Ligand Development

Establishing and validating a human lung organoid system

Premature babies make up one of the largest pediatric patient groups in Germany. Every tenth child is born prematurely and requires medical assistance. As the lungs are not fully developed, infant respiratory distress syndrome is the most common complication and cause of death among premature babies. The development of new therapies and active agents is therefore highly relevant to society and is the subject of intense research. 2D cell culture models and animal models are currently available as model systems for the development and preclinical testing of new therapies. The aim of this project is to develop three-dimensional lung organoids. As opposed to conventional cell cultures, organoids do a much better job of depicting the physiological situation and enable more specific statements to be made on influences and interactions during organogenesis. New therapies and active agents can therefore be developed in a much more targeted manner and the number of animal experiments required can also be reduced.

This project is co-financed by tax revenues on the basis of the budget approved by members of the Saxon state parliament.

Mapping of allergen epitopes in sera

Soybeans
© S.Piyaset – Fotolia

Soybeans.

Currently immunodiagnostics for diseases are usually based on proteins or extracts, which are directly obtained from the pathogenic organism or produced with biotechnological methods. The disadvantage of this approach is that variants, as they are for example commonly observed for the influenza virus, are difficult to distinguish. We have established protocols to exactly identify the antibody binding sites (epitopes) of patient antibodies, which are also directly applicable to sera. This allows a reliable identification of the pathogen, the causative antigen of allergies or many indications such as (auto)-immune or infectious disease as well as novel approaches for therapy and research.

Food allergies have been one focus of our research over many years. A steady increase of patients could be observed in the recent years. Skin prick tests diagnostics are only of limited use because many plant proteins are very similar in their architecture. Epitope-based diagnostics are most likely the only alternative to elaborate clinical investigations. These usually require venous blood collection, although only the provocation with the food is regarded as proof of an allergy, which has to be carried out under medical supervision in a clinic. An efficient diagnosis, appropriate treatment and adjustment of the food is therefore not available for many patients.

In an initial project we could show as an example for soy allergies that indeed only a few epitopes are sufficient for safely identifying sensitized persons as well as such with clinical symptoms. We are working to use these peptides in a simple test that could detect antibodies in a single drop of blood. Such a test would also be a model for tests on infectious diseases, vaccine efficacy or autoimmune diseases.

A particularly large project is now being funded by the Fraunhofer-Zukunftsstiftung, which is being followed nationally and internationally by allergologists with great interest. In cooperation with several other Fraunhofer Institutes and hospitals, the FoodAllergen project is working on a holistic approach to deal with food allergies. This also includes identifying allergens in foods and new processes of producing food ingredients with reduced allergenic potential. Meanwhile, the epitopes for a wide variety of plant allergens have been identified. An application in tests for patients is in preparation.

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.

 

Enzymatic treatment replacing plasma: Biocompatible polymer surfaces

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
© 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.