Your browser doesn't support javascript.
loading
Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR.
Simons, Jan Fredrik; Lim, Yoong Wearn; Carter, Kyle P; Wagner, Ellen K; Wayham, Nicholas; Adler, Adam S; Johnson, David S.
Afiliação
  • Simons JF; GigaGen, Inc ., South San Francisco, CA, USA.
  • Lim YW; GigaGen, Inc ., South San Francisco, CA, USA.
  • Carter KP; GigaGen, Inc ., South San Francisco, CA, USA.
  • Wagner EK; GigaGen, Inc ., South San Francisco, CA, USA.
  • Wayham N; GigaGen, Inc ., South San Francisco, CA, USA.
  • Adler AS; GigaGen, Inc ., South San Francisco, CA, USA.
  • Johnson DS; GigaGen, Inc ., South San Francisco, CA, USA.
MAbs ; 12(1): 1803646, 2020.
Article em En | MEDLINE | ID: mdl-32744131
ABSTRACT
IN VITRO affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis

methods:

random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study.
Assuntos
Palavras-chave

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Mutagênese / Anticorpos de Cadeia Única / Afinidade de Anticorpos Tipo de estudo: Guideline Limite: Humans Idioma: En Revista: MAbs Assunto da revista: ALERGIA E IMUNOLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Estados Unidos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Mutagênese / Anticorpos de Cadeia Única / Afinidade de Anticorpos Tipo de estudo: Guideline Limite: Humans Idioma: En Revista: MAbs Assunto da revista: ALERGIA E IMUNOLOGIA Ano de publicação: 2020 Tipo de documento: Article País de afiliação: Estados Unidos