A Novel Chimeric Antigen Receptor T Cells Therapy Strategy That Dual Targeting CD19 and CD123 to Treat Relapsed Acute Lymphoblastic Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Tu Sanfang , Deng Lan , Huang Rui , Zhou Xuan , Yang Jilong , Zhou Weijun , Li Meifang , Yue Chunyan , Wu Shaojie , Guo Zhenling , Chang Lung-Ji , Li Yuhua 
Published: October 2019

Abstract
BACKGROUND: The prognosis of patients with relapsed B-lineage acute lymphocyte leukemia (B-ALL) after allogeneic hematopoietic stem cells transplantation(HSCT) is poor. It is difficult to obtain disease-free remission and long-term survival in these patients based on chemotherapy, donor leukocyte infusion (DLI) or molecular targeted therapy. Donor-derived anti-CD19 CAR-T(CAR 19) cells may obtain remission in these patients, but many still encounter relapse, especially CD19-negative relapse. CD123 is a surface marker not only associated with the progenitors of acute myeloid leukemia, but also found in progenitors of acute lymphoblastic leukemia. Recent studies have shown that CD123 is highly expressed in the patients with CD19-negative relapsed after CAR19 cells therapy. we developed a novel therapeutic strategy for to administer pooled donor-derived fourth generation CART cells targeting CD19 and CD123 respectively, to patients who relapsed following allogeneic HSCT to avoid CD19-negative relapse. In this study, we report three patients who received combination CARTs therapy achieved disease-free survival that at least more than 6 months (www.clinicaltrials.gov; #NCT03125577).
PATIENTS AND METHODS: Three patients with relapsed B-ALL after HLA-matched sibling HSCT have been enrolled in the study to date, and all of their leukemia cells highly expressed CD19 and CD123 antigens. The first and second patients experienced relapse 6 months and one year after all-HSCT respectively; their B-ALLs were p190-positive and carried T315I mutation, and resisted to bonatinib, chemotherapy and DLI. The third patient was also refractory to chemotherapy and DLI. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m2, d1-3; CTX 300mg/m2, d1-3) before CART infusions. Donor T cells were apheresis collected and transduced with an apoptosis-inducible, safety-engineered lentiviral CD19 or CD123 scFv CAR fused with intracellular signaling domains: CD28/CD27/CD3ζ-iCasp9 (4SCAR19 and 4SCAR123). CAR-T cells were infused at dose range of 0.26-1.38x106 cells/kg. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR T infusion dose were quantitatively scored and documented.
RESULTS: All three patients achieved minimal residual disease (MRD) negative remission within 1 month after CAR-T infusions. Monthly follow-ups of the first and second patients indicated that they achieved stable MRD-negative and p190-negative remission, and remained disease-free for 7 months and 11 months, respectively. The third patient was MRD-positive but achieved bone marrow morphological remission at 7 month follow-up time. Flow cytometry analysis of the MRD cells detected CD19 positive and CD123 partial positive ALL clones, and note that the third patient received the lowest dose of CART infusion, 0.26x10e6/kg. The first patient developed grade 1 cytokine release syndrome (CRS) after CAR-T cell infusions. The second patient developed grade 1 oral acute graft-versus-host disease (aGVHD) and pulmonary infection. The third patient developed grade 2 CRS, with hypoxemia and unilateral massive pleural effusion. We detected high IL-6 in his pleural fluid >5000 IU/L, but serum level IL-6 is normal. There was no significant absorption of pleural effusion after treatment with anti-interleukin 6 receptor monoclonal antibody. The patient improved after 5 days of treatment with chest drainage and dexamethasone 10mg/qd. None of the three patients developed central nervous system toxicity, and there was no greater than grade 2 CRS and severe myelosuppression, consistent with the safety profile of the 4SCAR design.
CONCLUSIONS: We have successfully treated three relapsed allo-HSCT B-ALL patients using donor-derived 4SCAR19 and 4SCAR123 T cells. All three patients achieved long-term disease-free survival without severe CRS and GVHD. Thus, the administration of double 4SCAR19/4SCAR123 T cells may overcome CD19 escape and prolong disease-free survival.

Disclosures
No relevant conflicts of interest to declare.