Novel Antibody Hinge Regions for Efficient Production of CH2 Domain-deleted Antibodies

Scott M Glaser,Ina E Hughes,Jennifer R Hopp,Karen Hathaway,Daniel Perret,Mitchell E Reff

doi:10.1074/jbc.m508739200

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 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.

Highlights

Gastric, pancreatic, lung, and ovarian [9, 10]
Hinge-engineered HuCC49⌬CH2 Antibodies, Expression, and Western Blot Analysis—To examine the effect that hinge region modifications have on the secreted fraction of HuCC49⌬CH2 form A and form B isoforms, a series of genetically engineered HuCC49⌬CH2 hinge variants were constructed and the proteins expressed in CHO cells
Expression levels among the entire set of hinge variants ranged from 300 to 1200 ng/ml with no set of variants showing preferentially high or low expression. This would imply that the hinge modifications do not have a major effect on protein expression. 3 ng of total antibody protein from each isolate was analyzed by nonreducing SDS-PAGE followed by Western blot with an anti-human ␬ chain antibody to detect HuCC49⌬CH2 form A and form B isoforms using methods shown in Fig. 1

Summary

Introduction

Gastric, pancreatic, lung, and ovarian [9, 10]. In human tumor mouse xenograft models, radiolabeled HuCC49⌬CH2 was shown to accumulate and be retained to appreciable levels in tumor, exhibit favorable tumor to normal tissue ratios, but demonstrate rapid serum clearance compared with full-length HuCC49 IgG [9]. The atypical hinge region in HuCC49⌬CH2 is similar to that described in the anticarcinoembryonic antigen minibody [15], whereas the MH proline at position 243 (Kabat numbering system [16]) and the entire CH2 domain, including the LH residues, APELLGGP (the first eight amino-terminal residues of the IgG1 CH2 domain), are deleted and replaced by the 10-amino acid peptide Gly/Ser spacer. For HuCC49⌬CH2, one isoform, referred to as form A, contains covalent interchain disulfide bonds at heavy chain MH positions 239 and 242, Kabat numbering system. We hypothesized that generation of the two HuCC49⌬CH2 antibody isoforms is a consequence of hinge heterogeneity because of variation in disulfide bond formation. It follows, that stabilization of the hinge region should favor production of the desired form A isoform. By using a protein engineering strategy, we describe here a series of variant hinge-connecting peptides that were found to improve significantly the homogeneity and yield of CH2 domain-deleted antibodies

Methods

Results

Conclusion