TGF-β Inhibitor SD-208 Enhances the Anti-Tumor Efficacy of CD19 CAR-T Cells through a Reduction of CAR-Treg in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background: Acute lymphoblastic leukemia (ALL) is an incurable hematological malignancy with poor prognosis. Chimeric antigen receptor T cells (CAR-T) therapy is a milestone in the immunotherapy of relapsed or refractory B cells ALL (r/r B-ALL), but most cases are inevitably relapsed after CAR-T therapy and the causes are complex. Our preliminary work, a phase II clinical trial (#NCT02735291) involving pediatrics and adults with r/r B-ALL who were treated with CD19 CAR-T, exhibited that the overall survival (OS) and relapse-free survival (RFS) in patients with higher Treg are lower, and higher Treg may preclude the efficacy of CAR T therapy in r/r B-ALL. Further analysis of the frequencies of Treg within CD4 ⁺ cells at CD19 CAR-T therapy in r/r B-ALL patients investigated that higher circulating Treg, especially 1 week after CD19 CAR-T cells infusion, was a poor predict indicator for CD19 CAR-T therapy in r/r B-ALL patients.

Methods: In the present study, we investigated the significance of CAR-Treg in CD19 CAR-T cells in clinical response and prognosis, and explored an inhibitive effect of TGF-β inhibitor SD-208 on the proliferation and lethality of CAR-Treg. Regulatory T cells (Treg) in CD19 CAR-T cells were tested in 34 patients with r/r B-ALL received CD19 CAR-T cells therapy, and then its significance on the complete remission (CR) rate, relapse-free survival (RFS) and overall survival (OS) was validated respectively. Next, TGF-β inhibitor SD-208 was added or not added in the culturing protocols of CD19 CAR-T cells, then the proportion of Treg and CAR-Treg as well as the anti-tumor ability of CD19 CAR-T cells were analyzed and compared in vitro.

Results: 1) Higher proportion of Treg (≥7.2%) in CAR-T cells was a negative factor affecting RFS (p=0.0120) and OS (p=0.0162) in the r/r B-ALL patients with CAR-T therapy. 2) CD4 ⁺CD25 ⁺CD127 ^low/- Treg proliferated from 7.01% to 25.26% after anti-CD3 and anti-CD28 Abs activation. PBMCs cultured with SD-208 exhibited lower Treg levels than those of control groups (15.73% vs. 29.08%). 3) CD4 ⁺CD25 ⁺CD127 ^low/- Treg levels stimulated by SD-208 in CD19 CAR-T cells were lower than those in control groups both 24 and 48 hours (38.68% vs. 40.47% in 24 hours, 23.58% vs. 36.19% in 48 hours), and the treatment for 48 hours was most obvious. CAR ⁺CD4 ⁺CD25 ⁺CD127 ^low/- CAR-Treg levels in CD19 CAR-T cells decreased by SD-208 stimulating compared with controls especially 48 hours (35.07% vs. 37.31% in 24 hours, 20.94% vs. 34.41% in 48 hours). The results exhibit that about 90% Treg in CD19 CAR-T cells are CAR-Treg, and inhibiting TGF-β by TGF-β/SMAD inhibitor SD-208 reduce the proliferation of Treg especially CAR-Treg in CD19 CAR-T cells. 4) To determine the function to CD19 CAR-T cell, LDH activity was utilized for tumor cell damage assay. Human Burkitt's lymphoma cell line Raji (CD19 ⁺) was co-cultured with CD19 CAR-T cell for 24 hours before LDH assay. The percent of cytotoxicity calculated depending on the LDH activities showed that the inhibition rate of SD-208 treated CD19 CAR-T cell was significantly higher than that of control. To further determine the function of CD19 CAR-T cell Granzyme B and Perforin were tested. The results exhibited that SD-208 treated CD19 CAR-T cell had higher rates of Granzyme B and Perforin in CD3 ⁺, CD3 ⁺CD4 ⁺, CD3 ⁺CD8 ⁺ cells than controls, and achieved more functional.

Conclusions: Inhibition of TGF-β by TGF-β/SMAD inhibitor SD-208 inhibit the proliferation of Treg in PBMCs of r/r B-ALL patients. TGF-β inhibitor SD-208 might improve the anti-tumor ability of CD19 CAR-T cells via decreasing Treg and CAR-Treg in r/r B-ALL.