Christian Doppler Laboratory for Antibody Engineering - Modul 1
- Biotechnologie
- Forschungscluster "Bioindustrielle Technologien"
Abstract
The CD-Laboratory for Antibody Engineering is going to work on topics related to the Modular Antibody Technology which was developed initially at the Dpt. of Biotechnology at BOKU. This technology, which is now being further developed and applied commercially by f-star GmbH (Vienna), is based on the observation that, similar to CDR loops, so-called structural loops that connect ß-strands of immunoglobulin constant domains can be used to engineer new, additional antigen binding sites into immunoglobulin constant domains. For example, it has been shown that by Modular Antibody Technology the C-terminal structural loops of the CH3 domain of human IgG1 can be engineered such that binding sites to diverse antigens such as lysozyme, CD20, or integrin are created. Another attractive application of the technology will be to engineer binding sites into structural loops of the constant domains of the Fab fragment, paving the way to bispecific or bivalent Fabs. Manyfold improvements to the technology are conceivable. Among these, stabilizing the domain scaffold by targeted mutations is one of the most attractive options, since a structurally more stable scaffold will have an increased probability to retain the original overall wildtype structure, independently of which sequences are making up the structural loops. Another strategy is to construct structural loop libraries in which loop stem sequences as well as sequences in the randomized parts of the loops are designed to have an increased propensity to fold in such a way that the basic structure of the domain is not affected. Both these strategies will be investigated in Module 1 of the proposed CD-Laboratory. Targeted mutations that lead to increased stability of the immunoglobulin constant domains will be designed based on computational predictions, and the effect of these mutations on the folding and stability of the domains will be tested. In parallel, in vitro directed evolutionary methods will be employed by introducing random or semi-random mutations into the domain scaffold and subsequently selecting for stability-enhanced mutants using yeast display under stress conditions, e.g. at elevated temperature or under protease stress. Optimized loop sequences will also be designed using computational approaches. The domains under investigation will include the CH3 domain (in the format of an Fc), as well as the CL / CH1 heterodimer of human IgG1/kappa (in the format of an Fab). By making combinations of mutations that have been found to lead to enhanced stability, second generation mutants will be made and characterized, which will subsequently be used as scaffolds for new structural loop libraries. In Module 2 of the CD-Laboratory, two attractive applications of Modular Antibody Technology to improve Fab fragments will be investigated. Fab fragments are of limited clinical use today, due to their inherent short half-lives on the one hand and due to the fact that they do not display effector functions on the other hand. Both these issues will be addressed. A prolonged half-life can be engineered into proteins by endowing them with a binding site directed to a protein that already has a long half-life, such as serum albumin. CL/CH1 heterodimers with albumin binding sites engineered into the structural loops of the CL domain will be selected from yeast display libraries, characterized in vitro for their binding properties and subsequently tested for their in vivo half-life in mice. In the attempt to engineer the ability to display effector functions into Fab fragments, CL domains with binding sites specific to FcgammaR receptors such as FcgammaRIII will be selected from yeast display libraries. In order to test whether the selected engineered Fabs can actually elicit ADCC, they will be characterized both in vitro (binding to polymorphonuclear (PMN) cells and to natural killer (NK) cells; IL-2 and IFN-gamma production assays and in vitro ADCC tests).
Publications
Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif.
Autoren: Traxlmayr, MW; Wozniak-Knopp, G; Antes, B; Stadlmayr, G; Rüker, F; Obinger, C; Jahr: 2011
Journal articles
Directed evolution of Her2/neu-binding IgG1-Fc for improved stability and resistance to aggregation by using yeast surface display.
Autoren: Traxlmayr, MW; Lobner, E; Antes, B; Kainer, M; Wiederkum, S; Hasenhindl, C; Stadlmayr, G; Rüker, F; Woisetschläger, M; Moulder, K; Obinger, C; Jahr: 2013
Journal articles
Directed evolution of antibody fragments for improved stability and resistance to aggregation by using yeast surface display
Autoren: Lobner, E., Traxlmayr, M. W., Rüker, F., Obinger, C. Jahr: 2014
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Structures and thermodynamics of Fcab-antigen complexes.
Autoren: Lobner, E., Humm, A.-S., Göritzer, K., Mlynek, G., Hasenhindl, C., Traxlmayr, M., Djinoviv-Carugo, K., Obinger, C. Jahr: 2016
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.
