Specific inhibition of interleukin-13 activity by a recombinant human single-chain immunoglobulin domain directed against the IL-13 receptor α1 chain

2007 ◽  
Vol 388 (3) ◽  
pp. 325-330 ◽  
Author(s):  
Stefan Knackmuss ◽  
Sebastian Krause ◽  
Kathrin Engel ◽  
Uwe Reusch ◽  
J. Christian Virchow ◽  
...  
Keyword(s):  
Cell Activation ◽  
Binding Capacity ◽  
Specific Binding ◽  
Critical Role ◽  
Target Cells ◽  
Dependent Manner ◽  
Single Chain ◽  
Interleukin 13 ◽  
Single Chain Fv ◽  
Immunoglobulin Domain

Abstract Interleukin-13 (IL-13) is a T-cell-derived pleiotropic cytokine of particular medical importance because of its critical role in the development of allergic asthma. The effects of IL-13 on its target cells are mediated through a dimeric transmembrane receptor (IL-13R), which shares the IL-4Rα subunit with the IL-4R system, but contains as a specific component the IL-13Rα1 chain. We have generated a set of single-chain Fv fragments with specific binding capacity to the extracellular domain of the human IL-13Rα1 receptor. Bacteriophage clones displaying receptor-binding antibody domains were selected from both naive and synthetic libraries by repetitive panning on recombinant and cell surface-expressed recombinant IL-13Rα1. Their specific reactivity with native human IL-13Rα1 expressed on the surface of transfected cells was demonstrated by flow cytometry. One binder that specifically interfered with cell activation by IL-13 was extensively characterized. This scFv inhibited IL-13-driven gene transcription and cell proliferation in test cell lines, as well as IL-13-induced activation of primary human monocytes in a dose-dependent manner, with an IC50 below 300 nM. This novel reagent thus constitutes a valuable tool for the further elucidation of IL-13 function in disease and offers potential therapeutic perspectives.

scholarly journals NK cell-based cancer immunotherapy: from basic biology to clinical development

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Sizhe Liu ◽  
Vasiliy Galat ◽  
Yekaterina Galat4 ◽  
Yoo Kyung Annie Lee ◽  
Derek Wainwright ◽  
...  
Keyword(s):  
Cancer Immunotherapy ◽  
T Cell Activation ◽  
Cell Biology ◽  
Antigen Processing ◽  
Cell Activation ◽  
Nk Cell ◽  
Critical Role ◽  
Clinical Development ◽  
Target Cells ◽  
Immune Effector

AbstractNatural killer (NK) cell is a specialized immune effector cell type that plays a critical role in immune activation against abnormal cells. Different from events required for T cell activation, NK cell activation is governed by the interaction of NK receptors with target cells, independent of antigen processing and presentation. Due to relatively unsophisticated cues for activation, NK cell has gained significant attention in the field of cancer immunotherapy. Many efforts are emerging for developing and engineering NK cell-based cancer immunotherapy. In this review, we provide our current understandings of NK cell biology, ongoing pre-clinical and clinical development of NK cell-based therapies and discuss the progress, challenges, and future perspectives.

scholarly journals A scDb-based trivalent bispecific antibody for T-cell-mediated killing of HER3-expressing cancer cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nadine Aschmoneit ◽  
Sophia Steinlein ◽  
Lennart Kühl ◽  
Oliver Seifert ◽  
Roland E. Kontermann
Keyword(s):  
T Cell ◽  
Binding Site ◽  
Cancer Cell ◽  
Cancer Progression ◽  
T Cell Activation ◽  
Cell Activation ◽  
Egf Receptor ◽  
Bispecific Antibodies ◽  
Target Cells ◽  
Single Chain

