Abstract
The production potential of recombinant monoclonal antibody (mAb) expressing cell lines depends, among other factors, on the intrinsic antibody structure determined by the amino acid sequence. In this study, we investigated the influence of somatic mutations in the V(D)J sequence of four individual, mature model mAbs on the expression potential. Therefore, we defined four couples, each consisting of one naturally occurring mAb (2G12, Ustekinumab, 4B3, and 2F5) and the corresponding germline-derived cognate mAb (353/11, 554/12, 136/63, and 236/14). For all eight mAb variants, recombinant Chinese hamster ovary (CHO) cell lines were developed with mAbs expressed from a defined chromosomal locus. The presented workflow investigates critical parameters including productivity, intra- and extracellular product profile, XBP1 splicing, thermal stability, and in silico hydrophobicity. Significant differences in productivity were even observed between the germline-derived mAbs which did not undergo somatic mutagenesis. Accordingly, back-to-germline mutations of mature mAbs are not necessarily reflecting improved expression and stability but indicate opportunities and limits of mAb engineering. From our studies, we conclude that germinalization represents a potential to improve mAb properties depending on the antibody’s germline family, highlighting the fact that mAbs should be treated individually.
Highlights
Antibodies are among the key components involved in the adaptive immune system with a significant increase of monoclonal antibodies in therapeutic application (Kaplon and Reichert 2018)
The purified monoclonal antibody (mAb) were analyzed for thermal stability and we identified aggregation-prone regions on the antibody binding site as well as hydropathic regions
As mAb models, we focused on anti-HIV1 antibodies, as they show a higher somatic mutation rate compared to other IgGs (Scheid et al 2009; Xiao et al 2009a; Xiao et al 2009b). 2G12 (Buchacher et al 1994; Trkola et al 1996; Kunert et al 1998), 4B3 (Buchacher et al 1994), and 2F5 (Buchacher et al 1994; Purtscher et al 1994; Kunert et al 1998) were selected as model antibodies
Summary
Antibodies are among the key components involved in the adaptive immune system with a significant increase of monoclonal antibodies (mAbs) in therapeutic application (Kaplon and Reichert 2018). Appl Microbiol Biotechnol (2019) 103:7505–7518 sites, and somatic (hyper)mutation (Kim et al 1981; Maizels 2005) Antibody properties, such as productivity/availability in the plasma and thermal stability, are influenced by the choice of the VH and VL families (Ewert et al 2003). We adapted the recombinase-mediated cassette exchange (RMCE) concept in CHO K1 cells to allow insertion of the gene of interest into a pre-defined chromosomal locus with invariable gene copy numbers (Schlake and Bode 1994; Seibler et al 1998) Such single-copy recombinant cell lines are defined as isogenic (Mayrhofer et al 2014) and enable the investigation of the expression of different mAbs during cell propagation. We chose Ustekinumab (Bartlett and Tyring 2008; Leonardi et al.2008; Papp et al 2008), a therapeutic antibody that exhibits 17 VH gene somatic mutations (Table 1)
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