Modified Dual CLL1-CD15 and CLL1-CD16 Icar-T Cells for Mitigating Granulocytopenia Toxicities in the Treatment of Acute Myeloid Leukemia
Authors: Zhang Rui , Zhao Yifan , Zhao Mingfeng 
Published: December 2024

Objective

CLL1 Chimeric antigen receptor T-cell (CAR-T) therapy, as a promising immunotherapeutic approach, has demonstrated its potential to enhance the prognosis of patients diagnosed with acute myeloid leukemia (AML). However, due to the overexpression of CLL1 on neutrophils, CAR-T cells not only eliminated tumor cells but also eradicated neutrophils simultaneously, resulting in severe granulocytopenia and subsequent infections. Considering the distinct expression levels of CD15 and CD16 on neutrophils and AML blasts, we have devised novel modified CLL1-CD15 and CLL-CD16 iCAR structures incorporating diverse inhibitory elements.

Methods

Firstly, we engineered CLL1-CD15 iCAR constructs incorporating four distinct inhibitory domains (PD-1, CSK, PD-1 combined with 2B4, and PD-1 combined with BTLA). The cellular characteristics, including activation, differentiation, exhaustion, and cytotoxicity of diverse iCAR-T cells were evaluated to identify the most suitable inhibitory combination. The CLL1-CD16 iCAR structures were subsequently designed based on the prior screening of inhibitory domains, and the CD16-VHH fragments were creatively employed as the recognizing domain. Then, the cellular characteristics and functionality were compared through in vitro experiments. Ultimately, the efficacy and safety of CLL1-CD16 iCAR-T cells were evaluated in vivo.

Results

Our findings suggest that CLL1-CD15 iCAR-T cells with PD1 plus 2B4 inhibitory domains exhibit a greater potential for primitive differentiation, reduced exhaustion, and enhanced activation. Meanwhile, CD15-CLL1 iCAR-T cells with PD1/2B4 inhibitory domains demonstrate superior resolution capability by exhibiting the highest cytotoxicity towards CD15-negative tumor cells and the lowest cytotoxicity towards CD15-positive tumor cells. Furthermore, the CLL1-CD16 iCAR-T cells, based on PD1/2B4 with a truncated VHH recognition domain, demonstrate an increased transfection rate and enhanced elimination of CD16-negative blasts while minimizing potential side effects on CD16-positive neutrophils. Additionally, coculture experiments reveal that the activation and cytotoxicity of CLL1-CD16 iCAR-T cells remain unimpeded in the presence of coexisting iCAR-T cells, tumor cells, and neutrophils requiring protection. Moreover, CLL1-CD16 iCAR-T cells significantly prolonged the survival of xenograft models while reducing the occurrence rate of granulocytopenia compared to the group treated with CLL1 CAR-T cells.

Conclusion

We have developed a novel CD16-CLL1 iCAR structure that takes advantage of the significantly elevated expression level of CD16 in neutrophils, while demonstrating remarkably low expression in AML blasts. This design allows for precise targeting of AML tumor cells while minimizing any unintended harm to neutrophils. Additionally, we have employed a single VHH fragment to replace the entire scFv, thereby enhancing transfection efficiency, improving efficacy, and reducing potential side effects. In our upcoming clinical trials, our objective is to utilize this innovative CD16-CLL1 iCAR-T therapy for the treatment of patients with refractory and relapsed AML, with the aim of determining its effectiveness and safety as a promising approach towards improving the prognosis of AML patients.