- Hi , I am an expert scientist in Discovery at Roche Innovation Center Munichedit
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A new format of therapeutic proteins is bispecific antibodies, in which two different heavy chains heterodimerize to obtain two different binding sites. Therefore, it is crucial to understand and optimize the third constant domain... more
A new format of therapeutic proteins is bispecific antibodies, in which two different heavy chains heterodimerize to obtain two different binding sites. Therefore, it is crucial to understand and optimize the third constant domain (CH3-CH3) interface to favor heterodimerization over homodimerization, and to preserve the physicochemical properties, as thermal stability. Here, we use molecular dynamics simulations to investigate the dissociation process of 19 CH3-CH3 crystal structures that differ from each other in few point mutations. We describe the dissociation of the dimeric interface as a two-steps mechanism. As confirmed by a Markov state model, apart from the bound and the dissociated state, we observe an additional intermediate state, which corresponds to an encounter complex. The analysis of the interdomain contacts reveals key residues that stabilize the interface. We expect that our results will improve the understanding of the CH3-CH3 interface interactions and thus advance the developability and design of new antibodies formats. Open in a separate window
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Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed... more
Antibodies have emerged as one of the fastest growing classes of biotherapeutic proteins. To improve the rational design of antibodies, we investigate the conformational diversity of 16 different germline combinations, which are composed of 4 different kappa light chains paired with 4 different heavy chains. In this study, we systematically show that different heavy and light chain pairings strongly influence the paratope, interdomain interaction patterns and the relative VH-VLinterface orientations. We observe changes in conformational diversity and substantial population shifts of the complementarity determining region (CDR) loops, resulting in distinct dominant solution structures and differently favored canonical structures. Additionally, we identify conformational changes in the structural diversity of the CDR-H3 loop upon different heavy and light chain pairings, as well as upon changes in sequence and structure of the neighboring CDR loops, despite having an identical CDR-H3 ...
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The rise of antibodies as biotherapeutic proteins has attracted the interest in characterizing and understanding the antibody binding interface. Structure-based antibody design and accurate predictions of antibody-antigen interactions... more
The rise of antibodies as biotherapeutic proteins has attracted the interest in characterizing and understanding the antibody binding interface. Structure-based antibody design and accurate predictions of antibody-antigen interactions remain major challenges in computational biology. By using molecular dynamics simulations in combination with enhanced sampling techniques, we show that a single static X-ray structure is not sufficient to identify determinants of antibody recognition and to characterize functionally relevant conformational rearrangements. Thus, we suggest that antibodies should be described as ensembles of paratope states in solution, which are defined by a combination of correlated loop movements, which favor distinct interface orientations and elbow angles. In this study, we focus our attention on characterizing paratope states in solution of antibodies which undergo substantial conformational changes upon antigen binding. Because of their conformational changes, they have been classified as “difficult” cases for antibody-antigen docking in the Critical Assessment of PRediction of Interactions (CAPRI) blind docking challenge. Here, we present transition kinetics and thermodynamics of these conformational rearrangements of the paratope and show that kinetically relevant states can be used to improve antibody-antigen docking. We use the Rosetta SnugDock docking algorithm to show that by even using the unbound antibody X-ray structure as starting structures for molecular dynamics simulations, a binding competent conformation substantially different to the unbound antibody X-ray structure, can be retained. We also observe that the kinetically dominant antibody conformations are chosen by the bound antigen conformation with the highest probability. Thus, we show that kinetically relevant antibody conformations obtained by a combination of molecular dynamics simulations and Markov-state models can improve antibody-antigen docking and structure prediction.
