Therapy Validation

Staff of the department of therapy validation
© Photo Fraunhofer IZI

The department covers the following topics:

1) Planning and execution of preclinical efficacy and safety studies for new drug candidates (especially ATMPs) and medical devices (ISO 10993) under GLP or GLP-analogous conditions. This includes the development and validation of suitable in-vitro and in-vivo models.

2) Developing and optimising methods and processes for the diagnostic analysis of secretory and cellular protein biomarkers. This includes the development and production of specific monoclonal antibodies for detecting these biomarkers, as well as the development and validation of the corresponding diagnostic assays (e.g. ELISA, Luminex®, multicolour flow cytometry).

3) Identifying and validating new protein biomarkers for diagnosis and therapy of chronic inflammatory and tumour diseases, as well as for the sector of veterinary medicine / animal husbandry.

4) Developing human therapeutic monoclonal antibodies for the treatment of tumour and autoimmune diseases, as well as passive vaccines against bacterial toxins and pathogenic viruses, and their advancement to drug candidates.

5) Small-scale GMP production of therapeutic monoclonal antibodies for preclinical animal studies and clinical trials (Phase I and II).

  • GLP certified since 2009
  • Testing Category 9: Immuntoxicity / immunogenity in vitro / in vivo
  • Safety tests for ATMPs – biodistribution, tumorigenicity and immungenity / immunotoxicity

Development and production of human monoclonal antibodies in a humanized mouse model

This project focuses on developing and producing new kinds of fully humanized monoclonal antibodies for the treatment of tumor diseases (pilot project: triple-negative breast cancer). The development and production of such antibodies can be divided into several steps: The first step focuses on establishing a humanized mouse model which can be used to generate human monoclonal antibodies against known and yet unknown tumor antigens. A variety of immunization strategies are developed for this purpose in immune-deficient NSG or NOG mice which have developed a humanized immune system after the transfusion of human haematopoietic stem cells from cord blood. Tumor-specific human monoclonal antibodies are generated by fusing tumor-specific human B cells from these humanized mice with human plasmocytoma cells, and selected using suitable strategies. Selected candidates are finally tested in an established tumor mouse model in preclinical studies and modified if required (e.g. antibody-drug conjugates), in order to continue to improve therapeutic efficacy.

Project managers
Dr. Jörg Lehmann / Dr. Ulla Schwertassek

Establishing a flexible small-scale GMP manufacturing facility for therapeutic monoclonal antibodies – creating a technology platform

The increasing number of therapeutic monoclonal antibody (mAb) candidates under preclinical and clinical development requires flexible and efficient GMP production of such drug candidates. Large scale industrial GMP-manufacturing facilities are often not able to provide small quantities of test samples for late preclinical animal studies and clinical phase I and II trials.

This niche can be filled by small-scale GMP facilities established by non-profit institutions such as the Fraunhofer-Gesellschaft. The Fraunhofer Institute for Cell Therapy and Immunology IZI in Leipzig has established such a GMP facility. An initial pilot process for the GMP production of a monoclonal antibody is currently in progress.

The GMP clean room facility has an area of 180 m2 and covers the entire production process of a monoclonal antibody (upstream, downstream, aseptic filling). Several portable devices and the use of single-use materials will allow easy implementation of process changes and a quick product change-over. The maximal production volume of the facility amounts to 200 L and enables the production of 5–10 mg mAb per batch, depending on the antibody. Promising drug candidates can therefore be transferred from research to clinical development in a client-specific process.

Project manager
Dr. Jörg Lehmann (comm.)

Companion diagnostic for a clinical trial (Phase III) to evaluate the immunotherapeutic DCVax®-L for the treatment of Glioblastoma Multiforme

DCVax®-L (Northwest Biotherapeutics GmbH) is a therapeutic agent based on endogenous immune cells that will be used for treating Glioblastoma Multiforme after standard therapy. The treatment is based on immune cells of the peripheral blood which are differentiated in vitro to specific antigen-presenting cells, dendritic cells (DCs). The immature DCs are subsequently brought into contact with a tumor lysate, and thereby "trained" to recognize tumor cells. The maturated DCs are injected into the patient and can now trigger an immune response against remaining tumor cells. As part of a clinical trial (Phase III) to test the efficacy of DCVax®-L, cells from patients' peripheral blood (peripheral blood mononuclear cells; PBMCs) are isolated, quality controlled and cryopreserved for long-term storage. The aim of the project is to develop suitable diagnostic assays which can demonstrate a functional immune response in patients after treatment with DCVax®-L to support clinical data.

