A novel heavy domain antibody library with functionally optimized complementarity determining regions

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A novel heavy domain antibody library with functionally optimized complementarity determining regions. / Mandrup, Ole Aalund; Friis, Niels Anton; Lykkemark, Simon; Just, Jesper; Kristensen, Peter.

I: PLOS ONE, Bind 8, Nr. 10, e76834, 08.10.2013.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

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Mandrup, Ole Aalund ; Friis, Niels Anton ; Lykkemark, Simon ; Just, Jesper ; Kristensen, Peter. / A novel heavy domain antibody library with functionally optimized complementarity determining regions. I: PLOS ONE. 2013 ; Bind 8, Nr. 10.

Bibtex

@article{93318ae9a3bf41659b544e13be0517a1,
title = "A novel heavy domain antibody library with functionally optimized complementarity determining regions",
abstract = "Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.",
author = "Mandrup, {Ole Aalund} and Friis, {Niels Anton} and Simon Lykkemark and Jesper Just and Peter Kristensen",
year = "2013",
month = "10",
day = "8",
doi = "10.1371/journal.pone.0076834",
language = "English",
volume = "8",
journal = "P L o S One",
issn = "1932-6203",
publisher = "public library of science",
number = "10",

}

RIS

TY - JOUR

T1 - A novel heavy domain antibody library with functionally optimized complementarity determining regions

AU - Mandrup, Ole Aalund

AU - Friis, Niels Anton

AU - Lykkemark, Simon

AU - Just, Jesper

AU - Kristensen, Peter

PY - 2013/10/8

Y1 - 2013/10/8

N2 - Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

AB - Today a number of synthetic antibody libraries of different formats have been created and used for the selection of a large number of recombinant antibodies. One of the determining factors for successful isolation of recombinant antibodies from libraries lies in the quality of the libraries i.e. the number of correctly folded, functional antibodies contained in the library. Here, we describe the construction of a novel, high quality, synthetic single domain antibody library dubbed Predator. The library is based on the HEL4 domain antibody with the addition of recently reported mutations concerning the amino acid composition at positions critical for the folding characteristics and aggregation propensities of domain antibodies. As a unique feature, the CDR3 of the library was designed to mimic the natural human immune response by designating amino acids known to be prevalent in functional antibodies to the diversity in CDR3. CDR randomizations were performed using trinucleotide synthesis to avoid the presence of stop codons. Furthermore a novel cycle free elongation method was used for the conversion of the synthesized single stranded DNA containing the randomized CDRs into double stranded DNA of the library. In addition a modular approach has been adopted for the scaffold in which each CDR region is flanked by unique restrictions sites, allowing easy affinity maturation of selected clones by CDR shuffling. To validate the quality of the library, one round phage display selections were performed on purified antigens and highly complex antigen mixtures such as cultured eukaryotic cells resulting in several specific binders. The further characterization of some of the selected clones, however, indicates a reduction in thermodynamic stability caused by the inclusion the additional mutations to the HEL4 scaffold.

U2 - 10.1371/journal.pone.0076834

DO - 10.1371/journal.pone.0076834

M3 - Journal article

C2 - 24116173

VL - 8

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 10

M1 - e76834

ER -