Biomedical Engineering

From molecular design to therapeutic applications

Overview

Our group is working on the development of novel protein-based strategies for therapy of cancer, inflammatory and neurodegenerative diseases as well as neuroregenerative approaches. Besides antibodies, these proteins include bispecific and bifunctional fusion proteins. The bispecific and bifunctional molecules are generated by combining different building blocks, including antigen-binding sites of antibodies, homo- and heterodimerization modules, effector molecules such as co-stimulatory, immunoregulatory and apoptosis-inducing members of the TNF superfamily, as well as, if necessary, half-life extension modules. The aim of this combinatorial and modular approach is to design molecules for a selective, efficient and safe treatment by selecting appropriate targets and by adapting size, valency, flexibility, stability, and pharmacokinetic properties. Our research activities cover a broad methodical spectrum, from genetic engineering, recombinant protein expression and purification, biochemical assays, to functional characterization in vitro and in vivo.
 

Antibody generation

The generation of antibodies recognizing suitable targets is essential for the development of efficient targeting strategies. Together with external collaboration partners we are generation human antibodies against novel targets, but also apply antibody humanization by CDR grafting to convert non-human hydridoma-derived antibodies into humanized antibodies. These antibodies and their antigen binding sites form the basis for the generation of therapeutic antibodies and antibody derivatives, such as bispecific antibodies and bifunctional antibody fusion proteins. One focus is on antibodies targeting surface structures expressed by tumor cells, including receptors and cell adhesion molecules, but also antibodies targeting cells of the tumor microenvironment, such as tumor endothelial cells and cancer-associated fibroblasts. Furthermore, we are generating antibodies against regulatory receptors on immune cells and other cell types, such as members of the TNF receptor family.
An example of an antibody for cancer therapy recently developed in our laboratory is a novel human antibody directed against the tyrosine kinase receptor HER3, a member of the EGF receptor family. This antibody, IgG 3-43, is capable of efficiently inhibiting ligand-dependent and ligand-independent activation of HER3 by heterodimerization with other members of the EGFR family and inhibition of downstream signaling and cellular repsonses such as cell proliferation. Here, we closely collaborate with the group of Prof. Monilola Olayioye.
Furthermore, we have generated antibodies against cells of the tumor microenvironment, including humanized versions of FAP-targeting antibodies previously developed at the institute and a human antibody directed against endoglin (CD105), these antibodies are developed by the Spanish company Oncomatryx as antibody drug conjugates for attacking the tumor stroma and tumor vasculature of solid tumors.
 

Cancer therapy with bispecific antibodies for dual targeting strategies

Often, tumor cells utilize different pathways as proliferation and survival signals, but can also activate alternative receptor pathways as compensatory signals, e.g. as resistance mechanisms to treatment. Simultaneous targeting and inhibition of various receptor pathways (dual targeting) has, therefore emerged as novel therapeutic strategy. We are developing bispecific antibodies, e.g. based on our single-chain diabody (scDb) technology, but also novel formats developed in our laboratory, for such dual targeting strategies. These include bispecific antibodies targeted different members of the EGF receptor family, e.g. EGFR and HER3. Here we have generated a scDb-Fc fusion protein combining bivalent, bispecific scDb, composed of the variable domains of two antibodies, with an Fc region, resulting a tetravalent, bispecific molecules with IgG-like size and properties.
 

Cancer therapy with bispecific antibodies retargeting immune effector cell

The immune system comprises an arsenal of different and potent effector cells, such as cytotoxic T-lymphocytes and natural killer (NK) cells. Malignant cells often escape elimination by these effector cells by various mechanisms. Bispecific antibodies are capable of redirecting effector cells and to trigger killing of target cells, thus bypassing the escape mechanisms. In the past years, we have developed novel bispecific antibody formats, including the so-called single-chain diabodies (scDb) format, composed of the variable antigen-binding domains of two different antibodies. This format was successfully evaluated for a variety of applications, including retargeting of effector molecules, effector cells and viral gene vectors. Current work is focusing on the development of single-chain diabodies and various derivatives thereof as well as novel bi- and trivalent antibody formats for the retargeting of cytotoxic T-lymphocytes and their evaluation in relevant tumor models.
 