Project manager
Dr. Ulla Schwertassek

Development of a small animal model and in-vivo studies for evaluating the vitality of human cryopreserved adipose tissue transplants using modern imaging procedures

Autologous adipose tissue is used in reconstructive and plastic-aesthetic surgery as an ideal filling material for tissue augmentation. The vitality of adipocytes is an elementary prerequisite for successful engraftment of adipose tissue at the site of transplantation. The goal of the project includes obtaining the manufacturing authorization for the cryotechnology procedure for preparing a vital and safe-for-human-use adipose tissue product by the project partner Vita 34 AG. To evaluate the safety and efficacy of the novel adipose tissue product, comprehensive in-vitro and in-vivo studies for testing the vitality and functionality of human fat cells obtained by water-jet assisted liposuction (WAL) are performed in the frame of a publicly funded AiF project as part of the ZIM-KF program. Magnetic resonance imaging (MRI) scans are performed to examine the characteristics of cryopreserved fat cells, previously treated with different cryoprotectants, in immune-deficient mice. Furthermore, the xenotransplants are macroscopically and histologically examined in terms of vascularization, vitality and signs of possible degradation or rejection processes.

Project managers
Dr. Jörg Lehmann, Dr. Anke Hoffmann (Vita 34)

Preclinical animal models for the development of new IBD therapies

Inflammatory bowel diseases (IBDs) are multifactorial diseases of the gastrointestinal tract, the incidence and prevalence of which have been rising constantly in industrial and newly industrialized countries. The etiology of IBD is unknown but is discussed to be a combination of genetic predisposition, environmental factors and a dysregulated immune response to the gut microbiota. Current therapeutic approaches, such as immunosuppressives and biologics, often show severe side effects. Thus, there is an urgent need for the development of new therapies based on improved knowledge of the etiological factors of IBDs.

The goal of the in-house research project is to develop and characterize different IBD animal models for testing new therapies and elucidating mechanisms of pathogenesis. Thus, a model of chronic DSS colitis was established which shows typical symptoms such as weight loss and chronic bloody diarrhea. Moreover, the colon tissue of the animals features a continuous immune reaction with resulting ulcerations. In addition, the bacteria-induced chronic colitis has been established as an alternative animal model for IBDs which mainly focuses on the role of the microbiota in disease pathogenesis and / or as a target for novel treatment strategies. Both models can be used for preclinical studies as well as elucidating IBD pathogenesis and developing new therapies; in the latter case, the focus is currently on evaluating the therapeutic potential of phytochemicals.

We are also doing a functional analysis of the gut microbiota in the chronic DSS colitis model using BALB/c mice, together with the Helmholtz Centre for Environmental Research UFZ. The goal of this project is to better understand the interactions of the microbiota and the mucosal immune system in IBDs.

Project manager
Dr. Ulla Schwertassek

Immunomodulatory role of the arylhydrocarbon receptor (AhR)

Within the frame of this project, the immunomodulatory effect of the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) was characterized in collaboration with the Federal Institute for Risk Assessment (BfR). While the carcinogenic effect of BaP has been known for decades, little attention has been paid so far to the immunomodulatory effect of BaP, particularly of concentrations classified as uncritical. The immunomodulatory effects as well as the decomposition of BaP are mainly regulated by its binding to the arylhydrocarbon receptor (AhR) with subsequent receptor activation. A murine salmonella infection model is used to investigate the impact of BaP on an ongoing immune response. Analysis of the underlying mechanisms is carried out using suitable in-vitro models. Previous investigations have mainly been restricted to the maturation and functional activation of murine macrophages. By using AhR-deficient mice or cells from these animals, the AhR dependence of the observed effects could be demonstrated. Interestingly, the initial findings in our model showed an AhR-dependent immunomodulatory effect of BaP which had a positive impact on the course of disease. In further experiments, selected non-toxic AhR ligands are analyzed for their therapeutic potential in different disease models.

Project managers
Dr. Jörg Lehmann / Sina Riemschneider

LowAllergen / FoodAllergen

The frequency of newly developed food allergies (incidence) increases worldwide. At the same time, a rise in non-conventional food ingredients – particularly plant-based ingredients – has been registered, following the modern trend of resource-conscious food. This trend has also been noted by European directives with the purpose of making consumers aware of the risk of allergies as well as protecting them from the consequences of allergies (LMIV). As a result, the demand for reliable methods to analyze allergenic ingredients as well as for the technology to reduce allergenic ingredients has grown significantly over the past few years.

