Publications by authors named "Stefan Dengl"

14 Publications

  • Page 1 of 1

Format chain exchange (FORCE) for high-throughput generation of bispecific antibodies in combinatorial binder-format matrices.

Nat Commun 2020 10 2;11(1):4974. Epub 2020 Oct 2.

Roche Pharma Research and Early Development (pRED), Large Molecule Research, Roche Innovation Center Munich, Penzberg, Germany.

Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.
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http://dx.doi.org/10.1038/s41467-020-18477-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532213PMC
October 2020

Understanding the Half-Life Extension of Intravitreally Administered Antibodies Binding to Ocular Albumin.

Pharmaceutics 2020 Aug 26;12(9). Epub 2020 Aug 26.

Roche Pharma Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland.

The burden associated with frequent injections of current intravitreal (IVT) therapeutics may be reduced by long-acting delivery strategies. Binding to serum albumin has been shown to extend the ocular half-life in rabbits, however, the underlying molecular mechanisms and translational relevance remain unclear. The aim of this work was to characterize the in vitro and in vivo formation of complexes between human serum albumin (HSA) and an antigen-binding fragment of a rabbit antibody linked to an anti-HSA nanobody (FabA). The ocular and systemic pharmacokinetics of H-labeled FabA (0.05 mg/eye IVT) co-formulated with HSA (1 and 15 nmol/eye) were assessed in Dutch belted rabbits. Next, FabA was incubated in vitreous samples from cynomolgus monkeys and human donors (healthy and diseased) supplemented with species-specific serum albumin. Finally, the FabA-albumin complexes formed in vitro and in vivo were analyzed by radio-size exclusion chromatography. A 3-fold increase in FabA vitreal exposure and half-life was observed in rabbits co-administered with 15 nmol HSA compared to 1 nmol and a control arm. The different pharmacokinetic behavior was explained with the formation of higher molecular weight FabA-albumin complexes. The analysis of vitreous samples revealed the existence of predominantly 1:1 complexes at endogenous or low concentrations of supplemented albumin. A shift towards 1:2 complexes was observed with increasing albumin concentrations. Overall, these results suggest that endogenous vitreal albumin concentrations are insufficient for half-life extension and warrant supplementation in the dosing formulation.
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http://dx.doi.org/10.3390/pharmaceutics12090810DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7559355PMC
August 2020

The Contorsbody, an antibody format for agonism: Design, structure, and function.

Comput Struct Biotechnol J 2020 14;18:1210-1220. Epub 2020 May 14.

Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Nonnenwald 2, 82377 Penzberg, Germany.

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor 'cis'-dimerization without 'trans'-bridging of cells or mere receptor blockade.
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http://dx.doi.org/10.1016/j.csbj.2020.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283085PMC
May 2020

Interplay between reversible phosphorylation and irreversible ADP-ribosylation of eukaryotic translation elongation factor 2.

Biol Chem 2019 03;400(4):501-512

Roche Pharma Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Nonnenwald 2, D-82377 Penzberg, FRG, Germany.

The functionality of eukaryotic translation elongation factor 2 (eEF2) is modulated by phosphorylation, eEF2 is simultaneously the molecular target of ADP-ribosylating toxins. We analyzed the interplay between phosphorylation and diphthamide-dependent ADP-ribosylation. Phosphorylation does not require diphthamide, eEF2 without it still becomes phosphorylated. ADP-ribosylation not only modifies the H715 diphthamide but also inhibits phosphorylation of S595 located in proximity to H715, and stimulates phosphorylation of T56. S595 can be phosphorylated by CDK2 and CDK1 which affects EEF2K-mediated T56-phosphorylation. Thus, ADP-ribosylation and S595-phosphorylation by kinases occur within the same vicinity and both trigger T56-phosphorylation. Diphthamide is surface-accessible permitting access to ADP-ribosylating enzymes, the adjacent S595 side chain extends into the interior. This orientation is incompatible with phosphorylation, neither allowing kinase access nor phosphate attachment. S595 phosphorylation must therefore be accompanied by structural alterations affecting the interface to ADP-ribosylating toxins. In agreement with that, replacement of S595 with Ala, Glu or Asp prevents ADP-ribosylation. Phosphorylation (starvation) as well as ADP-ribosylation (toxins) inhibit protein synthesis, both affect the S595/H715 region of eEF2, both trigger T57-phosphorylation eliciting similar transcriptional responses. Phosphorylation is short lived while ADP-ribosylation is stable. Thus, phosphorylation of the S595/H715 'modifier region' triggers transient interruption of translation while ADP-ribosylation arrests irreversibly.
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http://dx.doi.org/10.1515/hsz-2018-0280DOI Listing
March 2019

Oxidation in the complementarity-determining regions differentially influences the properties of therapeutic antibodies.

MAbs 2016 Nov/Dec;8(8):1525-1535. Epub 2016 Sep 9.

a Roche Pharma Research and Early Development (pRED), Large Molecule Research, Roche Innovation Center Munich , Germany.