Cancer immunotherapy with bifunctional fusion proteins with costimulatory activity

Immuno-oncology has emerged as promising approach to induce and foster an anti-tumor response of the body’s own immune system, interfering at different levels of the cancer immunity circle. Besides mediating a recognition of cancer cells by T cells using bispecific antibodies (see above), additional approaches include the inhibition of immunosuppressive activities, e.g. with check-point inhibitors, and the stimulation of immune responses by providing co-stimulatory signals. Our research activities have a focus on generating novel molecules to boost an immune response through co-stimulatory members of the TNF superfamily. For example, together with the group of Prof. Klaus Pfizenmaier, we have established dual acting molecules, so-called duokines, composed of two different co-stimulatory ligands, capable of acting either in cis (i.e. on the same cell) or in trans (between two cells) and, thus enhancing an anti-cancer immune response, e.g. by T cells. These approaches complement further developments led by Dr. Dafne Müller’s group on targeted (co)-stimulation in cancer immunotherapy.
 

Cancer therapy with apoptosis-inducing activity fusion proteins

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of cell death through activation of death receptors expressed by tumor cells, with no or little activity against normal cells. However, soluble TRAIL, which is a homotrimeric molecule, showed minimal activity in clinical trials, mainly due to inefficient activation of death receptors, especially death receptor 5 (DR5), which requires receptor clustering by multivalent binding. Together with the group of Prof. Klaus Pfizenmaier, we have developed next-generation TRAIL molecules implementing two design principles: i) converting homotrimeric TRAIL into a single-chain derivative (scTRAIL) and ii) fusing scTRAIL to dimerisation modules, thus generating hexavalent molecules, which efficiently activate all death receptors. Furthermore, we have added a targeting moiety, e.g. a single-chain Fv (scFv) fragment for redirecting the molecules to target cells. These modifications resulted in non-targeted and targeted multivalent scTRAIL molecules, which showed highly efficient target cell killing in vitro and in various tumor models.
 

Antagonistic antibodies and TNFR2-selective agonists for treatment of inflammatory and neurodegenerative diseases and the induction of neuroregenerative processes

Tumor necrosis factor (TNF) is a pleiotropic cytokine and a key regulator of the immune responses. The two TNF receptors (TNFR1, TNFR2) induce dichotomic biological responses. Thus, TNFR1 promotes inflammation and tissue degeneration, while TNFR2 contributes to immune suppression, tissue homeostasis and regeneration. Of note, TNFR1 can be activated by membrane and soluble TNF, while TNFR2 requires clustering by binding to membrane TNF. Specific TNF receptor-mediated responses can be induced with selective reagents. Together with the group of Prof. Klaus Pfizenmaier, we have developed antagonistic TNFR1-specific antibodies and derivatives thereof, as well as TNFR2-selective agonistic molecules based on multivalent single-chain TNF molecules specifically binding to TNFR2, mimicking the biological activities of mTNF. Together with various collaboration partners, these molecules showed promising results in various disease models in mice (e.g. rheumatoid arthritis, experimental autoimmune encephalomyelitis, NMDA-induced acute neurodegeneration), supporting further development as therapeutic reagents.
 

Half-life extension strategies

Many therapeutic proteins, including small antibody fragments, are of small size and are, therefore, rapidly cleared from circulation. In order to increase the half-life of these molecules we have established and compared various half-life extension strategies (HLEs), including PEGylation, hyper-glycosylation by post-translational modifications, fusion human serum albumin or an albumin-binding domain (ABD), as well as fusion to an immunoglobulin-binding domain (IgBD), exemplified e.g. for a single-chain diabody for T-cell retargeting (see above). The applied strategies increase i) the hydrodynamic volume of the molecule, and ii) can implement a recycling process by the neonatal Fc-receptor (FcRn), e.g. for molecules binding to albumin or IgG immunoglobulins. These modifications resulted in a strongly prolong circulation and can, therefore, be implemented in therapeutic strategies.
 