The "LowAllergen" project and its follow-up project, "FoodAllergen", are concerned with plant-based allergenic food ingredients. One part of the project is geared towards changing proteins to the extent that their allergenic potential is decreased. The requirements for this are innovative technologies for modifying proteins which are developed in our partner institute Fraunhofer IVV. As part of the "Food Allergen" project, the Protein Biomarker Unit is responsible for developing antibody-based test systems which enable a quantitative analysis of allergenic ingredients and reveal any remaining allergenic potential after application of allergen-reducing procedures. Using soy as an example, in the "LowAllergen" project, a decrease of allergenic components could be observed as a result of applying certain microorganisms in fermentation processes and hydrolysis using high pressure. In the "FoodAllergen" project, the processes and analytical methods will be expanded to other plant-based protein ingredients, in order to put technologies for modifying allergens as well as analytical methods for detecting successful reduction of allergens into practice as soon as possible.

Project managers
Dr. Jörg Lehmann / Dr. Elke Ueberham

Development of new on-farm processes for testing health and fertility in German Holstein cattle

The joint project aims towards creating new innovative processes for testing the performance of German Holstein cattle with respect to fertility and health, to be recorded directly on the breeding farms (on-farm). Breeding processes were established based on previously defined key characteristics. The goal of the project is to determine and use breeding-related parameters for fat mobilization dynamics, female fertility and health characteristics, based on a partially automatic infrared image analysis for inflammatory markers on limbs and udders, as well as a partially automatic analysis of immunological parameters in the milk. The part of the project done at Fraunhofer IZI involves selecting immunological parameters in 10 animals, validating these immunological parameters in 100 animals, and developing and validating laboratory tests for immunological parameters for bulk investigation and field validation of the tests on 3 selected farms. In the meantime, a cellular biomarker (CD25 in neutrophilic granulocytes) and two protein biomarkers have been identified and validated. For both protein biomarkers, verification assays have been developed and are currently in the evaluation phase.

Project managers
Dr. Katharina Zoldan / Dr. Jörg Lehmann

Project "Bronchial Carcinoma" as part of the BMBF Program "Innovative Regional Growth Cores", "GC Potential" module

Every year, 50,000 people in Germany fall ill with lung cancer. Lung cancer is the third most frequently occurring type of cancer in Germany. The Leipzig-based GC Potential Bronchial Carcinoma is therefore working on a technologically innovative solution for early lung cancer diagnosis.

The currently available examination methods, such as x-rays and bronchoscopy, are associated with considerable burden for the patients. Analyzing specific protein biomarkers for bronchial carcinoma in breath condensation represents an alternative to these conventional diagnostic procedures. As part of the GC Potential Initiative, a device platform for routine application of this method in laboratory diagnostics should be developed, and the prototype of such a device should be tested. The project is carried out in collaboration with the University of Leipzig, Institute for Clinical Immunology, along with two regional companies, Compart Umwelttechnik GmbH in Weißenfels, and GESA Automation GmbH in Teuchern.

Project managers
Dr. Jörg Lehmann / Norbert Lidzba

Completed reference projects

  • Study of the therapeutic efficacy of a transglutaminase inhibitor in an acute DSS colitis model
  • Study of the therapeutic efficacy of an ion-based immunomodulator in a chronic DSS colitis model
  • Testing of a local bactericidal treatment after tick bites in a Borrelia infection model
  • Analyzing the mechanisms of action and identifying clinically relevant effects of colocynth and sage-based phytopharmaceuticals
  • Companion diagnostics to determine the immune status of patients with acute diarrhea after treatment with Arhama® Tinktur N
  • Companion diagnostics to determine Mucin-1 reactive antibodies and immune cells in patients' blood after treatment with CVac™

