Tedeschi, Fabiana (2024) Development of nanobody-based synthetic modulators to counteract T-cell exhaustion for enhanced T-cell-based immunotherapies. [Tesi di dottorato]

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Abstract

T-cell exhaustion is a dysfunctional state of CD8+ T cells that occurs during chronic infections or cancer due to chronic antigen stimulation and persistent inflammation. Exhausted T (exT) cells are characterized by progressive loss of effector functions, sustained co-expression of multiple inhibitory receptors (IRs), transcriptional and epigenetic modifications that ultimately result in the inability to mount effective immune responses. Inhibitory receptors, particularly ‘Cytotoxic T lymphocyte-associated molecule-4’ (CTLA-4) and ‘Programmed cell death-1’ (PD-1), play a crucial role in the landscape of T-cell exhaustion by negatively regulating T-cell functions. In the past decades, immune checkpoint therapy (ICT) has become the start-of-the-art treatment for T-cell exhaustion and numerous anti CTLA-4 and anti PD-1 monoclonal antibodies (mAbs) have been identified showing promising results in enhancing the anti-tumor immune response and tumor growth control. Nevertheless, only a subset of patients responded to ICT and drug resistance along with immune-related adverse effects have limited the clinical benefits, mostly in solid tumors. Furthermore, mAb-related issues, such as their large size and poor tissue penetration, restricted therapeutic efficacy and applicability of this strategy bringing out the necessity to develop new antibody-based therapeutic agents with improved characteristics for in vivo applications in cancer immunotherapy. Recently, camelid-derived single-domain antibodies (sdAbs), also called nanobodies, have emerged as a promising alternative to mAbs for immunotherapy due to their unique features, such as small size, high solubility, high stability, and excellent tissue penetration in vivo, which make them suitable for therapeutic applications. My PhD project proposes a nanobody-based synthetic biology approach to neutralize T-cell exhaustion and recover full cytotoxic activity for improving T-cell therapies in solid cancers. Specifically, a synthetic system was creating by coupling a sensing module, represented by a synthetic promoter activated by exhaustion-specific transcription factors (exTF), with an actuator module, including genetically encoded anti-exhaustion modulators (Figure 1a). In detail, my project was focused on the design of actuator modules based on sdAbs targeting CTLA-4 and PD-1 immune checkpoints to restore anti-tumor T-cell activity. T-cell engineering with the synthetic system will allow to achieve a fine regulation of modulator expression and a locally secretion of sdAbs in tumor site avoiding adverse and off-target effects usually due to systemic injection of mAbs. Our approach for T cell reprogramming has the potential to transform T cell-based immunotherapies since T-cell exhaustion poses a major hurdle in successful anti-tumor treatments limiting clinical efficacy and preventing long-lasting disease control.

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