Optimization of Blood Donor Selection for the Production of Therapeutic Gamma Delta T Lymphocytes in Treatment – SELCELL

The aim of the project is to develop the foundation for creating a standardized, allogeneic Advanced Therapy Medicinal Product (ATMP) based on γδ T lymphocytes, intended for the treatment of glioblastoma multiforme (GBM). GBM is one of the most aggressive brain tumors, for which conventional oncological therapies (surgical resection, radiotherapy, and chemotherapy) show limited effectiveness. This is reflected in the exceptionally poor survival rates: 37.2% at one year and only 5.1% at five years. Due to the low efficacy of current treatments, new therapeutic strategies are being actively developed, including immunotherapies that leverage the natural ability of the immune system to selectively destroy cancer cells.

One of the most advanced forms of immunotherapy is CAR-T cell therapy—autologous T lymphocytes that, after genetic engineering, produce a chimeric antigen receptor (CAR) capable of recognizing tumor antigens. Despite its groundbreaking success in treating certain hematologic cancers, the effectiveness of CAR-T therapy in solid tumors such as GBM remains significantly limited. Moreover, the need to use a patient’s own cells (autologous therapy), high production costs, limited availability, and the risk of adverse effects underscore the need to explore alternative cell-based immunotherapies. A promising direction is the development of allogeneic therapies based on effector cells obtained from healthy, unrelated donors, which could serve as off-the-shelf medicinal products ready for immediate use.

Among the most promising candidates are γδ T lymphocytes—a small subpopulation of T cells (1–10% of circulating T lymphocytes) characterized by a unique T-cell receptor (TCR) composed of γ and δ chains. These cells exhibit natural cytotoxic activity against tumor cells, mediated by both the γδ TCR and the NKG2D receptor. A key feature of γδ T lymphocytes is their activation independent of antigen presentation by the highly variable major histocompatibility complex (MHC), which allows for their use in allogeneic therapy in patients unrelated to γδ T-cell donors. Compared to conventional αβ T lymphocytes used in CAR-T therapy, γδ T cells have the potential to become a universal therapeutic product with broad application. Preclinical data, mainly from animal models, confirm the ability of γδ T cells to eliminate tumor cells of various types, as reflected in the growing number of clinical studies involving these cells.

Previous research conducted by the Immunotherapy Research Group at Łukasiewicz–PORT demonstrated that γδ T lymphocytes effectively destroy GBM cells under in vitro conditions. However, significant variability in cytotoxic activity was observed depending on the donor, suggesting that individual biological traits influence their therapeutic potential. This highlights the need for further, in-depth research aimed at identifying donors with a favorable immunological profile.

Accordingly, the project assumes the comprehensive characterization of γδ T lymphocytes (including Vδ1 and Vδ2 subtypes) isolated from leukocyte material obtained from healthy donors. Analyses will include both the original cells and those expanded in vitro. They will undergo molecular analyses—transcriptomic (using next-generation sequencing, NGS) and proteomic (using mass spectrometry). In parallel, a functional assessment of γδ T lymphocytes will be conducted to determine their ability to destroy glioma cells in cytotoxicity assays on various GBM cell lines, using biochemical methods and live-cell fluorescence microscopy.

Integrating the functional data with molecular profiles (transcriptome and proteome) will allow for the identification of prognostic markers of effective antitumor response, thus enabling the creation of a molecular “ideal donor profile.”

In the long term, the results will serve as the basis for developing a new generation of γδ T-cell-based immunotherapy for the treatment of glioblastoma multiforme and potentially other brain tumors.