Cysteine-Engineered CAR-T Cells for Antigen-Independent Targeting of B Cell Lymphoma

Abstract

Chimeric Antigen Receptor (CAR-) T cell therapy represents one of the newest, most promising treatments for cancer. Here, the patient’s own T lymphocytes are genetically modified with a cancer-specific CAR. CAR T cell therapy has shown significant clinical success, especially for the treatment of hematologic cancers by targeting the B cell antigen CD19. Nevertheless, selective pressure on the cancer cells frequently causes suppression or complete loss of the target antigen, leading to cancer relapse with fewer treatment options. This phenomenon, known as antigen escape, renders CAR-T cells ineffective representing a major clinical challenge. Consequently, there is an urgent need for targets that are less prone to antigen escape. In this thesis, a novel type of CAR-T cells is described, employing cysteine-engineered receptors that interact with altered extracellular redox states of B cell non-Hodgkin lymphoma (B-NHL). First, the redox-reactive nanobody CB2 was used for the generation of CB2-CAR-T cells, interacting directly with B-NHL redox states via a non-canonical cysteine in the complementarity-determining region (CDR) 3. The activation and specific cytotoxicity of CB2-CAR-T cells were verified in vitro against different subtypes of B-NHL, including antigen escape models. Next, the universality of the approach was confirmed by cysteine-engineering of a non-cancer-related nanobody-CAR. A single amino acid substitution with cysteine was sufficient to redirect these CAR-T cells to specifically target B-NHL, rendering cysteine-engineered CAR-T cells (or CysCAR-T cells) a universal strategy to enable antigen-independent targeting. Additionally, it was shown that cysteine engineering of state-of-the-art CD19-CAR-T cells enables co-targeting of both CD19-positive and -negative B-NHL. No systemic toxicity associated with these bifunctional CysCD19-CAR-T cells was observed in mice. Furthermore, T cells expressing the cysteine-engineered CAR delayed tumor growth and prolonged survival of mice engrafted with CD19-positive and -negative lymphoma. These findings introduce a novel type of bifunctional CAR-T cells that simultaneously target conventional antigens and altered redox states, potentially reducing the risk of antigen escape. It was shown that those CysCAR-T cells act through an antigen-independent mechanism and that cysteine engineering can be applied to CARs of diverse specificities. Altered redox states have been described for various cancers, including breast cancer and leukemia. Hence, these results indicate a broad therapeutic scope for CysCAR-T cells in preventing antigen escape associated with conventional targets.