TARGETING THE T‐CELL RECEPTOR Β‐CONSTANT DOMAIN FOR IMMUNOTHERAPY OF T‐CELL MALIGNANCIES

Abstract

Introduction: Mature T-cell lymphomas are typically aggressive, treatment-resistant and associated with poor prognosis. Immunotherapy has been limited by a lack of target antigens discriminating malignant from healthy T-cells. While B-cell cancer immunotherapy targets pan B-cell antigens, causing normal B-cell depletion, pan T-cell depletion would be prohibitively toxic. We propose a strategy for targeting a pan T-cell antigen without causing T-cell aplasia. The α/β T-cell receptor (TCR) is expressed on >90% of T-cell lymphomas and all normal T-cells. The β-constant region comprises 2 functionally identical genes: TRBC1 and TRBC2. Each T-cell expresses only one. Hence, normal T-cells are a mixture of individual cells expressing either TRBC1 or 2, while a clonal T-cell cancer expresses TRBC1 or 2 in its entirety. We hypothesised immunotherapy against either TRBC1 or 2 could target a whole cancer but preserve many normal T-cells. Methods: Standard techniques including flow cytometry, enzyme-linked immunosorbent assays, Cr51-release cytotoxicity assays, viral peptide T-cell stimulation, immunohistochemistry, and in vivo bioluminescence imaging. Results: Despite almost identical sequences, we identified an antibody with unique TRBC1 specificity. Flow cytometry in normal donors (n = 27) and T-cell cancer patients (n = 18) revealed median T-cell TRBC1 proportion of 35% (range 25–47%). Viral-specific T-cells for Epstein Barr virus, cytomegalovirus, or adenovirus contained similar TRBC1 proportion, suggesting depletion of either subset would not remove immunity. Conversely, TCR+ cell lines (n = 8) and primary T-cell cancers (n = 55) across many histological subtypes were restricted to one compartment (34% TRBC1). As proof of concept for TRBC-selective therapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T-cells. After retroviral transduction of healthy donor T-cells, comprising mixed TRBC1/2 populations, 90% expressed CAR. No detectable TRBC1 T-cells remained. Anti-TRBC1 CAR killed multiple TRBC1 cell lines (p < 0.001), autologous normal TRBC1 cells (p < 0.001), and primary cells from patients with TRBC1 tumours; and did not kill TRBC2 cell lines or autologous normal TRBC2 cells. We developed murine models of disseminated TRBC1 cancer by engrafting Jurkat or H9 cells in NSG mice. While control CAR recipients progressed, mice receiving anti-TRBC1 CAR had disease clearance (p < 0.0001) and prolonged overall survival (p < 0.05). Finally, mice were engrafted with equal proportions of TRBC1- and TRBC2-Jurkat cells. We observed specific eradication of TRBC1 and not TRBC2 cells by anti-TRBC1 CAR (p < 0.001). Conclusions: We describe a novel approach to treatment of T-cell malignancies distinguishing between 2 possible TCR β-chain constant regions. Using CART-cells targeting TRBC1 we have demonstrated proof of concept. Unlike strategies targeting the entire T-cell population, TRBC targeting could eradicate a T-cell tumour while preserving sufficient normal T-cells to maintain cellular immunity. Keywords: peripheral T-cell lymphomas (PTCL); T-cell receptor (TCR); T-cells.