scholarly journals Sequential class switching is required for the generation of high affinity IgE antibodies

2012 ◽  
Vol 209 (2) ◽  
pp. 353-364 ◽  
Author(s):  
Huizhong Xiong ◽  
Jayashree Dolpady ◽  
Matthias Wabl ◽  
Maria A. Curotto de Lafaille ◽  
Juan J. Lafaille
Keyword(s):  
Affinity Maturation ◽  
Class Switching ◽  
Ige Antibodies ◽  
High Affinity ◽  
Low Concentrations ◽  
Ige Response

IgE antibodies with high affinity for their antigens can be stably cross-linked at low concentrations by trace amounts of antigen, whereas IgE antibodies with low affinity bind their antigens weakly. In this study, we find that there are two distinct pathways to generate high and low affinity IgE. High affinity IgE is generated through sequential class switching (μ→γ→ε) in which an intermediary IgG phase is necessary for the affinity maturation of the IgE response, where the IgE inherits somatic hypermutations and high affinity from the IgG1 phase. In contrast, low affinity IgE is generated through direct class switching (μ→ε) and is much less mutated. Mice deficient in IgG1 production cannot produce high affinity IgE, even after repeated immunizations. We demonstrate that a small amount of high affinity IgE can cause anaphylaxis and is pathogenic. Low affinity IgE competes with high affinity IgE for binding to Fcε receptors and prevents anaphylaxis and is thus beneficial.

Shark Attack: High affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation

2014 ◽  
Vol 191 ◽  
pp. 236-245 ◽  
Author(s):  
Stefan Zielonka ◽  
Niklas Weber ◽  
Stefan Becker ◽  
Achim Doerner ◽  
Andreas Christmann ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Affinity Maturation ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity

scholarly journals Characterization of murine peritoneal macrophage receptors for fibrin(ogen) degradation products

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1224-1228 ◽  
Author(s):  
S Rajagopalan ◽  
SV Pizzo
Keyword(s):  
Degradation Products ◽  
Peritoneal Macrophages ◽  
Receptor System ◽  
Human Fibrinogen ◽  
Murine Peritoneal Macrophage ◽  
High Affinity ◽  
Macrophage Receptors ◽  
Low Concentrations ◽  
Mouse Peritoneal Macrophages

Abstract The binding of human fibrinogen degradation fragments D1, E, X, and Y, as well as fibrin fragment D1 dimer, to mouse peritoneal macrophages was examined. A Scatchard plot of fragment D1 binding was biphasic, suggesting two classes of receptors. Fragments D1, D1 dimer, X, and Y in low concentrations bound to macrophages with high affinity (Kd = 23 to 73 X 10(-11) mol/L). Fragment E bound specifically but at a much lower level than the other fragments. Fragment D1 was able to compete for the binding of radiolabeled fragments X and Y but not radiolabeled fragment E. These studies indicate that fragments D and E are recognized by separate receptor systems but that all of the fibrinogen degradation products that contain the D domain are recognized by the same receptor system.

scholarly journals Altered Affinity Maturation in Primary Response to (4-hydroxy-3-nitrophenyl) Acetyl (NP) after Autologous Reconstitution of Irradiated C57BL/6 Mice

2002 ◽  
Vol 9 (3) ◽  
pp. 119-125
Author(s):  
Carl De Trez ◽  
Annette Van Acker ◽  
Georgette Vansanten ◽  
Jacques Urbain ◽  
Maryse Brait
Keyword(s):  
Immune Responses ◽  
Single Gene ◽  
Gene Segment ◽  
Keyhole Limpet Hemocyanin ◽  
Primary Response ◽  
Affinity Maturation ◽  
Germinal Center Reaction ◽  
High Affinity ◽  
Primary Antibody Response ◽  
Vh Genes

Immune responses developing in irradiated environment are profoundly altered. The memory anti-arsonate response of A/J mice is dominated by a major clonotype encoded by a single gene segment combination called CRIA. In irradiated and autoreconstituted A/J mice, the level of anti-ARS antibodies upon secondary immunization is normal but devoid of CRIA antibodies. The affinity maturation process and the somatic mutation frequency are reduced. Isotype switching and development of germinal centers (GC) are delayed.The primary antibody response of C57BL/6 mice to the hapten (4-hydroxy-3-nitrophenyl) acetyl (NP)-Keyhole Limpet Hemocyanin (KLH) is dominated by antibodies encoded by a family of closely related VH genes associated with the expression of the λ1 light chain.We investigated the anti-NP primary response in irradiated and autoreconstituted C57BL/6 mice. We observed some splenic alterations as previously described in the irradiated A/J model. Germinal center reaction is delayed although the extrafollicular foci appearance is unchanged. Irradiated C57BL/6 mice are able to mount a primary anti-NP response dominated by λ1 positive antibodies but fail to produce high affinity NP-binding IgGl antibodies. Following a second antigenic challenge, irradiated mice develop enlarged GC and foci. Furthermore, higher affinity NP-binding IgG1 antibodies are detected.

scholarly journals Rapid Affinity Maturation of Novel Anti-PD-L1 Antibodies by a Fast Drop of the Antigen Concentration and FACS Selection of Yeast Libraries

