Optimizing C-Type Lectin-like Molecule 1 (CLL-1) Directed CAR T Cell Therapy of Acute Myeloid Leukemia

Abstract

Introduction Acute Myeloid Leukemia (AML) is a heterogeneous disease with continuous clonal evolution and frequent treatment-resistant relapses, often attributed to chemoresistant leukemic stem cells (LSCs). Since LSCs can be phenotypically and metabolically distinct from AML blast cells, any curative AML therapy has to target both LSC and AML blasts. Transcriptomic and proteomic analyses show that the CLL-1 antigen may fulfil these requirements and we previously showed (Tashiro et al) that T cells expressing a C-type lectin 1 (CLL-1)-specific CAR effectively recognized and eliminated both AML blasts and primitive AML colony-forming cells.We have now modified the structure of the CLL-1 CAR to optimize performance for clinical application in patients with AML. Material and Methods We utilized a CLL-1-specific single-chain fragment variable (scFV) to create a panel of CLL-1 CARs with modifications to the spacers, transmembrane and signaling endodomains (CD28z-CD8, CD28z-sh, CD28z-CH3, 4-1BBz-sh, 4-1BBz-CH3).To determine the functionally optimal construct, we compared the phenotype and cytolytic ability of T cells transduced with each construct. We measured 72 hr cytotoxicity against the target cell lines THP-1, HL60, Molm13 and KG1a expressing different levels of the target antigen.To measure exhaustion, we set up sequential co-culture assays, collecting the T cells every 3 days, counting them and then replating with fresh tumor.Finally, we tested the cytolytic activity of the constructs against FFLuc-modified HL 60 and THP- 1 AML cell lines in NOD.SCID IL-2Rg−/− (NSG) mice.The animals were engrafted with tumor cells by iv injection, and CLL-1 CAR T cells were injected 3 days later. IL-15 was co-transduced with CLL-1 CAR to sustain a positive signal 3. Results T cells expressing 4-1BBz-containing constructs expanded less than CD28z-containing constructs. T cells expressing CD28z constructs retained a mix of naive, minimally differentiated and central memory T cells similar to 4-1BBz constructs.CD28z with a short hinge and CD8 endodomains had the greatest anti-tumor activity in vitro against tumor cell lines.In sequential killing assays against HL60 and THP-1, both the above CD28z constructs had significantly prolonged anti-tumor activity compared to 4-1BBz, with sustained CAR T cell survival.In the in vivo xenograft NSG HL-60 mouse model CD28z constructs improved survival and co-expression of this optimized CLL-1 CAR with transgenic IL-15 (signal 3) further improved T-cell persistence and survival in THP-1 model. Conclusion The CD28z CAR with a short hinge and/or CD8 endodomains appears superior to other vector constructs against CLL-1 targets in terms of proliferation, functional persistence and in vitro and in vivo anti-tumor activity. Co-expression with transgenic IL-15 further improves these functional characteristics.