AbstractHER3 is a member of the EGF receptor family and elevated expression is associated with cancer progression and therapy resistance. HER3-specific T-cell engagers might be a suitable treatment option to circumvent the limited efficacy observed for HER3-blocking antibodies in clinical trials. In this study, we developed bispecific antibodies for T-cell retargeting to HER3-expressing tumor cells, utilizing either a single-chain diabody format (scDb) with one binding site for HER3 and one for CD3 on T-cells or a trivalent bispecific scDb-scFv fusion protein exhibiting an additional binding site for HER3. The scDb-scFv showed increased binding to HER3-expressing cancer cell lines compared to the scDb and consequently more effective T-cell activation and T-cell proliferation. Furthermore, the bivalent binding mode of the scDb-scFv for HER3 translated into more potent T-cell mediated cancer cell killing, and allowed to discriminate between moderate and low HER3-expressing target cells. Thus, our study demonstrated the applicability of HER3 for T-cell retargeting with bispecific antibodies, even at moderate expression levels, and the increased potency of an avidity-mediated specificity gain, potentially resulting in a wider safety window of bispecific T-cell engaging antibodies targeting HER3.

scholarly journals Bispecific T-Cell Engagers Targeting Membrane-Bound IgE

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1568
Author(s):  
Aleksandra Rodak ◽  
Gerhard Stadlmayr ◽  
Katharina Stadlbauer ◽  
Dominic Lichtscheidl ◽  
Madhusudhan Reddy Bobbili ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
T Cell Activation ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Single Chain ◽  
Ige Antibodies ◽  
Serum Ige ◽  
Single Chain Fv ◽  
Membrane Bound

The increased incidence of allergies and asthma has sparked interest in IgE, the central player in the allergic response. Interaction with its high-affinity receptor FcεRI leads to sensitization and allergen presentation, extracellular membrane-proximal domain in membrane IgE can act as an antigen receptor on B cells, and the interaction with low-affinity IgE receptor CD23 additionally influences its homeostatic range. Therapeutic anti-IgE antibodies act by the inhibition of IgE functions by interfering with its receptor binding or by the obliteration of IgE-B cells, causing a reduction of serum IgE levels. Fusion proteins of antibody fragments that can act as bispecific T-cell engagers have proven very potent in eliciting cytotoxic T-lymphocyte-mediated killing. We have tested five anti-IgE Fc antibodies, recognizing different epitopes on the membrane-expressed IgE, for the ability to elicit specific T-cell activation when expressed as single-chain Fv fragments fused with anti-CD3ε single-chain antibody. All candidates could specifically stain the cell line, expressing the membrane-bound IgE-Fc and bind to CD3-positive Jurkat cells, and the specific activation of engineered CD3-overexpressing Jurkat cells and non-stimulated CD8-positive cells was demonstrated for 8D6- and ligelizumab-based bispecific antibodies. Thus, such anti-IgE antibodies have the potential to be developed into agents that reduce the serum IgE concentration by lowering the numbers of IgE-secreting cells.

Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14

2002 ◽  
Vol 283 (3) ◽  
pp. G640-G645 ◽  
Author(s):  
Grace L. Su ◽  
Sanna M. Goyert ◽  
Ming-Hui Fan ◽  
Alireza Aminlari ◽  
Ke Qin Gong ◽  
...  
Keyword(s):  
Kupffer Cell ◽  
Kupffer Cells ◽  
Knockout Mice ◽  
Cell Activation ◽  
Critical Role ◽  
Dependent Manner ◽  
Liver Macrophage ◽  
Tnf Α ◽  
Presence And Absence ◽  
Lps Activation

Upregulation of CD14 in Kupffer cells has been implicated in the pathogenesis of several forms of liver injury, including alcoholic liver disease. However, it remains unclear whether CD14 mediates lipopolysaccharide (LPS) signaling in this specialized liver macrophage population. In this series of experiments, we determined the role of CD14 in LPS activation of Kupffer cells by using several complementary approaches. First, we isolated Kupffer cells from human livers and studied the effects of anti-CD14 antibodies on LPS activation of these cells. Kupffer cells were incubated with increasing concentrations of LPS in the presence and absence of recombinant human LPS binding protein (LBP). With increasing concentrations of LPS, human Kupffer cell tumor necrosis factor-α (TNF-α) production (a marker for Kupffer cell activation) increased in a dose-dependent manner in the presence and absence of LBP. In the presence of anti-human CD14 antibodies, the production of TNF-α was significantly diminished. Second, we compared LPS activation of Kupffer cells isolated from wild-type and CD14 knockout mice. Kupffer cells from CD14 knockout mice produced significantly less TNF-α in response to the same amount of LPS. Together, these data strongly support a critical role for CD14 in Kupffer cell responses to LPS.