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Motivation Co-evolution analysis can be used to accurately predict residue–residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift... more
Motivation Co-evolution analysis can be used to accurately predict residue–residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift from predicting binary contacts to predict distances between pairs of residues. These developments have significantly improved the accuracy of de novo prediction of static protein structures. With AlphaFold2 lifting the accuracy of some predicted protein models close to experimental levels, structure prediction research will move on to other challenges. One of those areas is the prediction of more than one conformation of a protein. Here, we examine the potential of residue–residue distance predictions to be informative of protein flexibility rather than simply static structure. Results We used DMPfold to predict distance distributions for every residue pair in a set of proteins that showed both rigid and flexible behaviour. Residue pairs that were i...
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3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all... more
3726 Poster Board III-662 CD20 is a specific cell surface marker found on normal as well as malignant B cells. Rituximab, a monoclonal antibody directed against CD20, has a major impact on treatment of malignant lymphomas. Although all therapeutic CD20 antibodies are directed against the two relatively small extracellular loops of CD20, such antibodies can be classified into Type I CD20 antibodies like Rituximab, Ofatumumab or Ocrelizumab or Type II CD20 antibodies like the novel glycoengineered humanized CD20 antibody GA101 or the murine antibody Tositumumab. Type I and Type II antibodies differ significantly in their mode of action and mechanisms of killing malignant B-cells. The molecular basis of this is not understood. We use data from epitope mapping, X-ray crystallography, isothermal titration calorimetry, and point mutagenesis i) to accurately map the epitopes of different anti-CD20 antibodies, in particular GA101, and ii) to compare the molecular interactions involved in th...
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The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal... more
The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal models of human diseases are often unsuitable for immunogenicity testing and for predicting adverse outcomes in human patients. Most human therapeutic antibodies trigger xenogeneic responses in wild-type animals and thus rapid clearance of the drugs, which makes in vivo toxicological testing of human antibodies challenging. Here we report the generation of Göttingen minipigs carrying a mini-repertoire of human genes for the immunoglobulin heavy chains γ1 and γ4 and the immunoglobulin light chain κ. In line with observations in human patients, the genetically modified minipigs tolerated the clinically non-immunogenic IgG1κ-isotype monoclonal antibodies daratumumab and bevacizumab, and elicited antibodies against the checkpoint inhibitor atezolizumab and ...
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Additional file 3. Additional materials.
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[This corrects the article DOI: 10.3389/fmolb.2022.812750.].
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As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new... more
As the current biotherapeutic market is dominated by antibodies, the design of different antibody formats, like bispecific antibodies and other new formats, represent a key component in advancing antibody therapy. When designing new formats, a targeted modulation of pairing preferences is key. Several existing approaches are successful, but expanding the repertoire of design possibilities would be desirable. Cognate immunoglobulin G antibodies depend on homodimerization of the fragment crystallizable regions of two identical heavy chains. By modifying the dimeric interface of the third constant domain (CH3-CH3), with different mutations on each domain, the engineered Fc fragments form rather heterodimers than homodimers. The first constant domain (CH1-CL) shares a very similar fold and interdomain orientation with the CH3-CH3 dimer. Thus, numerous well-established design efforts for CH3-CH3 interfaces, have also been applied to CH1-CL dimers to reduce the number of mispairings in th...
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Additional file 1. Additional figures.
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La presente invention concerne des anticorps diriges contre l'IL17A humaine (anticorps anti-IL17), des procedes pour leur production, des compositions pharmaceutiques contenant lesdits anticorps, et leurs utilisations.
La presente invention a pour objet les composes de formule (I), leurs sels de qualite pharmaceutique, leurs formes enantiomeres, leurs diastereoisomeres et leurs racemates, la synthese des composes susmentionnes, les medicaments les... more
La presente invention a pour objet les composes de formule (I), leurs sels de qualite pharmaceutique, leurs formes enantiomeres, leurs diastereoisomeres et leurs racemates, la synthese des composes susmentionnes, les medicaments les contenant et leur fabrication, ainsi que l'emploi des composes susmentionnes dans la regulation ou la prevention de maladies telles que le cancer.