2019 ◽  
Vol 2019 ◽  
pp. 1-22 ◽  
Author(s):  
Biancamaria Cembrola ◽  
Valentino Ruzza ◽  
Fulvia Troise ◽  
Maria Luisa Esposito ◽  
Emanuele Sasso ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Surface Display ◽  
Affinity Maturation ◽  
Yeast Surface Display ◽  
Antigen Concentration ◽  
Selection Scheme ◽  
High Affinity ◽  
Novel Approach ◽  
Yeast Surface

The affinity engineering is a key step to increase the efficacy of therapeutic monoclonal antibodies and yeast surface display is the most widely used and powerful affinity maturation approach, achieving picomolar binding affinities. In this study, we provide an optimization of the yeast surface display methodology, applied to the generation of potentially therapeutic high affinity antibodies targeting the immune checkpoint PD-L1. In this approach, we coupled a 10-cycle error-prone mutagenesis of heavy chain complementarity determining region 3 of an anti‐PD-L1 scFv, previously identified by phage display, with high-throughput sequencing, to generate scFv-yeast libraries with high mutant frequency and diversity. In addition, we set up a novel, faster and effective selection scheme by fluorescence-activated cell sorting, based on a fast drop of the antigen concentration between the first and the last selection cycles, unlike the gradual decrease typical of current selection protocols. In this way we isolated 6 enriched mutated scFv-yeast clones overall, showing an affinity improvement for soluble PD-L1 protein compared to the parental scFv. As a proof of the potency of the novel approach, we confirmed that the antibodies converted from all the mutated scFvs retained the affinity improvement. Remarkably, the best PD-L1 binder among them also bound with a higher affinity to PD-L1 expressed in its native conformation on human-activated lymphocytes, and it was able to stimulate lymphocyte proliferation in vitro more efficiently than its parental antibody. This optimized technology, besides the identification of a new potential checkpoint inhibitor, provides a tool for the quick isolation of high affinity binders.

scholarly journals Generation of high-affinity fully human anti-interleukin-8 antibodies from its cDNA by two-hybrid screening and affinity maturation in yeast

2010 ◽  
Vol 19 (10) ◽  
pp. 1957-1966 ◽  
Author(s):  
Ling Ding ◽  
Mark Azam ◽  
Yu-Huei Lin ◽  
James Sheridan ◽  
Shuanghong Wei ◽  
...  
Keyword(s):  
Affinity Maturation ◽  
Interleukin 8 ◽  
High Affinity ◽  
Two Hybrid ◽  
Hybrid Screening

scholarly journals Kinetics of binding, endocytosis, and recycling of EGF receptor mutants

1992 ◽  
Vol 117 (1) ◽  
pp. 203-212 ◽  
Author(s):  
S Felder ◽  
J LaVin ◽  
A Ullrich ◽  
J Schlessinger
Keyword(s):  
Point Mutation ◽  
Phorbol Ester ◽  
Egf Receptor ◽  
Receptor Internalization ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Low Concentrations ◽  
Internalization Rate ◽  
Kinetics Of

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.

scholarly journals Znu Is the Predominant Zinc Importer in Yersinia pestis during In Vitro Growth but Is Not Essential for Virulence

2010 ◽  
Vol 78 (12) ◽  
pp. 5163-5177 ◽  
Author(s):  
Daniel C. Desrosiers ◽  
Scott W. Bearden ◽  
Ildefonso Mier ◽  
Jennifer Abney ◽  
James T. Paulley ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Binding Protein ◽  
Bacterial Pathogens ◽  
Titration Calorimetry ◽  
Growth Defect ◽  
Uptake System ◽  
High Affinity ◽  
Zn Uptake ◽  
Low Concentrations

ABSTRACT Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.

scholarly journals Physical epistatic landscape of antibody binding affinity

2017 ◽  
Author(s):  
Rhys M. Adams ◽  
Justin B. Kinney ◽  
Aleksandra M. Walczak ◽  
Thierry Mora
Keyword(s):  
Free Energy ◽  
Binding Free Energy ◽  
Evolutionary Process ◽  
Null Model ◽  
High Specificity ◽  
Affinity Maturation ◽  
High Affinity ◽  
Antibody Sequence ◽  
Negative Epistasis ◽  
Insight Into

Affinity maturation produces antibodies that bind antigens with high specificity by accumulating mutations in the antibody sequence. Mapping out the antibody-antigen affinity landscape can give us insight into the accessible paths during this rapid evolutionary process. By developing a carefully controlled null model for noninteracting mutations, we characterized epistasis in affinity measurements of a large library of antibody variants obtained by Tite-Seq, a recently introduced Deep Mutational Scan method yielding physical values of the binding constant. We show that representing affinity as the binding free energy minimizes epistasis. Yet, we find that epistatically interacting sites contribute substantially to binding. In addition to negative epistasis, we report a large amount of beneficial epistasis, enlarging the space of high-affinity antibodies as well as their mutational accessibility. These properties suggest that the degeneracy of antibody sequences that can bind a given antigen is enhanced by epistasis — an important property for vaccine design.

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