scholarly journals Neutralizationof Enteric Coronaviruses with Escherichia coli CellsExpressing Single-Chain Fv-AutotransporterFusions

2003 ◽  
Vol 77 (24) ◽  
pp. 13396-13398 ◽  
Author(s):  
Esteban Veiga ◽  
Víctor de Lorenzo ◽  
Luis Angel Fernández
Keyword(s):  
Escherichia Coli ◽  
Infectious Agent ◽  
Target Cells ◽  
Single Chain ◽  
E Coli ◽  
Single Chain Antibodies ◽  
Single Chain Fv ◽  
Transmissible Gastroenteritis ◽  
Iga Protease

ABSTRACT We report here that fusions of single-chain antibodies (scFvs) to the autotransporter β domain of the IgA protease of Neisseria gonorrhoeae are instrumental in locating virus-neutralizing activity on the cell surface of Escherichia coli. E. coli cells displaying scFvs against the transmissible gastroenteritis coronavirus on their surface blocked in vivo the access of the infectious agent to cultured epithelial cells. This result raises prospects for antiviral strategies aimed at hindering the entry into target cells by bacteria that naturally colonize the same intestinal niches.

Activation of T Cells by Bispecific Single-Chain Construct MT103 (MEDI-538) Is Strictly Target Cell-Dependent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3889-3889
Author(s):  
Klaus Brischwein ◽  
Scott A. Hammond ◽  
Larissa Parr ◽  
Schlereth Bernd ◽  
Mathias Locher ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Target Cell ◽  
Cell Activation ◽  
Cell Lysis ◽  
Target Cells ◽  
Single Chain ◽  
Activation Markers ◽  
Human T Cells

Abstract Background: Bispecific antibodies have been extensively studied in vitro and in vivo for their use in redirected tumor cell lysis. A particular challenge of bispecific antibody constructs recognizing the CD3 signaling complex is to achieve a controlled polyclonal activation of T-cells that, ideally, is entirely dependent on the presence of target cells. If this is not the case, systemic production of inflammatory cytokines and secondary endothelial reactions may occur as side effects, as are observed with the murine anti-human CD3e antibody OKT-3 (muromab, Orthoclone®). Here we present evidence that MT103 (or MEDI-538), a bispecific single chain antibody of the BiTE class that targets CD19 and CD3, induces T-cell activation exclusively in the presence of target cells. Material and methods: Peripheral blood mononuclear cells from healthy donors were prepared by Ficoll density centrifugation. PBMC were incubated for 24 hours with MT103 in presence or absence of specific target cells. Target cell lysis was determined by measurement of adenylate kinase activity released from lysed cells. De novo expression of activation markers CD69 and CD25 on T-cells was assessed by flow cytometry using directly conjugated monoclonal antibodies, and the concentration of cytokines in the supernatant was determined by a commercial FACS-based bead array. Results: MT103 was analyzed for conditional T-cell activation. In the presence of target-expressing cell lines, low picomolar concentrations of MT103 were sufficient to stimulate a high percentage of peripheral human T-cells to express cytokines and surface activation markers, to enter into the cell cycle and to induce redirected lysis of target cells. However, in the absence of target cells, the BiTE molecules no longer detectably activated human T-cells even at concentrations exceeding the ED50 for redirected lysis and conditional T-cell activation by more than five orders of magnitude. Conclusion: Our data show that T-cell activation by MT103 is highly conditional in that it is strictly dependent on the presence.

