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Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.

ABSTRACT: Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.

SUBMITTER: Tabares-da Rosa S

PROVIDER: S-EPMC3193053 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.

Tabares-da Rosa Sofia S Rossotti Martin M Carleiza Carmen C Carrión Federico F Pritsch Otto O Ahn Ki Chang KC Last Jerold A JA Hammock Bruce D BD González-Sapienza Gualberto G

Analytical chemistry 20110829 18

Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten ...[more]

PMID: 21827167

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Project description:Therapeutic antibodies must have "drug-like" properties. These include high affinity and specificity for the intended target, biological activity, and additional characteristics now known as "developability properties": long-term stability and resistance to aggregation when in solution, thermodynamic stability to prevent unfolding, high expression yields to facilitate manufacturing, low self-interaction, among others. Sequence-based liabilities may affect one or more of these characteristics. Improving the stability and developability of a lead antibody is typically achieved by modifying its sequence, a time-consuming process that often results in reduced affinity. Here we present a new antibody library format that yields high-affinity binders with drug-like developability properties directly from initial selections, reducing the need for further engineering or affinity maturation. The innovative semi-synthetic design involves grafting natural complementarity-determining regions (CDRs) from human antibodies into scaffolds based on well-behaved clinical antibodies. HCDR3s were amplified directly from B cells, while the remaining CDRs, from which all sequence liabilities had been purged, were replicated from a large next-generation sequencing dataset. By combining two in vitro display techniques, phage and yeast display, we were able to routinely recover a large number of unique, highly developable antibodies against clinically relevant targets with affinities in the subnanomolar to low nanomolar range. We anticipate that the designs and approaches presented here will accelerate the drug development process by reducing the failure rate of leads due to poor antibody affinities and developability.Abbreviations: AC-SINS: affinity-capture self-interaction nanoparticle spectroscopy; CDR: complementarity-determining region; CQA: critical quality attribute; ELISA: enzyme-linked immunoassay; FACS: fluorescence-activated cell sorting; Fv: fragment variable; GM-CSF: granulocyte-macrophage colony-stimulating factor; HCDR3: heavy chain CDR3; IFN2a: interferon α-2; IL6: interleukin-6; MACS: magnetic-activated cell sorting; NGS: next generation sequencing; PCR: polymerase chain reaction; SEC: size-exclusion chromatography; SPR: surface plasmon resonance; TGFβ-R2: transforming growth factor β-R2; VH: variable heavy; VK: variable kappa; VL: variable light; Vl: variable lambda.

Project description:BackgroundMore than 3 million infections were attributed to Chikungunya virus (CHIKV) in the 2014-2016 outbreak in Mexico, Central and South America, with over 500 deaths directly or indirectly related to this viral disease. CHIKV outbreaks are recurrent and no vaccine nor approved therapeutics exist to prevent or treat CHIKV infection. Reliable and robust diagnostic methods are thus critical to control future CHIKV outbreaks. Direct CHIKV detection in serum samples via highly specific and high affinity anti-CHIKV antibodies has shown to be an early and effective clinical diagnosis.MethodsTo isolate highly specific and high affinity anti-CHIKV, Chikungunya virions were isolated from serum of a patient in Veracruz, México. After purification and characterization via electron microscopy, SDS-PAGE and binding to well-characterized anti-CHIKV antibodies, UV-inactivated particles were utilized as selector in a solid-phase panning in combination with ALTHEA Gold Libraries™, as source of antibodies. The screening was based on ELISA and Next-Generation Sequencing.ResultsThe CHIKV isolate showed the typical morphology of the virus. Protein bands in the SDS-PAGE were consistent with the size of CHIKV capsid proteins. UV-inactivated CHIKV particles bound tightly the control antibodies. The lead antibodies here obtained, on the other hand, showed high expression yield, > 95% monomeric content after a single-step Protein A purification, and importantly, had a thermal stability above 75 °C. Most of the antibodies recognized linear epitopes on E2, including the highest affinity antibody called C7. A sandwich ELISA implemented with C7 and a potent neutralizing antibody isolated elsewhere, also specific for E2 but recognizing a discontinuous epitope, showed a dynamic range of 0.2-40.0 mg/mL of UV-inactivated CHIKV purified preparation. The number of CHIKV particles estimated based on the concentration of E2 in the extract suggested that the assay could detect clinically meaningful amounts of CHIKV in serum.ConclusionsThe newly discovered antibodies offer valuable tools for characterization of CHIKV isolates. Therefore, the strategy here followed using whole viral particles and ALTHEA Gold Libraries™ could expedite the discovery and development of antibodies for detection and control of emergent and quickly spreading viral outbreaks.

Dataset Information

Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.

Publications

Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response.

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