Therapeutic antibodies can undergo a variety of chemical modification reactions in vitro. Depending on the site of modification, either antigen binding or Fc-mediated functions can be affected. Oxidation of tryptophan residues is one of the post-translational modifications leading to altered antibody functionality. In this study, we examined the structural and functional properties of a therapeutic antibody construct and 2 affinity matured variants thereof. Two of the 3 antibodies carry an oxidation-prone tryptophan residue in the complementarity-determining region of the V domain. We demonstrate the differences in the stability and bioactivity of the 3 antibodies, and reveal differential degradation pathways for the antibodies susceptible to oxidation.
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http://dx.doi.org/10.1080/19420862.2016.1231277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098445PMC
November 2017

Engineered hapten-binding antibody derivatives for modulation of pharmacokinetic properties of small molecules and targeted payload delivery.

Immunol Rev 2016 Mar;270(1):165-77

1Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Penzberg, Germany.

Hapten-binding antibodies have for more than 50 years played a pivotal role in immunology, paving the way to antibody generation (as haptens are very important and robust immunogens), to antibody characterization (as the first structures generated more than 40 years ago were those of hapten binders), and enabled and expanded antibody engineering technologies. The latter field of engineered antibodies evolved over many years and many steps resulting in recombinant humanized or human-derived antibody derivatives in multiple formats. Today, hapten-binding antibodies are applied not only as reagents and tools (where they still play an important part) but evolved also to engineered targeting and pretargeting vehicles for disease diagnosis and therapy. Here we describe recent applications of hapten-binding antibodies and of engineered mono- and bispecific hapten-binding antibody derivatives. We have designed and applied these molecules for the modulation of the pharmacokinetic properties of small compounds or peptides. They are also integrated as additional binding entities into bispecific antibody formats. Here they serve as non-covalent or covalent coupling modules to haptenylated compounds, to enable targeted payload delivery to disease tissues or cells.
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http://dx.doi.org/10.1111/imr.12386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755198PMC
March 2016

Hapten-directed spontaneous disulfide shuffling: a universal technology for site-directed covalent coupling of payloads to antibodies.

FASEB J 2015 May 10;29(5):1763-79. Epub 2015 Feb 10.

*Roche Pharma Research & Early Development, Large Molecule Research, Roche Innovation Center Penzberg, Penzberg, Germany; Roche Diagnostics GmbH, Penzberg, Germany; and Roche Discovery Technologies, Roche Innovation Center Basel, Basel, Switzerland

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a "universal" coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.
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http://dx.doi.org/10.1096/fj.14-263665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415024PMC
May 2015

PK modulation of haptenylated peptides via non-covalent antibody complexation.

J Control Release 2013 Oct 22;171(1):48-56. Epub 2013 Jun 22.

Roche Pharma Research & Early Development pRED, Large Molecule Research, Nonnenwald 2, D-82372 Penzberg, Germany.

We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide-IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide-antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody-hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.
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http://dx.doi.org/10.1016/j.jconrel.2013.06.021DOI Listing
October 2013

Aggregation and chemical modification of monoclonal antibodies under upstream processing conditions.

Pharm Res 2013 May 16;30(5):1380-99. Epub 2013 Jan 16.

Pharma Research and Early Development, Roche Diagnostics GmbH, Nonnenwald 2, 82372, Penzberg, Germany.

Purpose: To investigate antibody stability and formation of modified species under upstream processing conditions.

Methods: The stability of 11 purified monoclonal human IgG1 and IgG4 antibodies, including an IgG1-based bispecific CrossMab, was compared in downscale mixing stress models. One of these molecules was further evaluated in realistic bioreactor stress models and in cell culture fermentations. Analytical techniques include size exclusion chromatography (SEC), turbidity measurements, cation exchange chromatography (cIEX), dynamic light scattering (DLS) and differential scanning calorimetry (DSC).

Results: Sensitivity in downscale stress models varies among antibodies and results in formation of high molecular weight (HMW) aggregates. Stability is increased in cell culture medium and in bioreactors. Media components stabilizing the proteins were identified. Extensive chemical modifications were detected both in stress models as well as during production of antibodies in cell culture fermentations.

Conclusions: Protective compounds must be present in chemically defined fermentation media in order to stabilize antibodies against the formation of HMW aggregates. An increase in chemical modifications is detectable in bioreactor stress models and over the course of cell culture fermentations; this increase is dependent on the expression rate, pH, temperature and fermentation time. Consequently, product heterogeneity increases during upstream processing, and this compromises the product quality.
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http://dx.doi.org/10.1007/s11095-013-0977-8DOI Listing
May 2013

Allosteric antibody inhibition of human hepsin protease.

