DARPins against a functional IgE epitope

Baumann, Michael; Eggel, Alexander; Amstutz, Patrick; Stadler, Beda M.; Vogel, Monique

doi:10.1016/j.imlet.2010.07.005

Cited by 40 publications

(42 citation statements)

References 58 publications

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“…DARPins that have already been developed against several other targets, such as lactococcal phage TP901-1 (32), HIV gp120 (33), and human IgE (34), are potentially applicable in LAB-mediated therapy. We genetically fused DARPins I_07 and I_19 to the C terminus of the AcmA protein and to the Usp45 secretion signal and expressed the resulting fusion proteins in L. lactis by using nisin-controlled expression (NICE).…”

Section: Discussionmentioning

confidence: 99%

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Zadravec

Štrukelj

Berlec

2015

Appl Environ Microbiol

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bSafety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2-to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32-to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. T he ability to display heterologous proteins on bacterial surfaces is becoming increasingly important in various fields of biotechnology (1-3). Bacteria displaying heterologous proteins can be used as bioadsorbents, biosensors, biocatalysts, and oral vaccines and in antibody production, screening of peptide libraries, and detection of mutations (1-3). Lactic acid bacteria (LAB) are particularly attractive hosts due to their long-term usage in food, their industrial applicability, and the general acceptance of their probiotic properties (4). Several approaches to the display of heterologous proteins on the surfaces of LAB have been established, including the use of LPXTG-type domains (5), lysine motif (LysM) domains (6), surface layer proteins (7), and lipoprotein anchors (8). Display of heterologous proteins on the surfaces of LAB has been exploited for the preparation of mucosal vaccines (9, 10), for the delivery of binding molecules to the gastrointestinal tract (11), for the assembly of macromolecular enzyme complexes (12), and for bacterial immobilization (13).The nonrecombinant approach to surface display is preferred for therapeutic and food applications. This can be achieved by noncovalent binding of recombinant proteins to the surfaces of nonrecombinant bacteria. The feasibility of such an approach has already been demonstrated by using the C-terminal part of the lactococcal AcmA protein (cA) as the cell wall anchor in lactobacilli (14) and in nonviable Gram-positive enhancer matrix (GEM) particles (15). AcmA is an autolysin (N-acetylmuramidase) with a vital role in cell division in Lactococcus lactis. It comprises an enzymatic domain at the N terminus and a peptidoglycan-binding domain at the C terminus that comprises 3 LysM repeats (14,16,17). cA has been used as a fusion partner for noncovalent attachment of heterologous proteins to the surfaces of LAB,...

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Section: Discussionmentioning

confidence: 99%

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Zadravec

Štrukelj

Berlec

2015

Appl Environ Microbiol

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“…Small oligonucleotide aptamers 6,7 , phage-display selected peptides 8–10 , anti-IgE antibodies 11,12 , anti-FcεRI antibodies 13–15 , as well as designed ankyrin repeat proteins (DARPins) 16–18 have been identified as high-affinity inhibitors of IgE-receptor binding. However, only the anti-IgE antibody omalizumab (trade name Xolair ® ) is currently available for the treatment of moderate-to-severe persistent asthma.…”

Section: Introductionmentioning

confidence: 99%

Accelerated dissociation of IgE-FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells

Eggel

Baravalle

Hobi

et al. 2014

Journal of Allergy and Clinical Immunology

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125

114

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Background The remarkably stable interaction of immunoglobulin E (IgE) with its high-affinity receptor FcεRI on basophils and mast cells is critical for the induction of allergic hypersensitivity reactions. Due to the exceptionally slow dissociation rate of IgE:FcεRI complexes such allergic effector cells permanently display allergen-specific IgE on their surface and immediately respond to allergen challenge by releasing inflammatory mediators. We have recently described a novel macromolecular inhibitor that actively promotes the dissociation of IgE from FcεRI through a molecular mechanism termed facilitated dissociation. Objective Here, we assessed the therapeutic potential of this non-immunoglobulin based IgE inhibitor DARPin E2_79 as well as a novel engineered biparatopic DARPin bi53_79 and directly compared them to the established anti-IgE antibody omalizumab. Methods: IgE:FcεRI complex dissociation was analyzed in vitro using recombinant proteins in ELISA and surface plasmon resonance, ex vivo using human primary basophils with flow cytometry and in vivo using human FcεRI transgenic mice in a functional passive cutaneous anaphylaxis test. Results We show that E2_79 mediated removal of IgE from primary human basophils fully abrogates IgE-dependent cell activation and release of pro-inflammatory mediators ex vivo. Furthermore, we report that omalizumab also accelerates the dissociation of IgE from FcεRI albeit much less efficiently than E2_79. Using the biparatopic IgE targeting approach we further improved the disruptive potency of E2_79 by ~100 fold and show that disruptive IgE inhibitors efficiently prevent passive cutaneous anaphylaxis in mice expressing the human FcεRI alpha chain. Conclusion Our findings highlight the potential of such novel IgE inhibitors as important diagnostic and therapeutic tools to managing allergic diseases.

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“…The DE53-Fc fusion protein, described previously [15], consists of a human IgG 1 Fc-part (Uniprot: P01857, amino acids 106-330, comprising hinge-Cγ2-Cγ3) fused to an anti-IgE DARPin [17]. Amino acid substitutions in the Fc-part were introduced by site-directed mutagenesis by overlap extension using PCR [18].…”

Section: Methodsmentioning

confidence: 99%

Improved FcγRIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation

Buschor

Eggel

Zellweger³

et al. 2014

Int Arch Allergy Immunol

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Background: Mast cells and basophils express the high-affinity IgE receptor, FcεRI, as well as the low-affinity IgG receptor, FcγRIIb. While FcεRI is responsible for IgE-dependent degranulation upon coaggregation with allergens, FcγRIIb has been shown to downregulate degranulation through cross-linking with FcεRI. A previously developed fusion protein consisting of an anti-IgE DARPin linked to the human IgG₁-Fc part (DE53-Fc) has been shown to simultaneously target FcεRI and FcγRIIb with low affinity and to thereby prevent basophil activation. The affinity of a ligand for its receptor is known to be critical for the functional consequences of the binding. So we generated two mutated DE53-Fc molecules with either an improved (DE53-Fc mut+) or a reduced (DE53-Fc mut-) binding to FcγRIIb and assessed their potential to inhibit IgE-dependent basophil activation. Methods: DE53-Fc was modified by introducing single site-directed point mutations in the Fc part. The mutated constructs were used to assess kinetic parameters as well as the inhibitory capacity on basophil activation and the production of leukotriene C₄ (LTC₄) and IL-13. Results: DE53-Fc mut+ showed increased affinity for FcγRIIb as well as an enhanced potential to inhibit IgG₁ binding to FcγRIIb, resulting in improved efficacy in functional assays. Furthermore, DE53-Fc mut+ decreased de novo-synthesized LTC₄ as well as the cytokine IL-13, suggesting that it might be an inhibitor of the allergic late-phase reaction. Conclusion: Our data suggest that improved binding to FcγRIIb at constant low-affinity binding to IgE leads to more efficient coaggregation of FcεRI-FcγRIIb and results in the enhanced inhibition of basophil activation.

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DARPins against a functional IgE epitope

Cited by 40 publications

References 58 publications

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Accelerated dissociation of IgE-FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells

Improved FcγRIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation

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DARPins against a functional IgE epitope

Cited by 40 publications

References 58 publications

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

Accelerated dissociation of IgE-FcεRI complexes by disruptive inhibitors actively desensitizes allergic effector cells

Improved Fc&#947;RIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation

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Improved FcγRIIb Targeting Functionally Translates into Enhanced Inhibition of Basophil Activation