The Biomedical Engineering Team

Dr. Roman Fischer (Scientist)
Dr. Nadine Aschmoneit (Scientist)
Lennart Kühl (joint PhD student with the Olayioye lab)
Alexander Rau (joint PhD student with the Olayioye lab)
Doris Göttsch (Technician)
Nadine Heidel (Technician)
Sabine Münkel (Technician)
Kim Bui (Master student)

 

Funding

Our group has received funding from the following organisations:
BMBF, DFG, Deutsche Krebshilfe, Baden-Württemberg Stiftung, MRC, EC Eurostars,
and is funded by various industry collaborations.
 

Publications

Selected reviews on research topics
  • The making of bispecific antibodies. Brinkmann, U. & Kontermann, R.E. (2017) mAbs 7, 182-212.
  • Half-life extended biotherapeutics. Kontermann, R.E. (2016) Expert Opin. Biol. Ther. 16, 903-915.
  • Bispecific antibodies. Kontermann, R. and Brinkmann, U. (2015) Drug Discov. Today 20, 838-847.
  • Antibody-cytokine fusion proteins. Kontermann, R.E. (2012) Arch. Biochem. Biophys. 526, 194-205.
  • Dual targeting strategies with bispecific antibodies. Kontermann, R.E. (2012) mAbs 4, 182-197.
  • Antagonists of TNF action ‐ clinical experience and new developments. Kontermann, R.E., Scheurich, P. & Pfizenmaier, K. (2009) Exp. Opin. Drug Discovery 4, 279‐292.
  • Cellular cancer immunotherapy using recombinant bispecific antibodies. Müller, D. & Kontermann, R.E. (2007) Curr. Opin. Mol. Ther. 9, 319‐326.
 
Selected original publications
Antibody generation
  • Inhibition of HER3 activation and tumor growth with a human antibody binding to a conserved epitope within domain III and IV. Schmitt, L., Rau, A., Seifert, O., Honer, J., Schmid, S., Hutt, M., Zantow, J., Hust, M., Dübel, S., Olayioye, M.A. & Kontermann, R.E. (2017) mAbs 9, 831-843.
  • Murine endoglin‐reactive single‐chain Fv fragments for vascular targeting in mice.  Müller, D., Trunk, G., Sichelstiel, A., Zettlitz, K, Quintanilla, M. & Kontermann, R.E. (2008) J. Immunol. Meth. 339, 90‐98.
  • Isolation of endothelial cell‐specific antibodies from a fully synthetic scFv library. Völkel, T., Müller, R. & Kontermann, R.E. (2004) Biochem. Biophys. Res. Commun. 317, 515‐521.
 
Cancer therapy with bispecific antibodies for dual targeting strategies
  • Novel tetravalent and bispecific IgG‐like antibody molecules combining single‐chain diabodies with the immunoglobulin g1 Fc or CH3 region. Alt, M., Müller, R. & Kontermann, R.E. (1999) FEBS Lett. 454, 90‐94
 
Cancer therapy with bispecific antibodies retargeting effector cell
  • Optimized linker sequences for the expression of monomeric and dimeric bispecific single‐chain diabodies. Völkel, T., Korn, T., Bach, M., Müller, R. & Kontermann, R.E. (2001) Protein Eng. 14, 815‐823.
  • Bispecific single-chain diabody-mediated killing of endothelial cells by cytotoxic T lymphocytes.Korn, T., Müller, R. & Kontermann, R.E. (2004) J. Immunother. 27, 99-106.
 
Cancer immunotherapy with bifunctional fusion proteins with costimulatory activity
  • Duokines: a novel class of dual-acting co-stimulatory molecules acting in cis or trans. Fellermeier-Kopf S, Gieseke F, Sahin U, Müller D, Pfizenmaier K, Kontermann RE. (2018) Oncoimmunology. 7:e1471442.
  • Advancing targeted costimulation with antibody‐fusion proteins by introducing TNF superfamily members in a single‐chain format. Fellermeier, S., Beha, N., Meyer, J.E., Ring, S., Bader, S., Kontermann, R.E. & Müller, D. (2016) Oncoimmunology 5,e1238540.  
  • Combining antibody-directed presentation of IL-15 and 4-1BBL in a trifunctional fusion protein for cancer immunotherapy. Kermer, V., Hornig, N., Harder, M., Bondarieva, A., Kontermann, R.E. & Müller, D. (2014) Mol. Cancer Ther. 13, 112-121.
 
