cell Proliferation/drug effects , Class I Phosphatidylinositol 3-Kinases , Humans , Hypersensitivity/enzymology , Hypersensitivity/immunology , Immunity/drug effects , Immunologic Memory/drug effects , Immunologic Memory/immunology , Lymphocyte Activation/drug effects , Mice , Phosphoinositide-3 Kinase Inhibitors , Quinazolines/pharmacology , Receptors, Antigen, T-Cell/metabolism , Signal Transduction/drug effects
9.
Cell Signal ; 20(6): 1179-89, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18406577

ABSTRACT

Chemokines are known to regulate the chemotaxis of leukocytes and play an important role in immunological processes. Chemokine receptors are widely distributed in hematopoietic cells and are often co-localized with the hematopoietic-specific G(16) and its close relative, G(14). Yet, many chemokine receptors utilize pertussis toxin-sensitive G(i) proteins for signaling. Given that both G(16) and G(14) are capable of linking G(i)-coupled receptors to the stimulation of phospholipase Cbeta, we examined the capacity of six CC chemokine receptors (CCR1, CCR2a, CCR2b, CCR3, CCR5 and CCR7) to interact with G(14) and G(16) in a heterologous expression system. Among the CC chemokine receptors tested, CCR1, CCR2b, and CCR3 were capable of mediating chemokine-induced stimulation of phospholipase Cbeta via either G(14) or G(16). The G(14)/G(16)-mediated responses exhibited CC chemokine dose-dependency and were resistant to pertussis toxin (PTX) treatment. In contrast, CCR2a, CCR5 and CCR7 were unable to interact with G(14) and G(16). Under identical experimental conditions, all six CC chemokine receptors were fully capable of inhibiting adenylyl cyclase via G(i) as well as stimulating phospholipase Cbeta via 16z44, a G(16/z) chimera that possesses increased promiscuity toward G(i)-coupled receptors. Moreover, CCR1-mediated ERK1/2 phosphorylation was largely PTX-insensitive in THP-1 monocytic cells that endogenously express Galpha(16). In addition, CCR1 agonist was less efficacious in mediating chemotaxis of THP-1 cells following the knockdown of Galpha(16) by overexpressing siRNA, indicating the participation of Galpha(16) in CCR1-induced cell migration. These results show that different CC chemokine receptors can discriminate against G(14) and G(16) for signal transduction.


Subject(s)
Chemotaxis , Extracellular Signal-Regulated MAP Kinases/metabolism , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Phospholipase C beta/metabolism , Receptors, CCR/metabolism , Animals , Cell Line , Chemokines/pharmacology , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , Humans , Mice , Pertussis Toxin/pharmacology , Receptors, CCR1/metabolism , Receptors, CCR2/metabolism , Receptors, CCR3/metabolism , Recombinant Fusion Proteins/metabolism , Signal Transduction
10.
Eur J Immunol ; 34(3): 785-795, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14991608

ABSTRACT

Chemokines regulate the chemotaxis, development, and differentiation of many cell types enabling the regulation of routine immunosurveillance and immunological adaptation. CC chemokine receptor 1 (CCR1) is the target of 11 chemokines. This promiscuity of receptor-ligand interactions and the potential for functional redundancy has led us to investigate the selective activation of CCR1-coupled pathways by known CCR1 agonists. Chemokines leukotactin-1, macrophage inflammatory protein (MIP)-1alpha, monocyte chemotactic peptide (MCP)-3, RANTES, and MIP-1delta all inhibited adenylyl cyclase activity in cells transiently transfected with CCR1. In contrast, only MIP-1delta was unable to signal via G14-, G16- or chimeric 16z44-coupled pathways. In a stable cell line expressing CCR1 and Galpha14, all of these five chemokines along with hemofiltrate CC chemokine (HCC)-1 and myeloid progenitor inhibitory factor (MPIF)-1 were able to stimulate G(i/o)-coupled pathways, but MIP-1delta, HCC-1 and MPIF-1 were unable to activate G14-mediated stimulation of phospholipase Cbeta activity. In addition, MIP-1delta was unable to promote the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. This suggests that different chemokines are able to selectively activate CCR1-coupled pathways, probably because of different intrinsic ligand efficacies. CCR1 and Galpha14 or Galpha16 are co-expressed in several cell types and we hypothesize that selective activation of chemokine receptors provides a mechanism by which chemokines are able to fine-tune intracellular signaling pathways.


Subject(s)
Chemokines/pharmacology , GTP-Binding Protein alpha Subunits/metabolism , Receptors, Chemokine/agonists , Signal Transduction , Animals , COS Cells , Cell Line , Chemokine CCL3 , Chemokine CCL4 , Chemotaxis, Leukocyte , Chlorocebus aethiops , GTP-Binding Protein alpha Subunits/genetics , Humans , JNK Mitogen-Activated Protein Kinases , Ligands , Macrophage Inflammatory Proteins/pharmacology , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Receptors, CCR1 , Receptors, Chemokine/genetics , Receptors, Chemokine/metabolism , Transfection
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