Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor–modified T cells

Shared Expression of CD93 and Other Antigens By AML and Endothelial Cells Highlights a Need for Rational Combinatorial Targeting

TNFα Apoptotic Signaling By Antigen-Engaged CAR T-Cells Mediates Clearance of Target Antigen-Negative Acute Myeloid Leukemia

Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells targeting CD19 has shown remarkable therapeutic efficacy against B cell leukemia and lymphoma, and provided proof of concept for therapeutic potential in other hematologic malignancies. Acute myeloid leukemia (AML) is an entity with an unmet medical need for effective and curative treatments. Therefore, there is a strong desire for development of potentially curative CAR-T cell immunotherapy for AML treatment. FMS-like tyrosine kinase 3 (FLT3) is a homodimeric transmembrane protein expressed uniformly by AML blasts. FLT3 plays a vital role in the survival of AML blasts and is a key driver of leukemia-genesis in AML cases with internal tandem duplication (FLT3ITD) and tyrosine kinase domain (TKD) mutations. These attributes suggest that FLT3 could be an excellent target for CAR-T cell immunotherapy. Here, we engineered human CD4+ and CD8+ T cells to express FLT3-specific CARs and demonstrate that they confer potent reactivity against AML cell lines and primary AML blasts that express either wild-type FLT3 or FLT3-ITD. Further, we show that FLT3 CAR-T cells exert potent antileukemia activity in xenograft models of AML and induce complete remissions. We also demonstrate that FLT3-expression on FLT3-ITD+ AML cells can be augmented by FLT3 inhibitors, which lead to increased recognition by CARs and improved efficacy of FLT3 CAR-T cells. We confirmed this principle with three different FLT3 inhibitors which are at distinct stages of clinical development i.e. Phase II/III clinical trial (crenolanib, quizartinib) and clinically approved (midostaurin). Further, we observed the strongest anti-leukemia activity of FLT3 CAR-T cells in combination with crenolanib in vivo. FLT3 is known to be expressed by normal hematopoietic stem and progenitor cells. We evaluated FLT3-expression on normal hematopoietic stem cells (HSCs) using flow cytometry and confirmed lower level of FLT3-expression on HSCs and progenitors compared to AML cells. As anticipated, we found that FLT3 CAR-T cells recognize normal HSCs in vitro and in vivo, and compromise normal hematopoiesis, suggesting that adoptive therapy with FLT3 CAR-T cells will require successive CAR-T cell depletion and allogeneic HSC transplantation (HSCT) to reconstitute the hematopoietic system. Moreover, an FLT3 inhibitor treatment does not increase FLT3-expression on HSCs. Accordingly, we demonstrate that the depletion of FLT3 CAR-T cells is possible with inducible Caspase 9 (iCasp9) safety switch. Collectively, our data establish FLT3 as a novel CAR target in AML with particular relevance in high-risk FLT3-ITD+ AML. Our data demonstrate that FLT3 CAR-T cells act synergistically with FLT3 inhibitors in FLT3-ITD+ AML. i.e. FLT3 inhibitors-induced upregulation of FLT3 in FLT3-ITD+ AML cells enhances their recognition and elimination by FLT3 CAR-T cells. Due to recognition of normal HSCs, the clinical use of FLT3 CART cells is likely restricted to a defined therapeutic window and must be followed by CART cell depletion and allogeneic HSCT for hematopoietic reconstitution. The data provide rational to use FLT3 CAR-T cells in combination with FLT3 inhibitors to augment the anti-leukemia efficacy of FLT3 CAR-T cells in high-risk FLT3-ITD+ AML patients, and to mitigate the risk of relapse with FLT3-negative AML variants, which could otherwise develop under therapeutic pressure. The data provide proof of concept for synergistic use of CAR-T cell immunotherapy and small molecule targeted therapy and encourage the clinical evaluation of this combination treatment in high-risk patients with FLT3-ITD+ AML.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by the rapid expansion of undifferentiated myeloid progenitors, leading to impaired hematopoiesis and poor patient prognosis. Although chimeric antigen receptor (CAR) T-cell therapy using single-chain variable fragments has revolutionized immunotherapy, clinical application in AML remains limited by on-target, off-tumor toxicities, largely due to shared antigen expression on normal hematopoietic stem and progenitor cells. To address this challenge, we developed a nanobody-based CAR T-cell platform directed against C-type lectin-like molecule-1 (CLL-1), a myeloid-restricted surface antigen minimally expressed on healthy hematopoietic stem cells but consistently enriched on AML blasts and leukemic stem cells. Leveraging the high specificity, solubility, and reduced immunogenicity of llama-derived single-domain variable heavy-chain antibodies, we engineered both CLL-1 and CD33 nanobody CAR constructs and systematically compared their functional activity. Functional validation included real-time cytotoxicity monitoring using IncuCyte imaging of mKate2-labeled AML cells, serial tumor rechallenge assays to assess sustained killing, and NOD/SCID/IL2Rγnull xenograft models to evaluate in vivo efficacy under conditions of high leukemic burden. CLL-1 and CD33 CAR T cells demonstrated rapid and durable cytotoxicity, with significant killing efficiency at low effector-to-target ratios (0.33:1). Unlike CD33 CAR T cells, CLL-1–directed CARs spared normal hematopoietic progenitors, preserving colony-forming capacity. Importantly, CLL-1 CAR T cells retained a favorable memory phenotype with stable proliferation and viability, whereas cytokine release assays confirmed effective yet antigen-specific immune activation. In vivo, treatment with CLL-1 CAR T cells resulted in profound and sustained tumor regression in AML xenografts, accompanied by the persistence of functional CAR T cells. Together, these findings establish CLL-1–targeted nanobody-based CAR T cells as a precision-engineered immunotherapy with potent antileukemic activity, reduced off-target toxicity, and enhanced translational potential. This platform provides a promising therapeutic avenue to overcome current barriers in AML CAR T-cell development and improve patient outcomes.Significance:Nanobody-based CLL-1 CAR T-cell therapy balances potent antitumor activity with hematopoietic preservation, highlighting the potential of our CLL-1 CAR T-cell platform as a next-generation, safer, and clinically superior strategy for effective AML treatment.

