When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

Abstract

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

A Toolbox of IgG Subclass-Switched Recombinant Monoclonal Antibodies for Enhanced Multiplex Immunolabeling of Brain

“…This was evidenced by the overall efficiency of slightly below 50% for the successful projects tabulated in Table 2, and also by the projects that were unsuccessful on the first pass in which clones passed the colony PCR and restriction digest screening steps but failed in the COS-ICC screening assay for functional R-mAbs. Moreover, a lack of allelic exclusion at heavy and light chain IgG loci has been documented in hybridomas, resulting in expression of multiple IgG heavy and/or light chains at the mRNA and protein levels (8-10, 63). In these previous studies, while multiple combinations of heavy and light IgG chains were produced by monoclonal hybridoma cells, only one combination of heavy and light IgG chains present in the hybridoma produced an R-mAb capable of recognizing the target antigen.…”

“…Recombinant expression systems allow for the reliable production of an R-mAb not prone to loss by genetic instability of tetra-or hexa-ploid hybridoma cells or other factors (6, 7). Moreover, unlike hybridoma cell lines that can express multiple functional heavy and light immunoglobulin chains (8-10), recombinant expression ensures production of a single, molecularly defined R-mAb. Recombinant expression can also yield production levels 100s or 1000s of times higher than is possible with endogenous expressions of mAbs from hybridoma cells [ e.g.…”

Antibodies: Will Their Star Continue to Rise?

“…“Hybridomas are not stable and often not entirely clonal [11] so if you buy something that's made in a hybridoma today and if you buy something that is made in a hybridoma in a year, the end product that you receive can differ a lot. This can differ in the purity, it can differ in the fine composition, and it can also just be a completely different molecule,” commented Michael Fiebig (Absolute Antibody, Oxford, UK).…”

Animal-free alternatives and the antibody iceberg

“…Well-characterized antibodies have high specificity for their antigens and are essential tools used across all scientific disciplines. The research market is heavily dominated by animal-derived antibodies, yet is unable to supply high-quality reagents across the board, resulting in grave scientific concerns related to lack of reproducibility and specificity [5][6][7] . Fortunately, techniques that rely on animal immunization represent just the tip of the antibody iceberg: a vast expanse of additional molecules can be accessed by advanced recombinant methods that largely remain out of sight ( Fig.…”