Andrew R M Bradbury a,✉,*, Nathan D Trinklein b,*, Holger Thie c,*, Ian C Wilkinson d,*, Atul K Tandon e,*, Stephen Anderson d, Catherine L Bladen d, Brittany Jones e, Shelley Force Aldred b, Marco Bestagno f, Oscar Burrone f, Jennifer Maynard g, Fortunato Ferrara a, James S Trimmer h, Janina Görnemann i, Jacob Glanville j,**, Philipp Wolf k, Andre Frenzel l,p, Julin Wong m, Xin Yu Koh m, Hui-Yan Eng m, David Lane m, Marie-Paule Lefranc n, Mike Clark o, Stefan Dübel p,
https://doi.org/10.1080/19420862.2018.1445456
Posted: March 29, 2018
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.
NeoBiotechnologies’ products were used in this study:
A total of 116 laboratories assumed to be involved in hybridoma cloning work were contacted to determine whether they could contribute suitable datasets, and 15 positive responses were received. Sequence sets of 7 labs finally met the inclusion criteria, which, in addition to functional clones, required that a sequence set cover all hybridomas analyzed in that lab within a given timeframe without any preselection for “problematic” cases or cloning problems. All hybridomas studied were sequenced because of the need to either produce the antibodies as recombinants, or to gain access to their genes. Data for ∼90% of the analyzed hybridomas were contributed by three commercial antibody companies, Miltenyi Biotec, Absolute Antibody and NeoBiotechnologies, with roughly 1/3 of the sequences coming from each company. Smaller numbers (≤6 per set) were reported by the International Centre for Genetic Engineering and Biotechnology (Burrone lab), University of Heidelberg (Dübel lab), A*Star p53 laboratory (Lane lab) and University of Freiburg (Wolf lab).
Sequences were obtained over a 20-year period using using a number of different standard methods, including RACECitation26 or V region PCRCitation27 (49 hybridomas, Miltenyi and academic labs), while the majority (136 hybridomas) were analyzed by NGS within the past 3 years (Absolute Antibody and NeoBiotechnologies). In some cases, PCR-based methods included published measures to eliminate known unproductive aberrant light chains (A*Star).Citation20,Citation23,Citation38 which are not expected to have any major effect on the detection of other chains than the known aberrant light chain.
Keywords: hybridoma, monoclonal antibodies, specificity, paratope, recombinant antibodies
Publication History:
MAbs. 2018 Mar 29;10(4):539–546. doi: 10.1080/19420862.2018.1445456
