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Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

ABSTRACT: Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS method. However, the affinity was not similar to that of parental 528. For further affinity maturation, we aimed to isolate VL mutants that act in synergy with VH mutants. However, the OS method could not be applied for selecting VL fragments because the preparation of soluble VH fragments was hampered by their instability and insolubility. Therefore, we initially designed a modified OS method based on domain-swapping of VH fragments, from added soluble Fv fragments to phage-displaying VL fragments. Using this novel Fv-added OS selection method, we successfully isolated VL mutants, and one of the Fv comprising VH and VL mutants showed affinity almost equivalent to that of parental 528. This method is applicable for engineering other VL fragments for affinity maturation.

SUBMITTER: Sanada H

PROVIDER: S-EPMC5882652 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

Sanada Hideaki H Kobayashi Kazuki K Oyama Kenji K Maru Takamitsu T Nakanishi Takeshi T Umetsu Mitsuo M Asano Ryutaro R Kumagai Izumi I

Scientific reports 20180403 1

Affinity maturation is one of the cardinal strategies for improving antibody function using in vitro evolutionary methods; one such well-established method is phage display. To minimise gene deletion, we previously developed an open sandwich (OS) method wherein selection was performed using only phage-displaying VH fragments after mixing with soluble VL fragments. The decrease in anti-EGFR antibody 528 affinity through humanization was successfully recovered by selecting VH mutants using this OS ...[more]

PMID: 29615700

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Project description:BackgroundAffinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system.ResultsElution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml.ConclusionsAffinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be considered as a new phage purification method, appropriate for further investigations and development.

Project description:Antibodies isolated from human donors are increasingly being developed for anti-infective therapeutics. These antibodies undergo affinity maturation in vivo, minimizing the need for engineering of therapeutic leads for affinity. However, the affinities required for some therapeutic applications may be higher than the affinities of the leads obtained, requiring further affinity maturation in vitro. To improve the neutralization potency of natural human antibody MSL-109 targeting human cytomegalovirus (CMV), we affinity matured the antibody against the gH/gL glycoprotein complex. A phage display library where most of the six complementary-determining regions (CDRs) were allowed to vary in only one amino acid residue at a time was used to scan for mutations that improve binding affinity. A T55R mutation and multiple mutations in position 53 of the heavy chain were identified that, when present individually or in combination, resulted in higher apparent affinities to gH/gL and improved CMV neutralization potency of Fab fragments expressed in bacterial cells. Three of these mutations in position 53 introduced glycosylation sites in heavy chain CDR 2 (CDR H2) that impaired binding of antibodies expressed in mammalian cells. One high affinity (KD<10 pM) variant was identified that combined the D53N and T55R mutations while avoiding glycosylation of CDR H2. However, all the amino acid substitutions identified by phage display that improved binding affinity without introducing glycosylation sites required between two and four simultaneous nucleotide mutations to avoid glycosylation. These results indicate that the natural human antibody MSL-109 is close to a local affinity optimum. We show that affinity maturation by phage display can be used to identify and bypass barriers to in vivo affinity maturation of antibodies imposed by glycosylation and codon usage. These constraints may be relatively prevalent in human antibodies due to the codon usage and the amino acid sequence encoded by the natural human repertoire.

Dataset Information

Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

Publications

Affinity maturation of humanized anti-epidermal growth factor receptor antibody using a modified phage-based open sandwich selection method.

Similar Datasets

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