Biochem J 2012 Mar;442(3):483-94

Gene Center Munich, Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Hepsin is a type II transmembrane serine protease that is expressed in several human tissues. Overexpression of hepsin has been found to correlate with tumour progression and metastasis, which is so far best studied for prostate cancer, where more than 90% of such tumours show this characteristic. To enable improved future patient treatment, we have developed a monoclonal humanized antibody that selectively inhibits human hepsin and does not inhibit other related proteases. We found that our antibody, hH35, potently inhibits hepsin enzymatic activity at nanomolar concentrations. Kinetic characterization revealed non-linear, slow, tight-binding inhibition. This correlates with the crystal structure we obtained for the human hepsin-hH35 antibody Fab fragment complex, which showed that the antibody binds hepsin around α3-helix, located far from the active centre. The unique allosteric mode of inhibition of hH35 is distinct from the recently described HGFA (hepatocyte growth factor activator) allosteric antibody inhibition. We further explain how a small change in the antibody design induces dramatic structural rearrangements in the hepsin antigen upon binding, leading to complete enzyme inactivation.
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http://dx.doi.org/10.1042/BJ20111317DOI Listing
March 2012

A tandem SH2 domain in transcription elongation factor Spt6 binds the phosphorylated RNA polymerase II C-terminal repeat domain (CTD).

J Biol Chem 2010 Dec 6;285(53):41597-603. Epub 2010 Oct 6.

Gene Center Munich and Department of Biochemistry, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.

Spt6 is an essential transcription elongation factor and histone chaperone that binds the C-terminal repeat domain (CTD) of RNA polymerase II. We show here that Spt6 contains a tandem SH2 domain with a novel structure and CTD-binding mode. The tandem SH2 domain binds to a serine 2-phosphorylated CTD peptide in vitro, whereas its N-terminal SH2 subdomain, which we previously characterized, does not. CTD binding requires a positively charged crevice in the C-terminal SH2 subdomain, which lacks the canonical phospho-binding pocket of SH2 domains and had previously escaped detection. The tandem SH2 domain is apparently required for transcription elongation in vivo as its deletion in cells is lethal in the presence of 6-azauracil.
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http://dx.doi.org/10.1074/jbc.M110.144568DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3009887PMC
December 2010

Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.

Mol Cell 2009 Jun;34(6):710-21

Department of Chemistry and Biochemistry, Gene Center Munich and Center for Integrated Protein Science CIPSM, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.

We show that RNA polymerase (Pol) II prevents erroneous transcription in vitro with different strategies that depend on the type of DNARNA base mismatch. Certain mismatches are efficiently formed but impair RNA extension. Other mismatches allow for RNA extension but are inefficiently formed and efficiently proofread by RNA cleavage. X-ray analysis reveals that a TU mismatch impairs RNA extension by forming a wobble base pair at the Pol II active center that dissociates the catalytic metal ion and misaligns the RNA 3' end. The mismatch can also stabilize a paused state of Pol II with a frayed RNA 3' nucleotide. The frayed nucleotide binds in the Pol II pore either parallel or perpendicular to the DNA-RNA hybrid axis (fraying sites I and II, respectively) and overlaps the nucleoside triphosphate (NTP) site, explaining how it halts transcription during proofreading, before backtracking and RNA cleavage.
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http://dx.doi.org/10.1016/j.molcel.2009.06.002DOI Listing
June 2009

Torpedo nuclease Rat1 is insufficient to terminate RNA polymerase II in vitro.

J Biol Chem 2009 Aug 17;284(32):21270-9. Epub 2009 Jun 17.

Gene Center and Center for Integrated Protein Science Munich, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.

Termination of RNA polymerase (pol) II transcription in vivo requires the 5'-RNA exonuclease Rat1. It was proposed that Rat1 degrades RNA from the 5'-end that is created by transcript cleavage, catches up with elongating pol II, and acts like a Torpedo that removes pol II from DNA. Here we test the Torpedo model in an in vitro system based on bead-coupled pol II elongation complexes (ECs). Recombinant Rat1 complexes with Rai1, and with Rai1 and Rtt103, degrade RNA extending from the EC until they reach the polymerase surface but fail to terminate pol II. Instead, the EC retains an approximately 18-nucleotide RNA that remains with its 3'-end at the active site and can be elongated. Thus, pol II termination apparently requires a factor or several factors in addition to Rat1, Rai1, and Rtt103, post-translational modifications of these factors, or unusual reaction conditions.
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http://dx.doi.org/10.1074/jbc.M109.013847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755851PMC
August 2009

Structure and in vivo requirement of the yeast Spt6 SH2 domain.

J Mol Biol 2009 May 14;389(1):211-25. Epub 2009 Apr 14.

Gene Center and Center for Integrated Protein Science Munich, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

During transcription elongation through chromatin, the Ser2-phosphorylated C-terminal repeat domain of RNA polymerase II binds the C-terminal Src homology 2 (SH2) domain of the nucleosome re-assembly factor Spt6. This SH2 domain is unusual in its specificity to bind phosphoserine, rather than phosphotyrosine and because it is the only SH2 domain in the yeast genome. Here, we report the high-resolution crystal structure of the SH2 domain from Candida glabrata Spt6. The structure combines features from both structural subfamilies of SH2 domains, suggesting it resembles a common ancestor of all SH2 domains. Two conserved surface pockets deviate from those of canonical SH2 domains, and may explain the unusual phosphoserine specificity. Differential gene expression analysis reveals that the SH2 domain is required for normal expression of a subset of yeast genes, and is consistent with multiple functions of Spt6 in chromatin transcription.
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http://dx.doi.org/10.1016/j.jmb.2009.04.016DOI Listing
May 2009