CD39 Is Predominantly Expressed in Adverse AML Patients and Blockade By a New Anti-CD39 Nanobody Increases Lysis of AML Blasts

Introduction: The ATP-adenosine pathway has a decisive influence on the generation of the immunosuppressive tumor milieu. It has recently been shown that the ectoenzyme CD39 (ectonucleoside triphosphate diphosphohydrolase-1), the key enzyme of the pathway, regulates tumor-associated CD3⁺ T cells and NK cells in both their differentiation and cytotoxic potential. Recently we showed that CD39⁺CD8⁺ T cells represent a subpopulation that differs significantly between the bone marrow (BM) and peripheral blood (PB) of AML patients and healthy donors. In addition, CD39 has been described to be upregulated in solid and hematologic tumor cells and is increasingly recognized as a promising target for immunotherapeutic approaches. CD39 expression was evaluated on AML blasts and investigated for its functional relevance for acute myeloid leukemia (AML).

Methods: CD39 expression status was assessed on mononuclear cells derived from the BM of patients with AML. Multiparametric flow cytometry (MFC) was performed on age-matched patients with newly diagnosed AML (n=48), relapsed AML (n=9), patients in a stable remission (n=10), and non-malignant controls (n=10). T cell-mediated killing of leukemia cells was investigated by incubating AML cell lines with PBMCs from healthy donors and T cells isolated from the PB of AML patients with their corresponding autologous BM-derived AML blasts. Cytotoxicity was determined by MFC analyses using 7-AAD staining.

Results: Thirty six percent of AML blasts, being defined as CD33⁺CD117⁺ cells, expressed CD39 (median frequency 36.6%). To evaluate the prognostic relevance of CD39, we stratified the patients with pAML according to their European Leukemia Network (ELN) risk groups (favorable n=16, intermediate n=17 or adverse n=15). Patients with an adverse ELN risk showed the highest frequencies and median fluorescence intensity of CD39⁺ AML blasts compared to patients with intermediate and favorable risk (p=0.0002; p=0.0018 vs. intermediate and p=0.01; p=0.01 vs. favorable). Furthermore, patients with relapsed AML showed significantly higher rates of CD39⁺ AML blasts than patients newly diagnosed or in remission (p=0.008; p=0.0001).

Functional investigation was performed using the new developed anti-CD39 nanobody SB24. Blockade on CD3⁺ T cells resulted in the inhibition of ATP degradation, increased CD8⁺ and conventional CD4⁺ T cell proliferation and cytotoxicity (increased secretion of TFN-a, granzyme B and IFN-y). In allogeneic cocultures with the AML cell lines (OCI AML-3, IMS-M2, KG-1, and Kasumi-1, n=5 respectively), the 24h blockade of CD39 increased PBMC-mediated lysis of AML blasts. Stronger effects were observed in the CD39⁺ expressing cell line OCI AML-3 in comparison to IMS-M2, KG-1, and Kasumi-1 were only the effector CD3⁺ T cells expressed CD39 (anti-CD39 vs. isotype ctr: OCI-AML3 p=0.0009; IMS-M2 p=0.007; KG1-a p=0.01; Kasumi-1 p=0.02). Also, in the autologous kill assays using freshly isolated BM-derived AML blasts and PB-derived CD3⁺ T cells (n=5), blockade of CD39 resulted improved killing rates of AML blasts (anti-CD39 vs. isotype ctr: p=0.002). Again, higher lysis rates were observed in AML blasts displaying expression of CD39 >20%.

Conclusions: CD39 is expressed in (adverse) newly diagnosed and relapsed AML. The study demonstrates that blockade of CD39 represents a promising tool to overcome adenosine-mediated immune exhaustion. In vivo validation is currently ongoing to validate the efficacy of targeting CD39 in AML.