Ligand Development

The unit focuses on the interaction of biomolecules, in particular the identification of peptides for tumor targeting and antibody characterization. A new peptide phage display method is combined with modern devices and measurement methods. This allows in silico data evaluation for epitope mapping as well as the immunome of patient sera (e. g. allergy research) and the identification of peptide ligands for the characterization of complex structures (e. g. cell surfaces) as an alternative to antibodies. These applications range from the labeling of cancer cells / tissues to the characterization of (stem) cells in different culture and storage conditions.

Phage Display

In the past decades modern drug development has relied on the generation of enormous collections of small molecules. These millions of compounds are tested in the smallest possible scale. This approach is often limited by economic and physical factors and the expected success can usually only be reached after years of intense work.

The structural diversity of larger peptides, proteins and antibodies is orders of magnitude larger than the traditional drug compounds. It is impossible to generate all possible variants of these molecules with traditional chemical approaches. But with modern synthetic methods we can create complex gene mixtures, which are introduced in a special way into bacteriophage (virus of bacteria). Each bacteriophage then carries a gene coding for an individual peptide/protein variant and the gene product is displayed as one or multiple copies on the surface of the bacteriophage particle, hence the method’s name phage display. It is no problem to generate gene mixtures of billions of variants. It requires some larger effort to generate the corresponding bacteriophage mixtures by cloning the DNA, without losing the original complexity. Finally one milliliter can contain several billion variants of these bacteriophage. By binding to a target molecule individual phage can be fished from the original mixture, reamplified by growth in bacteria and enriched by repeating this step several times. It resembles the work of a gold-digger separating gold particles from the other stones and sand, and he would probably be glad to amplify the nuggets. Still, this comparison gave the name “panning” to the selection procedure in phage display.

The disadvantage of these libraries is still that they cover only a very tiny portion of all possible variants of a peptide. Ten amino acids, i.e. the size of small peptide hormones, give 2010 variants. These are 1013 possible sequences, which for practical and theoretical reasons cannot covered by a standard peptide phage library. We are therefore applying new methods:

  • (Re-)Combinatorial phage display with peptides
  • Next generation libraries

(Re-)Combinatorial phage display with peptides

In the evolution the fastest way to new proteins has been the recombination of genes and not random changes by individual mutations. This was already recognized many years ago by many researchers. Scientists like W.P.C. Stemmer (earlier at Affimax Inc.) introduced more or less directed methods for gene recombination. A procedure for the directed recombination of peptide genes was developed by Prof. John Collins and further developed at Cosmix GmbH (Brunswick. Germany) by Dr. Szardenings and colleagues to create methods also feasible for antibodies and proteins. These libraries do not only allow the generation of unusually large starting libraries (peptide libraries with > 1010 sequences) but also the exploration of a sequence space beyond 1015 sequences by incremental recombination. This allows at least theoretically to identify any variant of a peptide with 10 amino acids. Practical results indeed show homologies of 9-10 amino acid positions within selected binders.

(Re-)Combinatorial phage display with antibodies

Recombining antibody gene libraries requires special expression vectors and designated gene fragment cloning. We can use these methods for cDNA libraries as well as for naïve human Fab libraries with methods developed in Braunschweig earlier.

Next generation peptide phage display libraries

At Fraunhofer IZI we are turning decades of experience in the handling of peptide libraries into new tools and novel types of libraries. Phagemid vectors have been further optimized for cloning gene fragments and they allow further improvement in the selection efficacy. We have access to the best available DNA synthesis technologies of our partners and have abolished sequence bias and reduced sequence variability through intelligent design. These libraries are able to solve problems, which have not been tackled successfully by this technology.

Epitope / mimotope mapping

Peptide Phage Display libraries of the size available at Fraunhofer IZI allow a rapid and reliable identification of epitopes and mimotopes of monoclonal as well as polyclonal antibodies. The identified peptides allow a rapid identification of the exact binding site at the antigen. They can also be used in serological assays and for the purification of recombinant antibodies or specific antibodies from polyclonal mixtures.

This rapid routine work is very helpful in the early stages of antibody development as well as for the precise understanding of the spectrum of peptides bound by the individual antibody.

Equipment

All in all, the Fraunhofer IZI has excellent basic equipment that is employed in the highly diverse projects of this unit. The following devices are of particular significance for the Ligand Development Unit:

  • ÄKTA Avant – For process development for optimal protein purification from complex mixtures, e.g. antibodies from sera or allergens from raw materials, our group is using one of the best available chromatography systems.
  • Bioreactors – For the cultivation of large quantities of E.coli for accommodating phage display libraries in excellent quality.
  • Cell sorter / FACS – For the conduct of selection experiments in living cells and the validation of results.
  • BIACORE® – For the direct measurement of protein-protein interactions as part of our projects.

Epitope / mimotope mapping

Peptide Phage Display libraries of the size available at Fraunhofer IZI allow a rapid and reliable identification of epitopes and mimotopes of monoclonal as well as polyclonal antibodies. The identified peptides allow a rapid identification of the exact binding site on the antigen. They can also be used in serological assays and for the purification of recombinant antibodies or specific antibodies from polyclonal mixtures.

This rapid routine work is very helpful in the early stages of antibody development as well as for the precise understanding of the spectrum of peptides bound by the individual antibody.

