Immunoglobulin somatic hypermutation in a defined biochemical system recapitulates affinity maturation and permits antibody optimization.
Nucleic Acids Res
; 50(20): 11738-11754, 2022 11 11.
Article
em En
| MEDLINE
| ID: mdl-36321646
ABSTRACT
We describe a purified biochemical system to produce monoclonal antibodies (Abs) in vitro using activation-induced deoxycytidine deaminase (AID) and DNA polymerase η (Polη) to diversify immunoglobulin variable gene (IgV) libraries within a phage display format. AID and Polη function during B-cell affinity maturation by catalyzing somatic hypermutation (SHM) of immunoglobulin variable genes (IgV) to generate high-affinity Abs. The IgV mutational motif specificities observed in vivo are conserved in vitro. IgV mutations occurred in antibody complementary determining regions (CDRs) and less frequently in framework (FW) regions. A unique feature of our system is the use of AID and Polη to perform repetitive affinity maturation on libraries reconstructed from a preceding selection step. We have obtained scFv Abs against human glucagon-like peptide-1 receptor (GLP-1R), a target in the treatment of type 2 diabetes, and VHH nanobodies targeting Fatty Acid Amide Hydrolase (FAAH), involved in chronic pain, and artemin, a neurotropic factor that regulates cold pain. A round of in vitro affinity maturation typically resulted in a 2- to 4-fold enhancement in Ab-Ag binding, demonstrating the utility of the system. We tested one of the affinity matured nanobodies and found that it reduced injury-induced cold pain in a mouse model.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Hipermutação Somática de Imunoglobulina
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Anticorpos de Cadeia Única
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Anticorpos de Domínio Único
Tipo de estudo:
Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article