Novel antibody hinge regions for efficient production of CH2 domain-deleted antibodies.
J Biol Chem
; 280(50): 41494-503, 2005 Dec 16.
Article
en En
| MEDLINE
| ID: mdl-16221669
ABSTRACT
HuCC49 deltaCH2 is a heavy chain constant domain 2 domain-deleted antibody under development as a radioimmunotherapeutic for treating carcinomas overexpressing the TAG-72 tumor antigen. Mammalian cell culture biosynthesis of HuCC49 deltaCH2 produces two isoforms (form A and form B) in an approximate 11 ratio, and consequently separation and purification of the desired form A isoform adversely impact process and yield. A protein engineering strategy was used to develop a panel of hinge-engineered HuCC49 deltaCH2 antibodies to identify hinge sequences to optimize production of the form A isoform. We found that adding a single proline residue at Kabat position 243, immediately adjacent to the carboxyl end of the core middle hinge CPPC domain, resulted in an increase from 39 to 51% form A isoform relative to the parent HuCC49 deltaCH2 antibody. Insertion of the amino acids proline-alanine-proline (PAP) at positions 243-245 enhanced production of the form A isoform to 72%. Insertion of a cysteine-rich 15-amino acid IgG3 hinge motif (CPEPKSCDTPPPCPR) in both of these mutant antibodies resulted in secretion of predominantly form A isoform with little or no detectable form B. Yields exceeding 98% of the form A isoform have been realized. Preliminary peptide mapping and mass spectrometry analysis suggest that at least two, and as many as five, inter-heavy chain disulfide linkages may be present.
Buscar en Google
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Ingeniería de Proteínas
/
Radioinmunoterapia
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
J Biol Chem
Año:
2005
Tipo del documento:
Article
País de afiliación:
Estados Unidos