ZellFix – Enzymatic treatment replacing plasma: Biocompatible polymer surfaces

In a joint project with Fraunhofer IFAM (Bremen) we discovered an enzyme based reaction for covalent of peptide ligands to cell surface receptors. We initiated the Fraunhofer society sponsored project ZELLFIX to use a similar reaction to improve the attachment of cells to polymer surfaces. It is sometimes very difficult to achieve the attachment of cells to the surfaces of culture dishes. Usually polystyrene surfaces are treated with plasma which can result in toxic byproducts. With this new procedure we improve cell binding through peptides or proteins to the polymer surface or by direct binding of these proteins to the polymers just like to plasma activated surfaces. Within ZELLFIX we managed to obtain the expected results. Even local spots can be modified to enhance cell binding. Finally we have developed an enzymatic method that provides the same results as the plasma treatment of polymers without toxic side effects. This method is presently being tested with several partners from the industry and other Fraunhofer and research institutes. We have identified multiple applications in biological and medical areas and beyond.

LowAllergen – Reduktion der Allergenität von Lebensmitteln

The LowAllergen project is led by the food and nutrition specialists from the Fraunhofer IVV in Freising. They are collaborating with the Fraunhofer IZI, the Fraunhofer ITEM (Hanover), Fraunhofer IME (Aachen) and the dermatology department at the university hospital in Leipzig. LowAllergen will generate a basis for the production of food with reduced allergenic properties. Substances with allergenic potential in food mean a restrictions if not a threat for the life of allergy sufferers. The increased and often unnoticed usage of such substances leads to a growing exposition of consumers and increased risk for healthy people to acquire allergies.

The reduction of the allergenic potential of food ingredients could be a significant contribution to improve food safety. The principal requirements for this are processes suitable for the reduction of the allergenic features of food ingredients as well as detection systems, which are able to measure the allergenic potential safely and reproducibly.

Existing test systems for the application in the food industries are well suitable for the basic detection of allergenic components but they cannot reveal any information about the actual specific allergenicity. In addition, the production of hypoallergenic protein ingredients has so far relied on the usage inhomogeneous and difficult to obtain sera from allergic patients. The development of hypoallergenic food is therefore as sophisticated as unspecific and remains restricted to special groups of products, e.g. baby food. The main strategy for allergic patients and such being at risk acquiring allergies is the consequent omission of food with potentially allergenic substances. In this project we will be first to develop and test new diagnostic and food engineering technologies. With the soy bean as an example we want to detect and reduce the allergenic potential of food ingredients.

The molecular structures (epitopes), which are recognized by the allergy causing types of antibodies, will not only be investigated per protein. The project will characterize them in more detail on the molecular level of amino acids with three complementary approaches. This will enable the development of antibody based assays for each epitope as well as new concepts for the processes to generate hypoallergenic food ingredients. This requires special chemical, physical, and enzymatic treatments for the reduction of the allergenic potential while maintaining the sensory and functional characteristics of ingredients.

This project is supported by the Fraunhofer-Gesellschaft for three years with several million euros. The Ligand Development Unit is partially responsible for the purification of proteins and primarily the identification of epitopes and mimotopes of the allergic soy proteins.

New peptide technologies

The development of novel peptide ligands is not completed with the discovery of ligands for receptors. Together with partners we are actively testing new technologies. Despite, high specificity peptides are often suffering a relatively low affinity. We have developed procedures to couple peptides directly to their binding partners on cell surfaces. This is sufficient to use peptides in FACS experiments. Another approach comprises attempts to couple several peptides to larger structures. We are testing nanoparticles and polymers as well as the usage of peptides in proteomics and FACS analysis.

Peptide mapping and sera

Allergies are excessive defense responses of the immune system to otherwise harmless environmental substances, e.g. pollen.
© Photo MEV-Verlag, Germany

Allergies are excessive defense responses of the immune system to otherwise harmless environmental substances, e.g. pollen.

With the form of phage display applied at the Fraunhofer IZI we are able to identify peptide sequences binding specifically to antibodies from up to 1015 peptides. From many slightly different sequences discovered in such experiments we do not only identify the part of a protein that is recognized by a monoclonal antibody but we also learn about the allowed variability of the original sequence. From so many binders it is easy to select those that are most suitable for different applications. In addition, the cross reactivity of antibodies as well as their suitability for the recognition of mutant proteins can be predicted.

Presently, we use this technology to investigate sera from patients with allergies. This may provide novel opportunities to diagnose allergies at a very early stage. This would allow, in particular for children, an early treatment to prevent the onset of severe allergic symptoms.

  • Actualiti
  • AJ Roboscreen GmbH
  • AptaIT GmbH
  • ASA Spezialenzyme GmbH
  • Baxter Oncology GmbH
  • CNUHH (Chonnam National University Hospital Hwasun)
  • EUROIMMUN Medizinische Labordiagnostika AG
  • Fraunhofer Institute for Biomedical Engineering IBMT
  • Fraunhofer Institute for Ceramic Technologies and System IKTS
  • Fraunhofer Institute for Electron Beam and Plasma Technology FEP
  • Fraunhofer Institute for Manufacturing Technology and Advanced Materials IFAM
  • Fraunhofer Institute for Molecular Biology and Applied Ecology IME
  • Fraunhofer Institute for Process Engineering and Packaging IVV
  • Fraunhofer Institute for Toxicology and Experimental Medicine ITEM
  • Furtwangen University (HFU), Campus Schwenningen
  • Helmholtz Centre for Environmental Research UFZ
  • ibidi GmbH
  • PolyBatics, Ltd.
  • PolyQuant GmbH
  • Prima BioMed Ltd.
  • Radboud University Nijmegen
  • University Hospital Leipzig, Clinic and Polyclinic for Dermatology, Venerology and Allergology, Research Group Skin
  • University Hospital Leipzig, Clinic for Urology
  • University Hospital Regensburg
  • University of Leipzig, Translational Center for Regenerative Medicine (TRM) Leipzig