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

Abstract

Quantitation of individual mAbs within a combined antibody drug product is required for preclinical and clinical drug development. We have developed two antitoxins (XOMA 3B and XOMA 3E) each consisting of three monoclonal antibodies (mAbs) that neutralize type B and type E botulinum neurotoxin (BoNT/B and BoNT/E) to treat serotype B and E botulism. To develop mAb-specific binding assays for each antitoxin, we mapped the epitopes of the six mAbs. Each mAb bound an epitope on either the BoNT light chain (LC) or translocation domain (HN). Epitope mapping data was used to design LC-HN domains with orthogonal mutations to make them specific for only one mAb in either XOMA 3B or 3E. Mutant LC-HN domains were cloned, expressed, and purified from E. coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. Further engineering of domains allowed construction of ELISAs that could characterize the integrity, binding affinity, and identity of each of the six mAbs in XOMA 3B, and 3E without interference from the three BoNT/A mAbs in XOMA 3AB. Such antigen engineering is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that bind the same protein.

Potential therapeutic roles for antibody mixtures

“…One such example is using a cocktail of drugs including a protease inhibitor, a nucleoside-based reverse transcriptase inhibitor, a nonnucleoside reverse transcriptase inhibitor and/or an integrase inhibitor to treat patients infected with HIV [14]. Similar to these approaches, mixtures of rMAbs are now being developed as human therapeutics [15]. These antibody mixtures (10) contain two or more antibodies directed against either a single antigen or different antigens [16].…”

“…For example, Xoma is developing an antibody mixture containing three antibodies to target and neutralize the toxin from botulism poisoning [15][16][17]. Xoma 3AB, which is a lyophilized formulation of three different antibodies in equimolar proportions directed against three distinct domains on the BoNT/A neurotoxin protein, is an example of an antibody mixture against a single antigen [15][16][17].…”

“…Sym004 is a mixture of two chimeric IgG1 antibodies against non-overlapping epitopes on EGFR domain III (Figure 3) [15][16][17]. This antibody mixture seems to possess a unique mechanism of action which induces more rapid internalization and degradation of EGFR than single anti-EGFR antibodies (Figure 3).…”

“…Sym004 is currently in Phase II human clinical trials [17]. Sym013, or Pan-HER antibody mixture, contains six humanized antibodies targeting three different antigens, that is, EGFR, HER2 and HER3 [15][16][17]. The Pan-HER antibody mixture has been shown to simultaneously downmodulate the EGFR, HER2 and HER3 antigens and appears to circumvent the acquired resistance to anti-HER2 drugs due to upregulation of other receptor tyrosine kinases.…”

“…Several other similar antibody mixtures targeting a single antigen or multiple antigens are being developed by other companies (Table 2). Also, new platform technologies have been developed that makes it easier to produce antibody mixtures either using multiple cell lines or by using one cell line for each antibody molecule in order to reduce the effect of cost of goods (COGs) on the marketed products [15,16].…”