Fraunhofer Institute for Cell Therapy and Immunology IZIPreclinical Validation
Drug Repurposing
It takes a lot of time and money to develop new drugs. Drug candidates are identified for a particular indication based on findings from foundational research, which can take decades to evolve. These are first examined in great detail as to their efficacy and, above all, safety in various cell culture and animal models during the preclinical development phase. Only the few drug candidates whose benefit-risk profile justify being tested on human subjects make it into clinical development, where they are tested first on healthy subjects (phase I), then in smaller patient cohorts (phase II) and finally in trials involving as many patients as possible (phase III) before an application can be made for approval in the indication concerned.
In the case of drug repurposing, medicines which are already approved are tested for their suitability and possible applications in other indications. A prerequisite here is a thorough understanding of the disease mechanisms at a molecular and cellular level on the one hand and the precise characterization of the medicine’s principles of action on the other. If corresponding parallels are identified in different indications, it is worth taking development steps to expand the medicine’s area of application. This approach to medicine development is extremely efficient and takes much less time and effort as it can build on existing data and observations.
The REMEDI4ALL project plans to develop a Europe-wide competence platform to support European research institutes with drug repurposing in the future.
Fraunhofer IZI is contributing its competences in infection research to the consortium and will investigate the suitability of a previously approved oncology medicine for its application in the case of COVID-19 diseases.
The REMEDI4ALL consortium comprises 24 partners and will be supported over a number of years with funding in the value of 23 million euros from the European Union’s Horizon Europe research and innovation program under grant agreement ID 101057442.
MATURE NK – MAnufacturing TUmor-REactive Natural Killer cells
The MATURE-NK project provides future-oriented training in translational research in order to close the gap between basic research and the applied development of new cell-based therapeutic products by the biotech industry and their clinical application in incurable diseases. This is done using the example of activated genetically modified natural killer cells (NK cells), which are classified as advanced therapy medicinal products (ATMPs). Overall, fourteen partners from nine countries participate in the project which is sponsored by the EU under its Horizon 2020 programme.
Project coordination
Fraunhofer IZI
Grant Agreement No
765104
RSV Protect
RSV-F protein enables the binding of the virus to the host cell and fusion with its plasma membrane, therefore infecting the cell. Molecular inhibitors developed in the RSV-Protect project lock RSV-F in its inactive configuration, preventing the virus infection of host cells.
While mostly harmless in healthy adults, Respiratory Syncytial Virus (RSV) is a severe risk to the lives of infants, the elderly, or patients with weakened immune systems. Additionally, RSV disproportionately impacts heavily populated, developing countries such as India or China, and is an increasing risk globally, causing an estimated number of more than 200,000 infant deaths yearly. While risk factors such as pollution or immune deficiency are known, true therapeutic and preventative measures are nearly non-existent. No RSV vaccine has cleared clinical trials despite 5 decades of failed efforts, and the only prophylactic on the market – Palivizumab (Synagis®) – is an expensive monoclonal antibody that is only used in the most high-risk patients in wealthy countries.
The RSV Protect team (Dr. Thomas Grunwald, Dr. Mirko Buchholz, Dr. David M Smith) has been working since 2016 to develop small molecules and synthetic biologics as effective, inexpensive lead compounds to inhibit the virus’s ability to enter and infect host cells. By creating compounds that bind to the receptors on the surface of the virus that are responsible for adhering to and fusing with host cells in lungs, the overall infectivity of the virus can be eliminated.
Using this strategy, several small peptides shorter than 15 amino acids were produced, which were targeted to “lock” the main fusion protein of the virus, RSV-F, in a configuration that leaves it unable to inject its genetic material into the host cells. These peptides, which are more than 100 times smaller than a typical antibody, were effective in micromolar concentrations at preventing RSV infections in both cellular model systems and living animals. Replacing essential amino acids in the sequence with non-natural, synthetic derivatives ensures their usability, increases their efficacy, and reduces the risk of unwanted side-effects.
Similar to most viruses, the numerous surface receptors on RSV work together as a deadly team, where 3 identical protein units are geometrically arranged in trimeric form to cooperatively enhance fusion with host cells. Therefore, the principle of multivalence was utilized to arrange 3 of the RSV-blocking peptides on a small structural scaffold made from DNA strands, geometrically complementary to the virus proteins. Presentation on this trivalent, DNA-peptide conjugate was successful in enhancing the protective ability of the peptides 500-fold in cell models, equivalent to levels seen for the only commercially available prophylactic, Palivizumab. In a second approach to address RSV infection, a panel of novel small molecule inhibitors that interact with viral particles were designed and proved to be effective in inhibiting RSV infection in cell culture models at low nanomolar concentrations. This work has paved the way for a patent application and a broader, currently ongoing pre-clinical study.
