High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.

A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fuse...

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Main Authors: Audrey D McConnell, Minjee Do, Tamlyn Y Neben, Vladimir Spasojevic, Josh MacLaren, Andy P Chen, Laurence Altobell, John L Macomber, Ashley D Berkebile, Robert A Horlick, Peter M Bowers, David J King
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2012-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC3498135?pdf=render
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author Audrey D McConnell
Minjee Do
Tamlyn Y Neben
Vladimir Spasojevic
Josh MacLaren
Andy P Chen
Laurence Altobell
John L Macomber
Ashley D Berkebile
Robert A Horlick
Peter M Bowers
David J King
author_facet Audrey D McConnell
Minjee Do
Tamlyn Y Neben
Vladimir Spasojevic
Josh MacLaren
Andy P Chen
Laurence Altobell
John L Macomber
Ashley D Berkebile
Robert A Horlick
Peter M Bowers
David J King
author_sort Audrey D McConnell
collection DOAJ
description A method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.
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spelling doaj.art-b9ba0a64e8aa4557b73a1c4372b718692022-12-22T02:18:16ZengPublic Library of Science (PLoS)PLoS ONE1932-62032012-01-01711e4945810.1371/journal.pone.0049458High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.Audrey D McConnellMinjee DoTamlyn Y NebenVladimir SpasojevicJosh MacLarenAndy P ChenLaurence AltobellJohn L MacomberAshley D BerkebileRobert A HorlickPeter M BowersDavid J KingA method has been developed for the rapid generation of high-affinity humanized antibodies from immunized animals without the need to make conventional hybridomas. Rearranged IgH D(J) regions were amplified from the spleen and lymph tissue of mice immunized with the human complement protein C5, fused with a limited repertoire of human germline heavy chain V-genes to form intact humanized heavy chains, and paired with a human light chain library. Completed heavy and light chains were assembled for mammalian cell surface display and transfected into HEK 293 cells co-expressing activation-induced cytidine deaminase (AID). Numerous clones were isolated by fluorescence-activated cell sorting, and affinity maturation, initiated by AID, resulted in the rapid evolution of high affinity, functional antibodies. This approach enables the efficient sampling of an immune repertoire and the direct selection and maturation of high-affinity, humanized IgGs.http://europepmc.org/articles/PMC3498135?pdf=render
spellingShingle Audrey D McConnell
Minjee Do
Tamlyn Y Neben
Vladimir Spasojevic
Josh MacLaren
Andy P Chen
Laurence Altobell
John L Macomber
Ashley D Berkebile
Robert A Horlick
Peter M Bowers
David J King
High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
PLoS ONE
title High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
title_full High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
title_fullStr High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
title_full_unstemmed High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
title_short High affinity humanized antibodies without making hybridomas; immunization paired with mammalian cell display and in vitro somatic hypermutation.
title_sort high affinity humanized antibodies without making hybridomas immunization paired with mammalian cell display and in vitro somatic hypermutation
url http://europepmc.org/articles/PMC3498135?pdf=render
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