Preclinical Evaluation of CD64 As a Potential Target For CAR-T-cell Therapy For Acute Myeloid Leukemia

Abstract

The relapsed and refractory acute myeloid leukemia (AML) patients receiving traditional chemotherapies have poor survival rate. Chimeric antigen receptor (CAR)-modified T cells have demonstrated remarkable effectiveness against some malignancies. However, most of CAR-Ts targeting the candidate proteins on AML cells induce hematopoietic cell suppression. Because of extensive heterogeneity among different types of AML, it is essential to expand the choice of target antigen for the CAR-T treatment of AML. CD64 (FcγRI) is a transmembrane protein with broad expression on various types of AML cells, especially monocytic AML cells, but it is absent on hematopoietic stem cells (HSCs) and most of nonmonocytes. Here, we found that some types of AML patients showed the homogeneous high-level expression of CD64. So, we created a CAR-T targeting CD64 (64bbz) and further verified its high efficiency for eradicating CD64 + AML cells. In addition, 64bbz showed no cytotoxicity to HSCs. Overall, we developed a new treatment option for AML by using CD64 CAR-T cells while avoiding ablation of HSCs.

Immunophenotypical profiling of myeloid neoplasms with erythroid predominance using mass cytometry (CyTOF)

“…Genomic analyses and diagnostic criteria suggest that AEL can be characterized as a disease in the continuum of AML and MDS (Hasserjian et al, 2010). Here, high- detected aberrancies such as CD64, CD33, and CD47 might have therapeutic implications for AEL as they can be targeted (Majeti et al, 2009;Sun et al, 2021;Walter, 2014). Interestingly, an unsupervised approach using UMAP and FlowSOM (Figures S4 and S5) revealed similar erythroid-myeloid coexpression profiles on rare cell populations.…”

“…Aberrant expression levels of erythroid markers such as CD235a, CD49d and CD36 and expression of CD238 and CD71 on CD34 + CD38 − cells might support the notion that LSC phenotypes, characteristic of AEL, might potentially harbor the erythroid leukemogenic potential. Some detected aberrancies such as CD64, CD33, and CD47 might have therapeutic implications for AEL as they can be targeted (Majeti et al, 2009; Sun et al, 2021; Walter, 2014). Interestingly, an unsupervised approach using UMAP and FlowSOM (Figures S4 and S5) revealed similar erythroid‐myeloid coexpression profiles on rare cell populations.…”

Myeloid leukemia-derived galectin-1 downregulates CAR expression to hinder cytotoxicity of CAR T cells

“…CAR down-regulation was more pronounced at low E:T ratios in vitro and in vivo experiments. The U937 cell line, which naturally expresses CD33 and CLL-1, is widely used for in vitro experiments and in vivo mouse models in preclinical AML studies [ 30 – 32 ]. The Nalm6 cell line, which naturally expresses CD19 and CD22, is also widely used for in vitro experiments and in vivo mouse models in preclinical studies of B-cell malignancies [ 33 , 34 ].…”

“…Tumor cell lines overexpressing CD33, CLL-1, and CD123 antigens were constructed by lentiviral transduction, and they were further purified by cell sorting (FACS Aria II, BD Biosciences) or single-cell cloning. HEK293T cells and tumor cell lines were cultured as previously described [ 32 ]. T cells were cultured in T Cell Expansion Medium (Stem Cell Technologies), supplemented with 100 ng/ml recombinant human IL-2 (Peprotech).…”

Targets for chimeric antigen receptor T-cell therapy of acute myeloid leukemia

“…Blast % positive with CD64 is highest in the M5 subtype, but also present in M3, M2, and M4 with 80%, 58%, and 20% respectively. In both in vitro and in vivo models, anti-CD64 CAR T-cells demonstrate limited myeloid cell suppression and lack of toxicity to CD34+ HSCs ( 61 ). T-cells transduced with anti-CD64 CAR show approximately 80% specific cell lysis against U937 and THP-1 AML cell lines at E:T ratio 1:2 with control T-cells showing minimal killing effect ( 61 ).…”