TheraVision – platform technology for the development, production and testing of oncolytic herpes simplex viruses for tumor therapy of lung cancer
HSV-induced plaque formation in tumor tissue sections of mice after intratumoral virus therapy. DAPI (blue) depicts the cell nucleus and HSV (green) the viral proteins in infected lung cancer cells.
Viruses are able to penetrate cells and produce both foreign and viral proteins. Afterwards they multiply in order to kill the infected cells. Due to the fact that oncolytic (cancer-destroying) viruses selectively kill tumor cells, they have become an emerging hope in cancer therapy. The Herpes Simplex Virus (HSV) is one of those viruses.
We aimed to increase the efficacy of the oncolytic activity of an HSV-1 based vector by genetically introducing different genes for immune modulation and for targeting the optimization of tumor therapy. Thus, the virus-mediated oncolysis is combined with immunotherapy in one virus vector and an effective destruction of tumors as well as metastases is possible. The objective of the project ”TheraVision” is to establish a broadly applicable platform technology based on HSV for combinatorial oncolytic virus immunotherapy.
As a proof of concept, an oncolytic virus was developed for the therapy of non-small cell lung cancer (NSCLC), whereby the Fraunhofer IZI established the appropriate mouse model. The cells of the lung cancer tumors express the reporter Firefly-Luciferase in order to be detected by a highly sensitive light camera in vivo. The tumors showed a significant increase in bioluminescence intensity, which directly corresponds with an increase in size. The treatment of these tumors with an attenuated and neurotoxicity-deleted HSV vector led to a significant reduction in tumor growth and bioluminescence intensity compared to an untreated control group. Furthermore, the attenuated vector with deleted neurotoxicity genes caused a significant reduction of the viral load in the brain in comparison to an unmodified HSV vector.
To analyze the immunotherapeutic activity of novel functionalized oncolytic viruses, this tumor model must still be transferred into humanized mice with the appropriate human tumor in an allogenic immune environment that mimics more the natural situation. Finally, a broadly applicable platform technology to test the efficacy of virus vector and immune therapies or combinations will be available for future endeavors.
Vaccination against asthma – MucoRSV
Approximately 235 million people worldwide suffer from asthma. Among children, asthma is the most widespread chronic disease, resulting in a severe impact on their quality of life. It has been shown that children who were infected with respiratory syncytial virus (RSV) in infancy or early childhood are more likely to develop asthma later. Glucocorticoids and beta-2 sympathomimetics are used as the standard therapy for asthma, but this treatment is often not sufficient in severe cases and can lead to significant side effects when used for long periods of time. An alternative treatment is therefore urgently required.
Due to the correlation between repeated RSV infections in early childhood and the development of asthma, preventive measures present the most effective mode of action. The MucoRSV project investigates whether a vaccination against RSV can protect from repeated infections and thus prevent asthma.
Currently there are no approved vaccines against RSV. Within the scope of this project, different vaccines will be tested and administered mucosally, i.e. via nasal spray or inhalation. One candidate is a killed vaccine containing virus particles which were inactivated by low-energy electron irradiation. The virus will be packaged in nanoparticles in order to enable an increased uptake via the mucosal membranes. Another vaccine consists of DNA coding for RSV-F, the most important RSV antigen. This DNA is packaged in non-human papillomavirus capsids and applied using these viral vectors.
A vaccine against RSV could thus not only prevent the viral disease itself, but also reduce serious chronic consequences such as asthma.
Efficacy of novel helicase-primase based therapy for human Herpes Simplex Virus (HSV)
Currently, human Herpes Simplex Virus (HSV) infection affects about 82 % of Germany’s population. The pathogen is categorized into two types, which differ in their predilection for the site of infection. HSV type 1 (HSV-1) is associated with a wide range of clinical manifestations including cold sores. In contrast, HSV type 2 (HSV-2) is linked to genital herpes. Both types are able to develop severe disease progression leading to fatal Herpes Simplex Encephalitis (inflammation of the brain).
Until now nucleoside analogues, such as Acyclovir and Valacyclovir, are still the treatment of choice for HSV infections. However, due to the existence of nucleoside-resistant viral strains alternative therapies are needed. Recently, this alternative has been represented by helicase-primase inhibitors (HPIs), which use a novel mechanism of action to inhibit viral replication. In a drug development trial we analyzed the antiviral efficacy of new drug candidates for the treatment of HSV infections in a mouse model.
Despite the lower dose, a better outcome in clinical parameters using HPI therapy in comparison to Valacyclovir control was observed. Toxic side effects were not detected during the monitoring period of 3 weeks post infection. The subsequent analysis showed that treated animals harbor a significantly lower viral load compared with placebo animals.
