Novel antibody hinge regions for efficient production of CH2 domain-deleted antibodies.
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 1:1 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.
Glaser
Scott M
SM
Department of Protein Engineering, Biogen Idec, Inc., San Diego, California 92122, USA. scott.glaser@biogenidec.com
Hughes
Ina E
IE
Hopp
Jennifer R
JR
Hathaway
Karen
K
Perret
Daniel
D
Reff
Mitchell E
ME
D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000912:Antibodies, Neoplasm / Q000737:chemistry; D001667:Binding, Competitive; D015153:Blotting, Western; D016466:CHO Cells; D002417:Cattle; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D000860:Hypoxia; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D009077:Mucins / Q000737:chemistry; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D016499:Radioimmunotherapy / Q000379:methods*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D004220:Disulfides; D009077:Mucins; D009841:Oligonucleotides; D010455:Peptides; D020033:Protein Isoforms