Recombinant production of IgM antibodies poses a special challenge due to the complex structure of the proteins and their not yet fully elucidated interactions with the immune effector proteins, especially the complement system. In this study, we present transient expression of IgM antibodies (IgM617, IgM012 and IgM012_GL) in HEK cells and compared it to the well-established stable expression system in CHO cells. The presented workflow investigates quality attributes including productivity, polymer distribution, glycosylation, antibody structure and activation of the classical complement pathway. The HEK293E transient expression system is able to generate comparable amounts and polymer distribution as IgM stably produced in CHO. Although the glycan profile generated by HEK293E cells contained a lower degree of sialylation and a higher portion of oligomannose structures, the potency to activate the complement cascade was maintained. Electron microscopy also confirmed the structural integrity of IgM pentamers produced in HEK293E cells, since the conventional star-shaped structure is observed. From our studies, we conclude that the transient expression system provides an attractive alternative for rapid, efficient and high-throughput production of complex IgM antibodies with slightly altered post-translational modifications, but comparable structure and function.
Humanized monoclonal antibodies (mAbs) are among the most promising modern therapeutics, but defined engineering strategies are still not available. Antibody humanization often leads to a loss of affinity, as it is the case for our model antibody Ab2/3H6 (PDB entry 3BQU). Identifying appropriate back-to-mouse mutations is needed to restore binding affinity, but highly challenging. In order to get more insight, we have applied molecular dynamics simulations and correlated them to antibody binding and expression in wet lab experiments. In this study, we discuss six mAb variants and investigate a tyrosine conglomeration, an isopolar substitution and the improvement of antibody binding towards wildtype affinity. In the 3D structure of the mouse wildtype, residue R94h is surrounded by three tyrosines which form a so-called ‘tyrosine cage’. We demonstrate that the tyrosine cage has a supporting function for the CDRh3 loop conformation. The isopolar substitution is not able to mimic the function appropriately. Finally, we show that additional light chain mutations can restore binding to wildtype-comparable level, and also improve the expression of the mAb significantly. We conclude that the variable light chain of Ab2/3H6 is of underestimated importance for the interaction with its antigen mAb 2F5.
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. Electronic supplementary material The online version of this article (10.1007/s00253-019-09998-3) contains supplementary material, which is available to authorized users.
Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs. But, due to their high oligomeric complexity,in vitroproduction, biochemical characterization, and biophysical characterization are challenging. In this study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (analytical ultracentrifugation, size exclusion chromatography coupled to multi-angle laser light scattering, mass photometry, and transmission electron microscopy). Each IgM construct is defined by a specific expression and purification pattern with different sample quality. Nevertheless, both purified IgMs were able to activate complement in a C1q-dependent manner. More importantly, BioLayer Interferometry (BLI) was used for characterizing the kinetics of C1q binding to recombinant IgMs. We show that recombinant IgMs possess similar C1q-binding properties as IgMs purified from human plasma.
The Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1q complex which strongly activates the classical complement pathway. This biological function is the base for the huge therapeutic potential of IgMs but due to their high oligomeric complexity, in vitro production as well as biochemical and biophysical characterizations are challenging. In the present study, we present new attempts of recombinant production of two IgM models (IgM617 and IgM012) and the evaluation of their polymer distribution using biophysical methods (AUC, SEC-MALLS, Mass Photometry, transmission EM). Each IgM has an individual specific expression yield with different protein quality likely due to intrinsic IgM properties and patterning. Despite the presence of additional oligomeric states, purified recombinant IgMs retain their ability to activate complement in a C1q dependent manner. More importantly, a new method to evaluate their functional quality attribute by characterizing the kinetics of C1q binding to recombinant IgM has been developed using BioLayer Interferometry (BLI). We show that recombinant IgMs possess similar C1q binding properties as IgMs purified from human plasma.
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