Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Bremer, Ewald T.J. van den; Beurskens, Frank J.; Voorhorst, Marleen; Engelberts, Patrick J.; Jong, Rob N. de; Boom, Burt G van der; Cook, Erika M.; Lindorfer, Margaret A.; Taylor, Ronald P.; Berkel, Patrick H. van; Parren, Paul W.H.I.

doi:10.1080/19420862.2015.1046665

Cited by 59 publications

(39 citation statements)

References 42 publications

(55 reference statements)

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“…[24] As the target point for this modification is located far away from any sites that play an essential role for the functionality of the antibody like the antigen binding region or the Fc-region involved in receptor binding, it was commonly believed for a long time that C-terminal lysine clipping does not affect the in vitro or in vivo activity of the molecule, especially the CDC activity. [24,61] This claim was supported by the fact that the presence or absence of the C-terminal Lys residue does not cause any conformational changes to the antibody's structure. [62] However, a recent study by van den Bremer et al contradicted these findings by reporting that antibody fractions that had undergone complete C-terminal lysine clipping showed a clearly increased CDC activity compared to their counterparts, which still contained lysine residues on both C-termini of the heavy chains.…”

Section: C-terminal Lysine Clippingmentioning

confidence: 49%

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Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

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Section: C-terminal Lysine Clippingmentioning

confidence: 49%

“…It was then proposed that previous studies may not have been able to identify this link due to a lack of samples containing separated variant fractions rather than mixtures. [61] …”

Section: C-terminal Lysine Clippingmentioning

confidence: 99%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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“…The fluorescent binding signal of CD80-Fc-mCherry was lower than expected, so we next re-examined our Fc-tag construct. In humans and all other mammals examined to date the IgG heavy chain has glycine-lysine (Gly-Lys) residues at the C-terminus 38 ; the initial devil IgG constant region sequence available to us had an incomplete C-terminus, and thus our initial CD80-Fc-mCherry vector did not have the C-terminal Gly-Lys. We subsequently made a new FAST-Fc construct with CTLA4-Fc-mCherry, which exhibited strong binding to both CD80 and CD86 transfected DFT cells (Figure 4D).…”

Section: Resultsmentioning

confidence: 99%

A novel system to map protein interactions reveals evolutionarily conserved immune evasion pathways on transmissible cancers

Flies

Darby

Lennard

et al. 2019

Preprint

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Immune checkpoint immunotherapy has revolutionized medicine, but translational success for new treatments remains low. Around 40% of humans and Tasmanian devils (Sarcophilus harrisii) develop cancer in their lifetime, compared to less than 10% for most species. Additionally, devils are affected by two of the three known transmissible cancers in mammals. Unfortunately, little is known about of immune checkpoints in devils and other non-model species, largely due to a lack of species-specific reagents. We developed a simple cut-and-paste reagent development method applicable to any vertebrate species and show that immune checkpoint interactions are conserved across 160 million years of evolution. The inhibitory checkpoint molecule CD200 is highly expressed on devil facial tumor cells. We are the first to demonstrate that co-expression of CD200R1 can block CD200 expression. The evolutionarily conserved pathways suggest that naturally occurring cancers in devils and other species can serve as models for understanding cancer and immunological tolerance.GRAPHICAL ABSTRACT

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“…Since complement dependent cytotoxicity (CDC) occurs only when antibody is injected into the bloodstream and the C‐terminal lysine is removed, both WT (due to function of carboxyl peptidases) and –GK versions of an antibody can equally accommodate this purpose. If however an effector‐less antibody is desired, an aglycosylated –GK antibody may be engineered . While our initial investigations suggested that deletion of C‐terminal lysine or glycine–lysine residues from HC does not affect antibody glycosylation, potency, PK, and bioavailability relative to the WT antibody, nevertheless, the immunogenicity of the glycine–lysine deletion in antibodies will need to be tested in order to ensure patient safety prior to its utilization for commercial production.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

Hu¹,

Tang²,

Misaghi³

et al. 2017

Biotechnology Progress

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Monoclonal antibodies (mAbs) have been well established as potent therapeutic agents and are used to treat many different diseases. During cell culture production, antibody charge variants can be generated by cleavage of heavy chain (HC) C-terminal lysine and proline amidation. Differences in levels of charge variants during manufacturing process changes make it challenging to demonstrate process comparability. In order to reduce heterogeneity and achieve consistent product quality, we generated and expressed antibodies with deletion of either HC C-terminal lysine (-K) or lysine and glycine (-GK). Interestingly, clones that express antibodies lacking HC C-terminal lysine (-K) had considerably lower specific productivities compared to clones that expressed either wild type antibodies (WT) or antibodies lacking HC glycine and lysine (-GK). While no measurable differences in antibody HC and LC mRNA levels, glycosylation and secretion were observed, our analysis suggests that the lower specific productivity of clones expressing antibody lacking HC C-terminal lysine was due to slower antibody HC synthesis and faster antibody degradation. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:786-794, 2017.

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Human IgG is produced in a pro-form that requires clipping of C-terminal lysines for maximal complement activation

Cited by 59 publications

References 42 publications

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

A novel system to map protein interactions reveals evolutionarily conserved immune evasion pathways on transmissible cancers

Evaluation of heavy chain C‐terminal deletions on productivity and product quality of monoclonal antibodies in Chinese hamster ovary (CHO) cells

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