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Conjugate comprising an antigen or hapten and an antibody that specifically binds antigen or hapten, wherein a covalent bond is between a cysteine residue in the 52b position or at position 53 according to Kabat variable domain heavy... more
Conjugate comprising an antigen or hapten and an antibody that specifically binds antigen or hapten, wherein a covalent bond is between a cysteine residue in the 52b position or at position 53 according to Kabat variable domain heavy chain CDR2 in the heavy chain antibody and antigen or hapten.
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ABSTRACT The rise of antibodies as a promising and rapidly growing class of biotherapeutic proteins has motivated numerous studies to characterize and understand antibody structures. In the past decades, the number of antibody crystal... more
ABSTRACT The rise of antibodies as a promising and rapidly growing class of biotherapeutic proteins has motivated numerous studies to characterize and understand antibody structures. In the past decades, the number of antibody crystal structures increased substantially, which revolutionized the atomistic understanding of antibody functions. Even though numerous static structures are known, various biophysical properties of antibodies (i.e., specificity, hydrophobicity and stability) are governed by their dynamic character. Additionally, the importance of high-quality structures in structure–function relationship studies has substantially increased. These structure–function relationship studies have also created a demand for precise homology models of antibody structures, which allow rational antibody design and engineering when no crystal structure is available. Here, we discuss various aspects and challenges in antibody design and extend the paradigm of describing antibodies with only a single static structure to characterizing them as dynamic ensembles in solution.
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An antibody that binds to human IL33R, which is characterized in that the heavy chain variable domain comprises Sec. ID No.: 7 and the light chain variable domain comprises Sec. ID No.: 8.
ABSTRACTCo-evolution analysis can be used to accurately predict residue-residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift from... more
ABSTRACTCo-evolution analysis can be used to accurately predict residue-residue contacts from multiple sequence alignments. The introduction of machine-learning techniques has enabled substantial improvements in precision and a shift from predicting binary contacts to predicting distances between pairs of residues. These developments have significantly improved the accuracy of de novo prediction of static protein structures. Here we examine the potential of these residue-residue distance predictions to predict protein flexibility rather than static structure. We used DMPfold to predict distance distributions for every residue pair in a set of proteins that showed both rigid and flexible behaviour. Residue pairs that were in contact in at least one reference structure were considered and classified as rigid, flexible or neither. The predicted distance distribution of each residue pair was analysed for local maxima of probability indicating the most likely distance or distances betwee...
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Fab consist of a heavy and light chain and can be subdivided into a variable (VH and VL) and a constant region (CH1 and CL). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable... more
Fab consist of a heavy and light chain and can be subdivided into a variable (VH and VL) and a constant region (CH1 and CL). The variable region contains the complementarity-determining region (CDR), which is formed by six hypervariable loops, shaping the antigen binding site, the paratope. Apart from the CDR loops, both the elbow angle and the relative interdomain orientations of the VH–VL and the CH1–CL domains influence the shape of the paratope. Thus, characterization of the interface and elbow angle dynamics is essential to antigen specificity. We studied nine antigen-binding fragments (Fab) to investigate the influence of affinity maturation, antibody humanization, and different light-chain types on the interface and elbow angle dynamics. While the CDR loops reveal conformational transitions in the micro-to-millisecond timescale, both the interface and elbow angle dynamics occur on the low nanosecond timescale. Upon affinity maturation, we observe a substantial rigidification ...
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Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific... more
Based on the unprecedented clinical efficacy of PD-1/PD-L1 pathway checkpoint inhibitors (CPI), non-redundant immune checkpoints like TIM-3, LAG-3, TIGIT or BTLA are currently being targeted, by combinatorial approaches using monospecific or bispecific antibodies. Up-regulation of TIM-3 has been described as an adaptive CPI resistance mechanism, and internal prevalence data on archival samples of CPI-naïve and -experienced patients showed co-expression of PD-1 and TIM-3 in various tumor types, consistent with literature reports. Here, we describe RG7769 (PD1-TIM3), a novel avidity driven heterodimeric PD-1/TIM-3 1+1 bispecific CrossMabVH-VL intentionally designed as high affinity PD-1 (KD 250 pM, 37°C) and low affinity TIM-3 (KD 130 nM, 37°C) Fab-moieties to specifically target PD-1+ and PD-1+ TIM-3+ T cells through avidity gain, while bypassing PD-1- TIM3+ myeloid and NK cells. In contrast to IgG4-based PD-1 antibodies and conventional IgG1-based TIM-3 Fc-effector function competen...