This project showed that treatment with the new HPI candidates can significantly reduce or prevent clinical symptoms. HPI‘s are at least one order of magnitude more potent and efficacious compared to Valacyclovir. Thus, candidates of the new drug class are promising inhibitors of HSV infections in vivo and should be translated into clinical trials.
Establishment of a rabbit model for the propofol infusion syndrome
The use of anesthetics can lead to unwanted and sometimes life-threatening side effects. One of the most commonly used anesthetics is propofol. During the use of propofol for longterm anesthesia and during the anesthesia of children, propofol can cause a rare but fatal side effect, the propofol infusion syndrome (PRIS). PRIS is a symptom complex that can lead to severe disorders of the cardiovascular system, kidney failure, a drastic reduction in blood pH (lactic acidosis) as well as the resolution of striated muscles (rhabdomyolysis). In most cases these disorders lead to fatal multi-organ failure.
In cooperation with a large industrial partner, a model system in the rabbit was established to investigate PRIS. Based on a publication from 2007 (Ypsilantis et al., 2007), a pilot study was carried out at the Fraunhofer IZI to adapt the described model to the questions. After intubation and successful initiation of propofol anesthesia, it was possible to keep the animals stable under anesthesia for a period of up to 48 hours. Meanwhile, the oxygen and carbon dioxide levels as well as the acid-base balance of the animals were closely monitored. In addition, reflex tests were carried out to ensure a safe depth of anesthesia and the heart functions as well as the temperature were monitored at regular intervals. The successful development of PRIS resulted in an irreversible lethal multiple organ failure. After each experiment, all organs of the animals were removed, fixated and stained for histological examinations. Furthermore, mass spectroscopic analyses of the bile fluid and detailed examinations of the blood work were performed. The pathological findings of most animals were normal.
However, a new biomarker could already be identified in this pilot study, which may be of possible use for monitoring anesthetized patients. This biomarker will be validated in human blood samples over the next few months.
Non-human papilloma pseudoviruses for DNA delivery in vitro and in vivo
DNA vaccines are gaining popularity due to their inexpensive production and good stability even at room temperature. While it was possible to overcome the initially unsatisfyingly low antigen-specific antibody response in larger animals and humans by use of electroporation, which greatly increases the cellular uptake of the DNA, this method is comparatively laborious and painful for the vaccinee. Novel delivery methods are thus necessary.
Being the DNA delivery specialists that viruses are, pseudoviruses (PsVs), which package the vaccine-plasmid inside their capsid, can mediate the delivery of the vaccine and ensure the efficient shuttling of the DNA vaccine into the cells. Different animal papilloma viruses were detected and analyzed for their ability to form PsV particles, package DNA in form of a reporter plasmid and transduce cells in vitro. While most of the tested non-human papilloma viruses bearly showed a transfer of DNA in vitro, two candidates – papillomaviruses that normally infect the puma (PcPV1) and the macaque (MfPV11) – transduced especially effectively. PcPV1 and MfPV11 PsVs were therefore studied further in in-vivo experiments. Both candidates mediated the transduction of a luciferase reporter plasmid after intramuscular application in mice, leading to the expression of firefly luciferase. This expression lasted several weeks after injecting PcPV1 PsVs. Further, in a vaccination including intramuscular and intranasal application, it was tested whether the papilloma PsV mediated the delivery of a DNA vaccine against the respiratory syncytial virus (RSV) in mice. Finally, the mice were infected with infectious RSV and the viral load was quantified. The application of PcPV1 and MfPV11 PsVs carrying a plasmid coding for RSV-F led to a significantly reduced viral load in the lungs of the vaccinated mice upon challenge.
Human papilloma PsVs have successfully been used for gene delivery in the past, but have limitations due to vector immunity, which would occur in all individuals that have previously been exposed to these viruses.
The project results show that non-human papilloma viruses have the potential of being promising gene delivery vectors and present a vaccine platform for intramuscular or mucosal application.
Project Manager
Dr. Thomas Grunwald
Development of a vaccine against Respiratory Syncytial Virus
Human respiratory syncytial virus (RSV) usually only leads to mild complaints such as cold, cough or hoarseness. However, this virus is the greatest infectiological issue affecting premature babies and babies younger than six months old. The virus exhibits a severe disease progression among these infants which often has to be treated in hospital. At present, there is neither a therapy nor a vaccine which effectively protects against the RSV infection. A test among children in the 1960s involving a chemically inactive vaccination had a contrary effect: The RSV infection was seen to strengthen when contracted naturally.
Genetic vaccines are currently being tested to be developed for a series of indications. An innovative vaccination method was investigated with the aid of genetic vaccines as part of a major study. This involved a circular DNA molecule being administered as a vaccine, followed by a pain-free vaccination sprayed into the throat. This combination of vaccinations demonstrated surprisingly complete protection against infection with the virus. These promising, preclinical successes are now to be further tested in humans and expanded to other vaccine candidates.