Autoren: Hasenhindl, C; Traxlmayr, MW; Wozniak-Knopp, G; Jones, PC; Stadlmayr, G; Rüker, F; Obinger, C; Jahr: 2013
Journal articles
Construction of pH-sensitive Her2 binding antibody fragment by directed evolution using yeast display
Autoren: Lobner, E., Traxlmayr, M. W., Rueker, F., Obinger, C. Jahr: 2014
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Christian Doppler Laboratory for Antibody Engineering - 5 years Evaluation (2009-2013)
Autoren: Obinger, C., Rüker, F. Jahr: 2013
Forschungsbericht (extern. Auftraggeber)
external links and characteristics of the publication:Two-faced Fcab prevents polymerization with VEGF and reveals thermodynamics and the 2.15 Å crystal structure of the complex.
Autoren: Lobner, E; Humm, AS; Mlynek, G; Kubinger, K; Kitzmüller, M; Traxlmayr, MW; Djinović-Carugo, K; Obinger, C; Jahr: 2017
Journal articles
Stability-engineering of human IgG1-Fc by using yeast display
Autoren: Traxlmayr, M. W., Faissner, M., Stadlmayr, G., Rüker, F., Obinger, C. Jahr: 2011
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Engineered human IgG1-Fc binding to integrin
Autoren: Traxlmayr, M., Wozniak-Knopp, G., Rüker, F., Obinger, C. Jahr: 2009
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Stability engineering of the Fc fragment of human IgG1 by targeted mutagenesis
Autoren: Stadlmayr, G., Wozniak-Knopp, G., Hasenhindl, C., Rüker, F., Obinger, C. Jahr: 2010
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Creating stable stem regions for loop elongation in Fcabs - insights from combining yeast surface display, in silico loop reconstruction and molecular dynamics simulations.
Autoren: Hasenhindl, C; Lai, B; Delgado, J; Traxlmayr, MW; Stadlmayr, G; Rüker, F; Serrano, L; Oostenbrink, C; Obinger, C; Jahr: 2014
Journal articles
Engineered IgG1-Fc--one fragment to bind them all.
Autoren: Lobner, E; Traxlmayr, MW; Obinger, C; Hasenhindl, C; Jahr: 2016
Journal articles
Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast.
Autoren: Traxlmayr, MW; Faissner, M; Stadlmayr, G; Hasenhindl, C; Antes, B; Rüker, F; Obinger, C Jahr: 2012
Journal articles
Directed evolution of proteins for increased stability and expression using yeast display.
Autoren: Traxlmayr, MW; Obinger, C; Jahr: 2012
Journal articles
Construction of a stability landscape of the CH3 domain of human IgG1 by combining directed evolution with high throughput sequencing.
Autoren: Traxlmayr, MW; Hasenhindl, C; Hackl, M; Stadlmayr, G; Rybka, JD; Borth, N; Grillari, J; Rüker, F; Obinger, C; Jahr: 2012
Journal articles
Fcab-HER2 Interaction: a Ménage à Trois. Lessons from X-Ray and Solution Studies.
Autoren: Lobner, E; Humm, AS; Göritzer, K; Mlynek, G; Puchinger, MG; Hasenhindl, C; Rüker, F; Traxlmayr, MW; Djinović-Carugo, K; Obinger, C; Jahr: 2017
Journal articles
Stability assessment on a library scale – A rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc.
Autoren: Hasenhindl, C., Traxlmayr, M.W., Wozniak-Knopp, G ., Jones, P.C., Stadlmayr, G., Rüker, F., Obinger, C. Jahr: 2014
Conference & Workshop proceedings, paper, abstract
external links and characteristics of the publication:Construction of pH-sensitive Her2-binding IgG1-Fc by directed evolution.
Autoren: Traxlmayr, MW; Lobner, E; Hasenhindl, C; Stadlmayr, G; Oostenbrink, C; Rüker, F; Obinger, C; Jahr: 2014
Journal articles
Project staff
Christian Obinger
Univ.Prof. Mag.rer.nat. Dr.rer.nat. Christian Obinger
christian.obinger@boku.ac.at
Tel: +43 1 47654-10011, 77273
BOKU Project Leader
01.03.2009 - 31.12.2016
Florian Rüker
Ao.Univ.-Prof.i.R. Dr. Florian Rüker
florian.rueker@boku.ac.at
Tel: +43 1 47654-79854
Sub Projectleader
01.03.2009 - 31.12.2016
Gordana Wozniak-Knopp
Dipl.-Biol. Dr.rer.nat. Gordana Wozniak-Knopp
gordana.wozniak@boku.ac.at
Tel: +43 1 47654-79868
Project Staff
01.03.2009 - 31.12.2016
BOKU partners
External partners
f-star GmbH
partner