Background

Acute myeloid leukemia (AML) is due to the clonal expansion of myeloid precursor cells driven by recurrent genetic alterations, leading to their uncontrolled proliferation and accumulation. The genomic heterogeneity of AML poses significant therapeutic challenges. Despite advances in treatment and the emergence of novel therapies in recent years, a substantial proportion of patients continue to experience disease relapse or progression. Immunotherapy has emerged as a promising approach in the treatment of acute leukemias. Among these strategies, chimeric antigen receptor (CAR) T cell therapy—where T cells are genetically engineered to express CARs that redirect their specificity toward antigens expressed on leukemic cells—has shown remarkable success. While several CAR T cell products have reached the market, none has been approved for AML. A major challenge in applying immunotherapy to AML lies in identifying an ideal surface antigen, that is highly and selectively expressed on leukaemia blasts but minimally on healthy tissue cells.

B7-H3 (CD276), a member of the B7 family of immune checkpoint molecules, has emerged as a promising therapeutic target across various type of cancers, including AML. Indeed, while minimally expressed in normal tissues, B7-H3 is particularly expressed in the monocytic subset of AML and in leukemic stem cells (LSCs), which play a key role in disease recurrence and chemotherapy resistance.

Methods

To support our choice of selecting B7-H3, we performed bioinformatic analyses in the context of AML. We demonstrated that elevated B7-H3 expression is significantly associated with adverse clinical outcomes in patients with AML. Specifically, high B7-H3 expression correlates with reduced overall survival and decreased disease-free survival compared to patients exhibiting low B7-H3 expression levels. These findings reinforce the prognostic significance of B7-H3 in AML and underscore its potential involvement in disease progression and resistance to therapy.

Results

To develop a CAR T cell approach targeting B7-H3, we screened for novel single-chain variable fragments with high affinity and specificity. Thanks to this approach, we generated two distinct third-generation CAR constructs incorporating different co-stimulatory domains: CD28.4-1BB and CD28.OX40. Both CARs demonstrated high membrane stability in vitro, with expression levels of 65.8% ± 12.3% and 86.8% ± 5.3%, respectively, at day 15 post-transduction.

In vitro functional assays showed that both CAR.B7H3.CD28.4-1BB and CAR.B7H3.CD28.OX40 T cells exhibited potent anti-leukemic activity, effectively eradicating the B7-H3+ THP-1 cell line in a 5-day co-culture assay across effector-to-target (E:T) ratios ranging from 1:1 to 1:8. At the 1:8 E:T ratio, residual THP-1 cells were reduced to 0.14% and 0.089%, respectively, compared to 96.1% in control conditions. Consistent with these findings, both CAR constructs also demonstrated robust and similar anti-leukemic activity against primary leukemia blasts obtained from the bone marrow of AML patients at diagnosis.

We also evaluated both constructs in a AML xenograft immunodeficient mouse model in which NSG mice were infused intravenously with 0.5x106 THP-1.FFluc+ cells. At leukaemia establishment, mice received 10x106 CAR+ T cells or control un-transduced T cells. IVIS imaging showed that both CAR constructs successfully eradicated leukemia cells within the first two weeks of treatment (2.07E6 ± 1.31E6 p/sec for B7-H3.CARCD28.OX40 and 3.36E6 ± 5.67E6 p/sec for B7-H3.CARCD28.4-1BB, compared to un-transduced T cells: 1.51E8 ± 1.099E8 p/sec in the control group; p=0.0156 and p=0.0163, respectively). The leukemia control translated also in an improved overall survival in treated mice compared to the control group given un-transduced T cells. Specifically, mice in the control cohort reached bioluminescence levels of 10¹⁰ photons/second (p/s) and either deceased or had to be euthanized by 31.4 ± 5.27 days post-treatment. In contrast, 90% of mice treated with CAR T cells (9 out of 10) remained alive for the duration of the experimental period (60 days).

Conclusions

Collectively, these preclinical results demonstrate the strong therapeutic potential of B7-H3-targeted CAR-T cells as a novel treatment approach for AML, supporting the rationale for advancing toward clinical trials that have the potential to significantly improve outcomes for both pediatric and adult AML patients.

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2025
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