GLP studies

  • Differential protein-biochemical and serological analysis of UV-irradiated and non-irradiated canine thrombocyte concentrates
  • Immunotoxicological in-vitro study of the plant-based immunostimulant mistellectin with intestinal epithelial cells
  • Safety and efficacy studies of MSC-generated cartilage products after autologous implantation in a large animal model (sheep)
  • Safety and efficacy studies of MSC-generated chondrocyte spheroids after implantation in a large animal model (sheep)
  • Preclinical safety study for evaluating the biodistribution and tumorigenicity of tissue-engineered human chondrocyte spheroids in an NSG mouse model
  • Preclinical safety study for evaluating the biodistribution and tumorigenicity of tissue-engineered human MSC-MACT in an NSG mouse model

  • Lehmann J, Schulz RM, Sanzenbacher R. [Strategic considerations on the design and choice of animal models for non-clinical investigations of cell-based medicinal products]. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz. 2015 Nov;58(11-12):1215-24. DOI German.
  • Riemschneider S, Herzberg M, Lehmann J. Subtoxic Doses of Cadmium Modulate Inflammatory Properties of Murine RAW 264.7 Macrophages. Biomed Res Int. 2015;2015:295303. DOI Epub 2015 Aug 3. Free PMC Article
  • Zscharnack M, Krause C, Aust G, Thümmler C, Peinemann F, Keller T, Smink JJ, Holland H, Somerson JS, Knauer J, Schulz RM, Lehmann J. Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP). J Transl Med. 2015 May 20;13:160. DOI Free PMC Article
  • Lehmann J, Härtig W, Seidel A, Füldner C, Hobohm C, Grosche J, Krueger M, Michalski D. Inflammatory cell recruitment after experimental thromboembolic stroke in rats. Neuroscience. 2014 Aug 26. pii: S0306-4522(14)00699-X. DOI [Epub ahead of print]
  • Wege AK, Schmidt M, Ueberham E, Ponnath M, Ortmann O, Brockhoff G, Lehmann J. Co-transplantation of human hematopoietic stem cells and human breast cancer cells in NSG mice: a novel approach to generate tumor cell specific human antibodies. MAbs. 2014 Jul-Aug;6(4):968-77. DOI Epub 2014 May 8.
  • Zoldan K, Moellmer T, Schneider J, Fueldner C, Knauer J, Lehmann J. Increase of CD25 expression on bovine neutrophils correlates with disease severity in post-partum and early lactating dairy cows. Dev Comp Immunol. 2014 Aug 11;47(2):254-263. DOI [Epub ahead of print]
  • Fueldner C, Mittag A, Knauer J, Biskop M, Hepp P, Scholz R, Wagner U, Sack U, Emmrich F, Tárnok A, Lehmann J. Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry. Arthritis Res Ther. 2012 Jan 17;14(1):R8. DOI
  • Pohler P, Lehmann J, Veneruso V, Tomm J, von Bergen M, Lambrecht B, Kohn B, Weingart C, Müller TH, Seltsam A. Evaluation of the tolerability and immunogenicity of ultraviolet C-irradiated autologous platelets in a dog model. Transfusion. 2012 Nov;52(11):2414-26. DOI Epub 2012 Mar 8.
  • Knauer J, Krupka I, Fueldner C, Lehmann J, Straubinger RK. Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model. J Antimicrob Chemother. 2011 Dec;66(12):2814-22. DOI Epub 2011 Sep 15.
  • Lehmann I, Sack U, Lehmann J. Metal ions affecting the immune system. Met Ions Life Sci. 2011;8:157-85. Review.
  • Roesler U, Szabo I, Matthies C, Albrecht K, Leffler M, Scherer K, Nöckler K, Lehmann J, Methner U, Hensel A, Truyen U. Comparing validation of four ELISA-systems for detection of Salmonella derby- and Salmonella infantis-infected pigs. Berl Munch Tierarztl Wochenschr. 2011 Jul-Aug;124(7-8):265-71.
  • Schubert K, Polte T, Bönisch U, Schader S, Holtappels R, Hildebrandt G, Lehmann J, Simon JC, Anderegg U, Saalbach A. Thy-1 (CD90) regulates the extravasation of leukocytes during inflammation. Eur J Immunol. 2011 Mar;41(3):645-56. DOI Epub 2011 Jan 25.
