Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

Abstract

Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development including pharmacokinetics (PK), toxicology, stability and biochemical characterization studies of such drugs. We have developed an antitoxin (XOMA 3AB) consisting of three recombinant monoclonal antibodies (mAbs) that potently neutralizes the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind non-overlapping BoNT/A epitopes with high affinity. XOMA3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. MAb specific domains were used to develop an ELISA for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay was also developed that is robust to interference from components in serum and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein and is superior to anti-idiotype approaches.

Engineered domain-based assays to identify individual antibodies in oligoclonal combinations targeting the same protein

“…Similarly, the BoNT/E (NCBI accession number YP_001920504) H C (amino acids G805-K1248) H N (amino acids S424-L804), or L C (amino acids M1-K423) were cloned into pYD2. LC-H N domains, which are comprised of LC and H N domains, were cloned by the yeast gap repair method inserting H N into a pYD2 plasmid that already had the LC (19). Plasmid DNA was used to transform Lithium Acetate-treated EBY100 cells.…”

“…Wild-type BoNT/B LC-H N domain (amino acids 1-861) and the wild-type BoNT/E LC-H N domain (amino acids 1-834) were both cloned from the pYD2 vector into the pET21d vector in the same way as previously described (19). In this vector, each domain construct has a SV5 epitope tag and a hexa-histidine tag at the C-terminal.…”

“…In this vector, each domain construct has a SV5 epitope tag and a hexa-histidine tag at the C-terminal. Mutations which knocked out individual mAb binding to the yeast-displayed BoNT domains were introduced into the BoNT/B or BoNT/E LC-H N , expression induced at small scale and the domains purified as described in Meng et al, 2012 (19) for BoNT/A domains. The purified mutant domains were tested for binding to mAbs B-a, B-b and B-e (for the BoNT/B LC-H N ) or for binding to mAbs E-a, E-b, and E-c (for the BoNT/E LC-H N ) using a Attana A100 Quartz Crystal Microbalance (QCM) (Attana AB, Stockholm, Sweden).…”

“…BoNT/B (mAbs B-a, B-b, and B-c) and BoNT/E (mAbs E-a, E-b, and E-c) IgG were expressed from stable Chinese Hamster Ovary (CHO) cell lines and purified by Protein A chromatography followed by flow-through anion exchange and hydrophobic interaction chromatographies as described in Meng et al, (19). …”

“…(19) This proved possible by expressing and purifying three different folded domains of BoNT/A, each recognized by only one of the three component mAbs. This is not possible for XOMA 3B and 3E since at least two of the three mAbs in each antitoxin bind the same BoNT domain.…”