Cancer therapy with apoptosis-inducing activity fusion proteins
  • Superior properties of Fc-comprising scTRAIL fusion proteins. Hutt, M., Marquart, L., Seifert, O., Siegemund, M., Müller, I., Kulms, D., Pfizenmaier, K. & Kontermann, R.E. (2017) Mol. Cancer Ther. 16, 2792-2802.
  • An optimized antibody-single-chain TRAIL fusion protein for cancer therapy.  Siegemund, M., Seifert, O., Zarani, M., Dzinic, T., De Leo, V., Göttsch, D., Münkel, S., Hutt, M., Pfizenmaier, K. & Kontermann, R.E. (2016) MAbs 8, 879-891.
  • Tetravalent antibody-scTRAIL fusion proteins with improved properties. Seifert, O., Plappert, A., Fellermeier, S., Siegemund, M., Pfizenmaier, K. & Kontermann, R.E. (2014) Mol. Cancer Ther. 13, 101-111.
 
Antagonistic antibodies and antibody fragments for treatment of inflammatory and neurodegenerative diseases
  • Monovalent TNF receptor 1-selective antibody with improved affinity and neutralizing activity. Richter F, Zettlitz KA, Seifert O, Herrmann A, Scheurich P, Pfizenmaier K, Kontermann RE. (2018) MAbs.
  • Antagonistic TNF receptor one-specific antibody (ATROSAB): receptor binding and in vitro bioactivity.  Richter, F., Liebig, T., Guenzi, E., Herrmann, A., Scheurich, P., Pfizenmaier, K. & Kontermann, R.E. (2013) PLOS ONE 8, e72156.
  • ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody. Zettlitz, K.A., Lorzenz, V., Landauer, K., Münkel, S., Herrmann, A., Scheurich, P., Pfizenmaier, K. & Kontermann, R.E. (2010) mAbs 2, 639-647.
 
TNFR2-selective agonists for neuroregeneration
  • Novel strategies to mimic transmembrane tumor necrosis factor-dependent activation of tumor necrosis factor receptor 2. Fischer, R., Marsal, J., Guttá, C., Eisler, S.A., Peters, N., Bethea, J.R., Pfizenmaier, K. & Kontermann, R.E. Sci. Rep.7, 6607.
  • Selective targeting tumor necrosis receptors in neurodegeneration: Essential protective role of TNFR2.  Dong, Y., Fischer, R., Naudé, P.J.W., Maier, O., Nyakas, C., Duffey, M., van der Zee, E.A., Dekens, D., Douwenga, W., Herrmann, A., Guenzi, E., Kontermann, R.E., Pfizenmaier, K. & Eisel, U.L.M. (2016) Proc. Natl. Acad. Sci. USA 113, 12304-12309.
 
Half-life extension strategies
  • A Fab-selective immunoglobulin-binding domain from streptococcal protein G with improved half-life extension properties. Unverdorben, F., Hutt, M., Seifert, O. & Kontermann, R.E. (2015) PLOS One, e0139838.
  • Plasma half-life extension of small recombinant antibodies by fusion to immunoglobulin-binding domains (IgBD). Hutt, M., Färber-Schwarz, A., Unverdorben, F., Richter, F. & Kontermann, R.E. (2012) J. Biol. Chem. 287, 4462-4469.
  • Biodistribution of a bispecific single-chain diabody and its half-life extended derivatives. Stork, R., Campigna, E., Robert, B., Müller, D. & Kontermann, R.E. (2009) J. Biol. Chem. 284, 25612-25619.
  • A novel tri‐functional antibody fusion protein with improved pharmacokinetic properties generated by fusing a bispecific single‐chain diabody with an albumin‐binding domain from streptococcal protein G. Stork, R., Müller, D. & Kontermann, R.E. (2007) Protein Eng. Des. Sel. 20, 569‐576.
  • Improved pharmacokinetics of recombinant bispecific antibody molecules by fusion to human serum albumin. Müller, D., Karle, A., Meißburger, B., Höfig, I., Stork, R. & Kontermann, R.E. (2007) J. Biol. Chem. 282, 12650-12660.
 
Books
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