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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Format: | Article |
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Public Library of Science (PLoS)
2012-01-01
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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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institution | Directory Open Access Journal |
issn | 1932-6203 |
language | English |
last_indexed | 2024-04-14T02:14:43Z |
publishDate | 2012-01-01 |
publisher | Public Library of Science (PLoS) |
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series | PLoS ONE |
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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