Reprogramming the antigen specificity of B cells using genome-editing technologies.
Antibodies, Neutralizing
/ immunology
Antibody Specificity
Antigen-Antibody Reactions
/ genetics
B-Lymphocytes
/ immunology
CRISPR-Cas Systems
Cell Line
Cytidine Deaminase
/ metabolism
Gene Editing
/ methods
HIV Antibodies
/ immunology
Humans
Immunoglobulin Heavy Chains
/ genetics
Receptors, Antigen, B-Cell
/ genetics
B cell
CAR-B
HIV
bnAb
engineering
human
immunology
infectious disease
inflammation
microbiology
vaccine
Journal
eLife
ISSN: 2050-084X
Titre abrégé: Elife
Pays: England
ID NLM: 101579614
Informations de publication
Date de publication:
17 01 2019
17 01 2019
Historique:
received:
19
10
2018
accepted:
31
12
2018
pubmed:
17
1
2019
medline:
28
5
2020
entrez:
17
1
2019
Statut:
epublish
Résumé
We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.
Identifiants
pubmed: 30648968
doi: 10.7554/eLife.42995
pii: 42995
pmc: PMC6355199
doi:
pii:
Substances chimiques
Antibodies, Neutralizing
0
HIV Antibodies
0
Immunoglobulin Heavy Chains
0
Receptors, Antigen, B-Cell
0
AICDA (activation-induced cytidine deaminase)
EC 3.5.4.-
Cytidine Deaminase
EC 3.5.4.5
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Subventions
Organisme : NIH HHS
ID : 5R01DE025167-05
Pays : United States
Organisme : NIAID NIH HHS
ID : UM1 AI100663
Pays : United States
Organisme : Medical Research Council
ID : MR/R008698/1
Pays : United Kingdom
Organisme : NCATS NIH HHS
ID : UL1 TR001114
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI073148
Pays : United States
Organisme : NIDCR NIH HHS
ID : R01 DE025167
Pays : United States
Organisme : FP7 People: Marie-Curie Actions
ID : FP7-PEOPLE-2013-IOF
Pays : International
Organisme : Ministerio de Ciencia, Innovacion y Universidades
ID : Ramón y Cajal Merit Award RYC-2016-21155
Pays : International
Organisme : NIAID NIH HHS
ID : R37 AI059714
Pays : United States
Organisme : Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades
ID : RYC-2016-21155
Pays : International
Organisme : Marie-Curie Fellowship
ID : FP7-PEOPLE-2013-IOF
Pays : International
Organisme : Bill and Melinda Gates Foundation
ID : OPP1183956
Pays : International
Informations de copyright
© 2019, Voss et al.
Déclaration de conflit d'intérêts
JV, AG, RA, RF, BM, LM, KP, DH, WL, DS, KL, BB, MC, GR, LH, AF, DN, PC, DB No competing interests declared
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