Siglec-6 is a novel target for CAR T-cell therapy in acute myeloid leukemia

Anti-CD123 Chimeric Antigen Receptor T Cells (CART-123) Provide A Novel Myeloablative Conditioning Regimen That Eradicates Human Acute Myeloid Leukemia In Preclinical Models

Minimal Residual Disease following Allogeneic Hematopoietic Stem Cell Transplantation

Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here, we isolated a high affinity (HA) folate receptor beta (FRβ)-specific scFv (2.48nM KD) for optimization of FRβ-redirected CAR T-cell therapy for AML. T-cells stably expressing the HA-FRβ CAR exhibited greatly enhanced antitumor activity against FRβ+ AML in vitro and in vivo compared to a low affinity (LA) FRβ CAR (54.3nM KD). Using the HA-FRβ IgG, FRβ expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34+ hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FRβ CAR T-cells lysed mature CD14+ monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FRβ CAR T-cells retained effective anti-tumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FRβ CAR T-cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

Optimizing the IFNγ Axis Improves CAR T-Cell Potency in AML but Not B-ALL

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Simple SummaryChimeric antigen receptor (CAR) therapy has increased treatment options for many patients who have failed standard chemotherapy. So far, CAR therapy has been used more frequently in B-cell mediated cancers due to unique challenges posed by patients with acute myeloid leukemia (AML) and concern for life-threatening side effects. This review discusses both challenges to creating effective and safe CARs for use in AML, as well as recent advances in CAR development both in pre-clinical and human studies. Overall, continued improvement in AML CAR therapy would be of great benefit to a disease that still has a high morbidity and mortality. The advent of chimeric antigen receptor (CAR) T-cell therapy has led to dramatic remission rates in multiple relapsed/refractory hematologic malignancies. While CAR T-cell therapy has been particularly successful as a treatment for B-cell malignancies, effectively treating acute myeloid leukemia (AML) with CARs has posed a larger challenge. AML not only creates an immunosuppressive tumor microenvironment that dampens CAR T-cell responses, but it also lacks many unique tumor-associated antigens, making leukemic-specific targeting difficult. One advantage of CAR T-cell therapy compared to alternative treatment options is the ability to provide prolonged antigen-specific immune effector and surveillance functions. Since many AML CAR targets under investigation including CD33, CD117, and CD123 are also expressed on hematopoietic stem cells, CAR T-cell therapy can lead to severe and potentially lethal myeloablation. Novel strategies to combat these issues include creation of bispecific CARs, CAR T-cell “safety switches”, TCR-like CARs, NK CARs, and universal CARs, but all vary in their ability to provide a sustained remission, and consolidation with an allogeneic hematopoietic cell transplantation (allo-HCT) will be necessary in most cases This review highlights the delicate balance between effectively eliminating AML blasts and leukemic stem cells, while preserving the ability for bone marrow to regenerate. The impact of CAR therapy on treatment landscape of AML and changing scope of allo-HCT is discussed. Continued advances in AML CAR therapy would be of great benefit to a disease that still has high morbidity and mortality.

PI3 Kinase p110 Delta Is Required for Leukemic Cell Survival and Self-Renewal in t(8;21) Acute Myeloid Leukemia

CD44‐targeted chimeric antigen receptor‐T eradicates acute myeloid leukaemic stem cells

Probing the AML Surfaceome for Chimeric Antigen Receptor (CAR) Targets

Structural Surfaceomics Reveals an AML-Specific Conformation of Integrin-β2 As an Immunotherapeutic Target