scholarly journals A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies

Blood ◽  
2012 ◽  
Vol 119 (3) ◽  
pp. 884-893 ◽  
Author(s):  
Kristi L. Allen ◽  
Fabio V. Fonseca ◽  
Venkaiah Betapudi ◽  
Belinda Willard ◽  
Jainwei Zhang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cell Activation ◽  
Transmembrane Protein ◽  
Critical Role ◽  
Fetal Loss ◽  
Annexin A2 ◽  
Dependent Manner ◽  
Protein Protein Interactions ◽  
Endothelial Cell Activation ◽  
Signaling Complex

Abstract Antiphospholipid Abs (APLAs) are associated with thrombosis and recurrent fetal loss. These Abs are primarily directed against phospholipid-binding proteins, particularly β2GPI, and activate endothelial cells (ECs) in a β2GPI-dependent manner after binding of β2GPI to EC annexin A2. Because annexin A2 is not a transmembrane protein, the mechanisms of APLA/anti-β2GPI Ab–mediated EC activation are uncertain, although a role for a TLR4/myeloid differentiation factor 88–dependent pathway leading to activation of NF-κB has been proposed. In the present study, we confirm a critical role for TLR4 in anti-β2GPI Ab–mediated EC activation and demonstrate that signaling through TLR4 is mediated through the assembly of a multiprotein signaling complex on the EC surface that includes annexin A2, TLR4, calreticulin, and nucleolin. An essential role for each of these proteins in cell activation is suggested by the fact that inhibiting the expression of each using specific siRNAs blocked EC activation mediated by APLAs/anti-β2GPI Abs. These results provide new evidence for novel protein-protein interactions on ECs that may contribute to EC activation and the pathogenesis of APLA/anti-β2GPI–associated thrombosis and suggest potential new targets for therapeutic intervention in antiphospholipid syndrome.

scholarly journals Endothelial cell activation by antiphospholipid antibodies is modulated by Krüppel-like transcription factors

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6383-6391 ◽  
Author(s):  
Kristi L. Allen ◽  
Anne Hamik ◽  
Mukesh K. Jain ◽  
Keith R. McCrae
Keyword(s):  
Endothelial Cells ◽  
Antiphospholipid Antibodies ◽  
Transcriptional Activity ◽  
Cell Activation ◽  
Critical Role ◽  
Endothelial Activation ◽  
Dependent Manner ◽  
Endothelial Cell Activation ◽  
Proinflammatory Response ◽  
Endothelial Response

Abstract Antiphospholipid syndrome is characterized by thrombosis and/or recurrent pregnancy loss in the presence of antiphospholipid antibodies (APLAs). The majority of APLAs are directed against phospholipid-binding proteins, particularly β2-glycoprotein I (β2GPI). Anti-β2GPI antibodies activate endothelial cells in a β2GPI-dependent manner through a pathway that involves NF-κB. Krüppel-like factors (KLFs) play a critical role in regulating the endothelial response to inflammatory stimuli. We hypothesized that activation of endothelial cells by APLA/anti-β2GPI antibodies might be associated with decreased expression of KLFs, which in turn might facilitate cellular activation mediated through NF-κB. Our experimental results confirmed this hypothesis, demonstrating markedly decreased expression of KLF2 and KLF4 after incubation of cells with APLA/anti-β2GPI antibodies. Restoration of KLF2 or KLF4 levels inhibited NF-κB transcriptional activity and blocked APLA/anti-β2GPI–mediated endothelial activation despite NF-κB p65 phosphorylation. Chromatin immunoprecipitation analysis demonstrated that inhibition of NF-κB transcriptional activity by KLFs reflects sequestration of the cotranscriptional activator CBP/p300, making this cofactor unavailable to NF-κB. These findings suggest that the endothelial response to APLA/anti-β2GPI antibodies reflects competition between KLFs and NF-κB for their common cofactor, CBP/p300. Taken together, these observations are the first to implicate the KLFs as novel participants in the endothelial proinflammatory response to APLA/anti-β2GPI antibodies.

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