  • Fricke S, Fricke C, Oelkrug C, Hilger N, Schönfelder U, Kamprad M, Lehmann J, Boltze J, Emmrich F, Sack U. Characterization of murine non-adherent bone marrow cells leading to recovery of endogenous hematopoiesis. Cell Mol Life Sci. 2010 Dec;67(23):4095-106. DOI Epub 2010 Jun 18.
  • Zoldan K, Arnold A, Stolzing A, Lehmann J. Automated harvest of induced pluripotent stem cell colonies and colony fractions using the cell separation robot CellCelector™. App. Note, Nature Methods, Online publication
  • Zoldan K, Arnold A, Stolzing A, Lehmann J. Visualizing of migration, interaction, proliferation or differentiation of cells in time lapse exposures using the cell separation robot CellCelector™. App. Note, Nature Methods, Online publication
  • Zoldan K, Füldner C, Schubert A, Lehmann J. Automated isolation of semi-adherent macrophage-like cells from a fibroblast contaminated culture using the cell separation robot CellCelector™. App. Note, Nature Methods, Online publication
  • Zoldan K, Knauer J, Lehmann J. Automated clonal selection of hybridoma colonies from methylcellulose-based, semi-solid medium using the CellCelector™. App. Note, Nature Methods, Online publication
  • Pfefferkorn C, Scholz U, Veneruso V, Nikolaus T, Madin K, Eichenlaub U, Schubert A, Lehmann J. Influence of Serum Deprivation on Adherence and Proliferation of Murine Mesenchymal Progenitor Cells Analysed with Roche´s xCELLigence System. Biochemica 2008 4:14-16
  • Treese C, Mittag A, Lange F, Tarnok A, Loesche A, Emmrich F, Lehmann J, Sack U. Characterization of fibroblasts responsible for cartilage destruction in arthritis. Cytometry A. 2008 Apr;73(4):351-60. DOI
  • Trepnau D, Ulrich E, Uhlig R, Lindner T, Selbitz HJ, Rösler U, Gabert J, Bergfeld U, Fehlhaber K, Brabetz W, Lehmann J. [Antibody response after immunization with a Salmonella Typhimurium live vaccine in dependence on the way of application]. Berl Munch Tierarztl Wochenschr. 2008 Sep-Oct;121(9-10):334-40. German.
  • Brumme S, Arnold T, Sigmarsson H, Lehmann J, Scholz HC, Hardt WD, Hensel A, Truyen U, Roesler U. Impact of Salmonella Typhimurium DT104 virulence factors invC and sseD on the onset, clinical course, colonization patterns and immune response of porcine salmonellosis. Vet Microbiol. 2007 Oct 6;124(3-4):274-85. Epub 2007 Apr 27.
  • Hemdan NY, Emmrich F, Faber S, Lehmann J, Sack U. Alterations of TH1/TH2 reactivity by heavy metals: possible consequences include induction of autoimmune diseases. Ann N Y Acad Sci. 2007 Aug;1109:129-37. Review.
  • Hemdan NY, Lehmann I, Wichmann G, Lehmann J, Emmrich F, Sack U. Immunomodulation by mercuric chloride in vitro: application of different cell activation pathways. Clin Exp Immunol. 2007 May;148(2):325-37. Epub 2007 Feb 14.
  • Ehlers J, Alt M, Trepnau D, Lehmann J. [Use of new immunoglobulin isotype-specific ELISA-systems to detect Salmonella infections in pigs]. Berl Munch Tierarztl Wochenschr. 2006 Nov-Dec;119(11-12):461-6. German.
  • Härtig W, Lehmann J, Stieler J, Singer D, Grosche J, Arendt T, Hoffmann R. Simultaneous detection of tau phospho-epitopes with haptenylated antibodies. Neuroreport. 2006 Jun 26;17(9):869-74.
  • Hemdan NY, Emmrich F, Sack U, Wichmann G, Lehmann J, Adham K, Lehmann I. The in vitro immune modulation by cadmium depends on the way of cell activation. Toxicology. 2006 May 1;222(1-2):37-45. Epub 2006 Feb 28.
  • Lehmann J, Springer S, Werner CE, Lindner T, Bellmann S, Straubinger RK, Selbitz HJ, Alber G. Immunity induced with a Salmonella enterica serovar Enteritidis live vaccine is regulated by Th1-cell-dependent cellular and humoral effector mechanisms in susceptible BALB/c mice. Vaccine. 2006 May 29;24(22):4779-93. Epub 2006 Mar 29.
  • Singer D, Lehmann J, Hanisch K, Härtig W, Hoffmann R. Neighbored phosphorylation sites as PHF-tau specific markers in Alzheimer's disease. Biochem Biophys Res Commun. 2006 Aug 4;346(3):819-28. Epub 2006 Jun 9.