Molecular Evolution of Antibody Affinity for Sensitive Detection of Botulinum Neurotoxin Type A

Razai, Ali; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Geren, Isin N.; Forsyth, Charles M.; Robles, Yetzi; Tsai, Richard; Smith, Theresa J.; Smith, Leonard A.; Siegel, Robert W.; Feldhaus, Michael J.; Marks, James D.

doi:10.1016/j.jmb.2005.06.003

Cited by 129 publications

(151 citation statements)

References 42 publications

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“…2B, mean ± standard deviation), which is lower than the monovalent affinity for the full-length IgG (K d = 0.35 nM) generated using the same variable regions (27). Even though scFv constructs are highly advantageous for imaging (37) and delivery (38)(39)(40), a 5 to 15-fold reduction in affinity upon conversion of IgG to scFv had been reported (38,41), potentially due to higher stability and functional concentration of full IgGs. To confirm that the yeast displayed scFv was properly folded, we generated alanine point mutations in the CDR residues that interacted with the phosphoepitope (Supp.…”

Section: Yeast Surface Display Of a Phospho-tau-specific Scfvmentioning

confidence: 87%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTMtargeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phospho-threonine 231 (pT231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinitymatured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved > 20-fold over that of the wild-type antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity towards the nonphosphorylated target site, but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phospho-tau antibody. In cell-and tissueimaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTMspecific antibodies, and highlight the importance of assessing the specificity in the antibody engineering process.

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Section: Yeast Surface Display Of a Phospho-tau-specific Scfvmentioning

confidence: 87%

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Wang

Maziuk

et al. 2018

Journal of Biological Chemistry

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“…Simple addition of an albumin binding peptide (34,36) can dramatically improve the serum stability of VNAs (34). Because VHHs can be obtained from preexisting libraries (37) and then affinity-matured for improved properties (38,39), new antitoxins can be rapidly developed as treatments for emerging threat agents. VHHs are known to be poorly immunogenic (5), and immunogenicity can by reduced further by introducing targeted mutations (40).…”

Section: Discussionmentioning

confidence: 99%

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

Moayeri

Leysath

Tremblay

et al. 2015

Journal of Biological Chemistry

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Background: Anthrax toxin is the primary cause of pathology from exposure to anthrax spores. Results: Two linked single domain antibodies (VHHs), each neutralizing anthrax toxicity by different mechanisms, potently protect mice from anthrax spore challenge. Conclusion: Linked, toxin-neutralizing VHHs (VNAs) are highly effective anthrax antitoxin agents. Significance: VNAs offer excellent versatility in developing novel therapeutics for many toxin-mediated diseases.

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“…Micromagnetically activated cell sorting (MACS) HA-tagged magnetic immunobead and CD20 MicroBeads were purchased from Miltenyi (Auburn, CA). The yeast display vector pYD2 was kindly provided by J. D. Marks (University of California at San Francisco [UCSF]) (21). IgG1-Abvec for full-length immunoglobulin G1 (IgG1) expression was kindly provided by P. Wilson (University of Chicago).…”

Section: Methodsmentioning

confidence: 99%

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

Keck

Enterlein

Howell

et al. 2016

J Virol

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Filoviruses cause highly lethal viral hemorrhagic fever in humans and nonhuman primates. Current immunotherapeutic options for filoviruses are mostly specific to Ebola virus (EBOV), although other members of Filoviridae such as Sudan virus (SUDV), Bundibugyo virus (BDBV), and Marburg virus (MARV) have also caused sizeable human outbreaks. Here we report a set of pan-ebolavirus and pan-filovirus monoclonal antibodies (MAbs) derived from cynomolgus macaques immunized repeatedly with a mixture of engineered glycoproteins (GPs) and virus-like particles (VLPs) for three different filovirus species. The antibodies recognize novel neutralizing and nonneutralizing epitopes on the filovirus glycoprotein, including conserved conformational epitopes within the core regions of the GP1 subunit and a novel linear epitope within the glycan cap. We further report the first filovirus antibody binding to a highly conserved epitope within the fusion loop of ebolavirus and marburgvirus species. One of the antibodies binding to the core GP1 region of all ebolavirus species and with lower affinity to MARV GP cross neutralized both SUDV and EBOV, the most divergent ebolavirus species. In a mouse model of EBOV infection, this antibody provided 100% protection when administered in two doses and partial, but significant, protection when given once at the peak of viremia 3 days postinfection. Furthermore, we describe novel cocktails of antibodies with enhanced protective efficacy compared to individual MAbs. In summary, the present work describes multiple novel, cross-reactive filovirus epitopes and innovative combination concepts that challenge the current therapeutic models. IMPORTANCEFiloviruses are among the most deadly human pathogens. The 2014-2015 outbreak of Ebola virus disease (EVD) led to more than 27,000 cases and 11,000 fatalities. While there are five species of Ebolavirus and several strains of marburgvirus, the current immunotherapeutics primarily target Ebola virus. Since the nature of future outbreaks cannot be predicted, there is an urgent need for therapeutics with broad protective efficacy against multiple filoviruses. Here we describe a set of monoclonal antibodies cross-reactive with multiple filovirus species. These antibodies target novel conserved epitopes within the envelope glycoprotein and exhibit protective efficacy in mice. We further present novel concepts for combination of cross-reactive antibodies against multiple epitopes that show enhanced efficacy compared to monotherapy and provide complete protection in mice. These findings set the stage for further evaluation of these antibodies in nonhuman primates and development of effective pan-filovirus immunotherapeutics for use in future outbreaks. F iloviruses consisting of Marburg virus (MARV), Ravn virus (RAVV), and five species of ebolavirus, Ebola virus (EBOV), Sudan virus (SUDV), Bundibugyo virus (BDBV), Reston virus (RESTV), and Taï Forest virus (TAFV), are causative agents of severe hemorrhagic fever in humans and nonhuman primates (NHPs) (1, ...

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Molecular Evolution of Antibody Affinity for Sensitive Detection of Botulinum Neurotoxin Type A

Cited by 129 publications

References 42 publications

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

Macaque Monoclonal Antibodies Targeting Novel Conserved Epitopes within Filovirus Glycoprotein

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