Developing a framework to enhance T-cell receptor functionality

Background.

The human immune system can identify and eliminate cancer cells by recognizing tumor-specific epitopes, which arise from genetic and epigenetic aberrations. These so-called neoantigens are presented as short peptides by major histocompatibility complex (MHC) class I molecules on the cell surface, enabling recognition by cytotoxic T cells via their T cell receptors (TCRs) [1]. However, tumors frequently evade immune surveillance by exploiting various immunosuppressive mechanisms within the tumor microenvironment [2].
To counteract these mechanisms, several immunotherapeutic strategies have been developed, including immune checkpoint inhibitors targeting PD-1 and CTLA-4, as well as chimeric antigen receptor (CAR)-T cells. While CAR-T cell therapies have demonstrated remarkable efficacy in treating B-cell malignancies [3], their application to solid tumors remains challenging due to factors such as limited antigen availability, tumor heterogeneity, and immune suppression [4, 5]. Furthermore, CAR-T cells require higher antigen densities than their endogenous TCR counterparts [6], increasing susceptibility to immune evasion through simply downregulating the recognized antigen, which is indeed a major cause of relapse in CAR-T cell-treated leukemia patients [7].

TCR-T cell therapy offers a promising alternative with distinct advantages. Unlike CAR-T cells, TCRs can target a broader range of antigens, including those derived from intracellular and membrane proteins, thereby tremendously expanding the targetable antigen repertoire [5]. Moreover, the unique structure of the TCR and its proximal signaling machinery allow for the highly sensitive detection of minimal antigen densities, down to a single peptide-MHC molecule [8]. However, thymic selection during T cell maturation results in the removal of a huge proportion of T cells that would otherwise display reactivity against the tumor[9].
TCR engineering aims to overcome the limitations of natural TCRs by enhancing the affinity and functionality of tumor-reactive TCRs. The primary goal is to enable T cells to detect and respond to neoantigens at lower densities than they naturally could. This enhancement involves but is not limited to affinity maturation, where TCRs are optimized to improve their binding strength to peptide-MHC. Such improvements have been shown to increase T-cell responsiveness and overcome the immune evasion tactics of tumors, such as downregulating antigen presentation [9].
However, the complex and poorly understood relationship between TCR affinity and functionality presents a challenge. Therefore, optimization strategies e.g., screening approaches should not only assess receptor affinity but also account for the complex signaling processes that determine TCR functionality [10].

Aims and methods.

The thesis will be part of the DART²OS project funded by the Emerging Fields Program of the Austrian Science Foundation (FWF). DART²OS aims to identify osteosarcoma-specific neoantigens and their corresponding TCRs, followed by the engineering of TCR-T cells to enhance therapeutic efficacy and safety. Initially, osteosarcoma-reactive TCRs will be deorphanized and subsequently optimized to generate enhanced TCRs (eTCRs) with superior single-molecule affinity and functional activity.
To achieve this, we will first establish a robust platform for TCR enhancement using model TCRs. Specifically, a low-performing TCR will be systematically engineered to meet or surpass the efficacy of a high-performing benchmark TCR. Optimized eTCRs will undergo comprehensive biochemical characterization, including surface plasmon resonance analysis, and functional validation in cellular systems.
Upon validation, the platform will be applied to patient-derived TCRs, enabling the generation of osteosarcoma-specific eTCRs with improved functionality and enhanced therapeutic efficacy.

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