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Solving the structure of an antibody-antigen complex gives atomic level information of the interactions between an antibody and its antigen, but such structures are expensive and hard to obtain. Alternative experimental sources include... more
Solving the structure of an antibody-antigen complex gives atomic level information of the interactions between an antibody and its antigen, but such structures are expensive and hard to obtain. Alternative experimental sources include epitope mapping and binning experiments which can be used as a surrogate to identify key interacting residues. However, their resolution is usually not sufficient to identify if two antibodies have identical interactions. Computational approaches to this problem have so far been based on the premise that antibodies with similar sequences behave similarly. Such approaches will fail to identify sequence-distant antibodies that target the same epitope.We present Ab-Ligity, a structure-based similarity measure tailored to antibody-antigen interfaces. Using predicted paratopes on model antibody structures, we assessed its ability to identify those antibodies that target highly similar epitopes. Most antibodies adopting similar binding modes can be identifi...
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Research Interests: Water, Molecular Biology, Biology, Medicine, Macromolecular X-Ray Crystallography, and 15 moreMolecular, Crystal structure, Humans, Sequence alignment, Proteins, Hydrogen Bonding, Substrate Specificity, Amino Acid Sequence, Protein Binding, Ligands, Biochemistry and cell biology, Focal Adhesion Kinase, Tyrosine Kinase, Molecular Sequence Data, and Protein tyrosine kinases
Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high... more
Driven by the potential to broaden the target space of conventional monospecific antibodies, the field of multi-specific antibody derivatives is growing rapidly. The production and screening of these artificial proteins entails a high combinatorial complexity. Antibody-domain exchange was previously shown to be a versatile strategy to produce bispecific antibodies in a robust and efficient manner. Here, we show that the domain exchange reaction to generate hybrid antibodies also functions under physiological conditions. Accordingly, we modified the exchange partners for use in therapeutic applications, in which two inactive prodrugs convert into a product with additional functionalities. We exemplarily show the feasibility for generating active T cell bispecific antibodies from two inactive prodrugs, which per se do not activate T cells alone. The two complementary prodrugs harbor antigen-targeting Fabs and non-functional anti-CD3 Fvs fused to IgG-CH3 domains engineered to drive cha...
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Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppres-sion of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific... more
Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppres-sion of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen–specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI–IgG). Here, we show that tumor antigen–expressing cancer cells, which are deco-rated with pMHCI–IgGs containing a HLA-A0201 molecule associated with a CMV-derived peptide, are specifically elim-inated through engagement of antigen-specific CD8þ T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8þ T cells act without additional expansion, preactivation, or provision of costimu-latory signals. Elimination of tumor cells is induced at sim...
In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as... more
In the last decades, antibodies have emerged as one of the most important and successful classes of biopharmaceuticals. The highest variability and diversity of an antibody is concentrated on six hypervariable loops, also known as complementarity determining regions (CDRs) shaping the antigen-binding site, the paratope. Whereas it was assumed that certain sequences can only adopt a limited set of backbone conformations, in this study we present a kinetic classification of several paratope states in solution. Using molecular dynamics simulations in combination with experimental structural information we capture the involved conformational transitions between different canonical clusters and additional dominant solution structures occurring in the micro-to-millisecond timescale. Furthermore, we observe a strong correlation of CDR loop movements. Another important aspect when characterizing different paratope states is the relative VH/VL orientation and the influence of the distinct CD...
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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... more
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 ...
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Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a... more
Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridg...