All-Trans Retinoic Acid and Interferon-Alpha Increase CD38 Expression on Adult T-Cell Leukemia Cells and Sensitize Them to T Cells Bearing Anti-CD38 Chimeric Antigen Receptors
Authors: Mihara Keichiro , Yoshida Tetsumi , Ishida Seiko , Takei Yoshifumi , Kitanaka Akira , Shimoda Kazuya , Morishita Kazuhiro , Takihara Yoshihiro , Ichinohe Tatsuo 
Published: October 2019

Abstract
The survival of patients with adult T-cell leukemia (ATL) has been improved by the introduction of anti-CCR4 monoclonal antibody and the expanding use of allogeneic hematopoietic stem cell transplantation. However, not all patients benefit from these treatment modalities, warranting development of a novel therapeutic strategy. CD38, a cell surface ectoenzyme that functions as cyclic ADP ribose hydrolase, is an attractive target of chimeric antigen receptor (CAR) therapy for lymphoid neoplasms because it is widely expressed on the cells of B- or T-lymphoid malignancies. We have previously demonstrated the prominent cytotoxicity of T cells engineered to express an anti-CD38-CAR against B-lymphoma cells and myeloma cells expressing CD38. To expand the applicability of anti-CD38-CAR against ATL cells that usually express undetectable or low levels of CD38, notably, we were successfully able to induce cell surface CD38 expression in HTLV-1-infected cell lines with all-trans retinoic acid (ATRA) (Yoshida T, et al. 2013 ASH Meeting). In ATL cells freshly isolated obtained from the patients, we were able to induce CD38 with ATRA in 60-80% of the cells; the remaining cells survived under the anti-CD38-CAR treatment. We hereby report our attempts in improving the efficacy of anti-CD38-CAR T cells against ATL cells from the patients through the expression of CD38 enhanced with the entry of agents, which are clinically used. Firstly, we investigated whether ATL cells from patients could be transduced with anti-CD38-CAR and what is the efficiency of transduction into T cells in our settings. ATL cells (CD4+ CD25+ GFP+) transduced with retroviral vector were little detected. CD4- CD25- GFP+ T cells alone were detected in our transduction methods. Transduction efficiency was over 40%. To increase the expression of CD38 on ATL cells, we took notice of the CD38 gene upstream region that contains binding sites for interferon regulatory factor-1 (IRF-1) and peroxisome proliferator-activated receptor (PPAR). We thus investigated whether IFN-α, IFN-γ or troglitazone, which is a PPAR-α and -γ agonist, could enhance CD38 expression in ATL cell lines (MT-4, Su9T, ED, and S1T cells), which are negative for CD38. IFN-α and IFN-γ efficiently enhanced CD38 expression in MT-4 cells in a dose-dependent manner but not in Su9T, ED, and S1T cells. As little as 2.5U/ml of IFN-α induced CD38 expression in MT-4 cells for 18 hours in vitr o (>95% at positivity of CD38). 10-25% increase in CD38 expression was observed in ED cells with 125-250 pM troglitazone after 18 hours of treatment, but not in MT-4, Su9T, and S1T cells. Prolonged exposure to troglitazone was toxic to cells. Combined treatment with 10nM ATRA and IFN-α, which induced higher expression of CD38 than IFN-γ, synergistically enhanced CD38 expression of ATL cells from the patients (>90%at positivity of CD38). We next co-cultured ATL cells form three patients with T cells transduced with mock or anti-CD38-CAR in the presence of both ATRA and IFN-α at effector (E): target (T) ratio of 1: 2 for 3 days. The treatment eradicated more than 95% of these ATL cells, demonstrating that ATL cells can be eliminated by T cells harboring anti-CD38-CAR in the presence of ATRA and IFN-α, which is actively used for ATL patients. CD38 targeting therapy is a feasible method, because an anti-CD38 antibody, daratuzumab, has been used to treat plasma cell myeloma. The safety regarding the clinical use of T cells bearing anti-CD38-CAR still needs to be established. As CAR therapy reportedly causes cytokine storm and can potentially be lethal, we envision an inducible immunotherapy with CAR to be a preferred modality with increased efficacy and safety. Our results provide a rationale for a novel therapeutic strategy involving T cells carrying anti-CD38-CAR in combination with ATRA and IFN-α for patients with ATL.

Disclosures
No relevant conflicts of interest to declare.