Preclinical development of ARD103, a CLL-1 CAR-T produced by a 3-day DashCAR technology, for acute myeloid leukemia.

Abstract

119 Background: Acute myeloid leukemia (AML) is the most common acute leukemia in adults. More than half of the AML patients experienced primary refractory or relapse after current treatments, such as chemotherapy, hematopoietic stem cell transplantation, and targeted therapy. AML patients are in urgent need of novel therapeutic strategies. Leukemic stem cells (LSCs), which cause the treatment failure and leukemia relapsed. Human C-type lectin-like molecule-1 (CLL-1) is a surface antigen expressed on both AML malignant blast cells and LSCs, but not on normal hematopoietic stem cells, suggesting that CLL-1 is a compelling target with low off-tumor toxicity for novel AML treatment. Methods: ARD103 is a second-generation chimeric antigen receptor (CAR)-T cell, expressing a novel extracellular anti-CLL-1 scFv fused to a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ activation domain. ARD103 was generated using lentiviral vector in an optimized 3-day DashCAR manufacturing process and demonstrated high percentage of CAR population (>80%), high percentages of memory T cells (Tscm and Tcm are >70%) and profound effector cytokine production (IFN-γ, granzyme A, perforin and granulysin). Results: ARD103, CLL-1 CAR-T exhibited specific killing on various CLL-1-expressing AML cell lines, such as U937, THP-1, and HL60, correlated to the levels of CLL-1 expression. In the AML U937-Luc xenograft mouse model, the CLL-1 CAR-T cells prolonged the survival time significantly (p < 0.0001), with superior CAR-T cell persistence, to the end point of the study at 48 days compared to the median survival time at 23 days in the untreated or control groups. Furthermore, the xenograft mice receiving as minimum as 3 x105 CAR-T cells per mouse were able to elicit effective recall responses after tumor rechallenge and survived for more than 60 days. The off-target binding interactions of ARD103 CAR-T cells using a human cell microarray showed a specific and strong interaction with CLL-1 but not with other human membrane/extracellular proteins. In the aspect of on-target off-tumor toxicity, the hematotoxicity assessment on ARD103 exerted modest (5%-23%) or no effect on colony formation inhibition of CD34+ cells derived from human normal bone marrow or peripheral blood, respectively. Furthermore, autologous ARD103 cells were successfully prepared from relapsed/refractoryAML patients and demonstrated to mediate specific cytotoxicity to primary AML blasts and LSCs derived from the same patients. Conclusions: Taken together, the ARD103 CLL-1 CAR-T candidate has demonstrated excellent anti-AML potency and efficacy with minimal safety concerns, and warrant to be further developed preclinically and clinically for the treatment of relapsed/refractory AML patients. Keywords: Acute myeloid leukemia; CLL-1; Chimeric antigen receptor-T cell; Immunotherapy.