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Blocking Inhibition of T-cell Co-stimulation for Anti-tumour Therapy

Periodic Reporting for period 2 - BITCAT (Blocking Inhibition of T-cell Co-stimulation for Anti-tumour Therapy)

Période du rapport: 2019-05-08 au 2020-05-07

Tumors escape proper immune response by propagating immune suppression. Immunotherapy aims to enhance anti-tumor immunity. This highly relevant research area arose from advantages in immunology, genetics and oncology. Currently, immunotherapeutics are either protein or cell-based and promising approaches target immune checkpoint control, a mechanism crucial to anti-tumor T cell activity. Here, development of a new class of immunotherapeutics is proposed. To this aim, gene therapeutics will be designed to modulate expression of receptor genes involved in immune control and thus block tumor-mediated T cell inhibition. Of special interest will be the exploration of combinations of immune-oncological gene therapeutic techniques such as the CAR technique with short oligonucleotide-based approaches. During the course of the project a special focus could be established on glioblastoma (GBM), a highly malignant brain tumor with up to date very poor outcomes often resulting in patient’s death within 1-2 years following diagnosis. Currently, there are no curative treatments for this devastating disease available. Following extensive training in GBM models and establishment of close collaborations with clinical partners such as neurosurgery departments at host and beneficiary locations, within the project several novel drug candidates targeting GBM could be signed and tested for functionality, efficacy and toxicity in vitro and in vivo. Novel online datamining and in-lab biopanning approaches revealed additional potential drug candidates, which might also be suitable candidates targeting GBM.
The project’s premise is the development of novel genetics-based therapeutics, which are able to overcome tumor-associated escape mechanisms, particularly immune suppression in the tumor microenvironment. In a first step potential immune checkpoint candidates on the surface of selected tumor cells and various immune cell populations were characterized by gene expression studies and protein-targeted flow cytometry. Suitable in vitro test models could be established in order to evaluate the functionality of the novel gene therapeutics and several candidates could be examined for their effects by qPCR and flow cytometry. Identification of suitable target molecules on the surface of tumor cells is often restricted to specific tumor types. Thus, special focus was put on a particularly relevant solid tumor disease: malignant glioblastoma (GBM). GBM is a highly malignant brain tumor that is currently subject to a particularly high unmet need. GBM tops the list of cancer-related causes of death in children and the general public is aware of the suffering of numerous affected patients and their relatives in the recent past, e.g. Beau Biden, son of the current democratic US president, Joe Biden. Two strategies to identify suitable target molecules were applied. On one hand, the analysis of various relevant, publicly available data sets for protein and RNA information (TCGA database, Ivy Atlas, etc.) was carried out using a novel datamining strategy developed by the project researcher. By means of biostatistical software, an informed evaluation of harmonized data sets with regard to defined, therapeutically relevant criteria was possible (see figure: datamining strategy). The comprehensive evaluation of the available data sets initially led to the identification of two new candidate target molecules. The relevance of the two candidates was subsequently validated in further data sets and subsequently through laboratory tests. Based on an invention of the researcher, a process for production of off-the-shelf cellular anti-cancer therapeutics could be developed and proof of principle could be achieved (see figure: NK irradiation). A patent application was filed accordingly in cooperation with Fraunhofer FEP in Germany. Overall, two highly efficient genetically engineered cell therapies for treatment of malignant glioblastoma were developed during the project. Preclinical testing was initiated and showed promising results but remains to be validated in GMP/GLP compliant settings. Furthermore, a number of additional ligands potentially suitable as a basis for drug development schemes was also identified by varies approaches. Especially concerning target 1, a so far unknown GBM associated protein, mechanisms underlying this association are of interest and should also be elucidated in the future.
One goal is the development of technical innovations which potentially increase the effectiveness of cancer immunotherapeutic agents and allow their use for the treatment of solid tumors. A special focus was on the development of new approaches for the treatment of malignant glioblastoma (GBM), a brain tumor with a particular bad outcome, which currently leads to the death of the affected patients within a few months despite maximum therapeutic intervention. Within the project, a general focus was on the development, testing and application of genetic engineering processes for the production and optimization of cancer immunotherapeutic agents. For example, control mechanisms are to be integrated that allow the therapeutic cells to be regulated and thus potentially expand the therapeutic range. Viewed retrospectively, there are two properties in particular that often form the basis for particularly successful cancer immunotherapeutic methods: 1) therapeutic targets are significantly associated with cancer, but not healthy tissue; and 2) these therapeutic targets are well accessible to a cancer immunotherapeutic agent.
With regard to the first point mentioned, expertise in the field of genome-wide biostatistical analyzes, a previous focus of the researcher, and experience in the fields of histopathology and protein engineering methods proved essential to the project and were further deepened as deemed necessary during the project’s course. A number of new and potentially promising therapeutic targets for the treatment of GBM have been identified through the efforts within the project. In addition to the identification of new target molecules, the greatest potential for successful utilization is primarily the development of new, highly specific ligands for these new targets. Such ligands are at the beginning of every pharmaceutical-biotechnological development pipeline and offer unique opportunities in the treatment of patients. In particular, methods for screening ligand libraries with high diversity are now of great importance in cancer immunotherapy. Access to one such library allowed the identification of additional ligands within the project. The large overlap in the manufacturing and validation methods allows an extremely attractive expansion to the project’s future outcome while relatively little additional effort is necessary. Such synergies have come into play concerning the still largely unmet need for off-the-shelf cancer immunotherapeutics, which has emerged as an attractive R&D field. This is also due to the fact that such methods have a potentially more competitive cost-benefit profile and can possibly be used much more broadly than current cell therapy methods allow. Results from this project’s main drug developments thus also served as a basis for testing innovative off-the-shelf approaches, resulting in filing and granting of a patent for producing of-the-shelf cellular cancer immunotherapeutics by electron beam irradiation.
animal model overview
biodistribution of ONTs
overview of production of genetically engineered new therapeutics
validation of novel ligand binding by staining
efficacy of novel therapeutics in vitro
measurement of significant reduction in both the total number of target gene-positive cells
novel therapeutic efficacy in vivo
novel datamining strategy to identify novel tumor specific therapeutic targets
modeling of novel ligand binding to targets