The combination of 225Ac-anti-CD33 radioimmunoconjugate and CD47 blockade enhances survival in a preclinical model of acute myeloid leukemia

Introduction: CD47, a macrophage checkpoint that suppresses phagocytosis, is upregulated in several cancers including acute myeloid leukemia (AML). The CD47 “don’t eat me” signal is a mechanism which enables AML cells to evade immunosurveillance. By contrast, calreticulin (CRT) is a pro-phagocytic “eat me” signal that enhances the innate immune response to kill tumor cells. Since CD33 is overexpressed in AML cells, we hypothesized that radiation-induced endoplasmic reticulum stress resulting from ²²⁵Ac-radiolabeled anti-CD33 (²²⁵Ac-anti-CD33) treatment will upregulate CRT and potentially synergize with a CD47 blocking agent such as magrolimab to trigger the innate immune response and improve antitumor efficacy. Here, we investigate the potential benefit of combining CD47 blockade with ²²⁵Ac-anti-CD33 (Actimab-A) in a preclinical model of human AML.

Methods: Anti-CD33 (lintuzumab) monoclonal antibody was radiolabeled with ²²⁵Ac via p-SCN-Bn-DOTA. The biological activity and cytotoxicity of ²²⁵Ac-anti-CD33 was evaluated using CD33 positive tumor cell lines. ²²⁵Ac-anti-CD33 mediated CRT upregulation was evaluated in vitro in a panel of heme cell lines using flow cytometry and in vivo in mice bearing U937 xenograft tumors using Positron Emission Tomography (PET). The ability of the ²²⁵Ac-anti-CD33 and anti-CD47 antibody (magrolimab) combination to enhance macrophage phagocytosis in vitro was assessed by flow cytometry. The efficacy resulting from the combination of ²²⁵Ac-anti-CD33 and anti-CD47 antibodies was evaluated in the human MV-4-11 disseminated AML model in vivo.

Results: ²²⁵Ac-anti-CD33 binds CD33 antigen similar to the native antibody and demonstrates specific cytotoxicity to receptor positive cells. ²²⁵Ac-anti-CD33 treatment led to the upregulation of CRT in vitro. Furthermore, treatment with ²²⁵Ac-anti-CD33 induced CRT upregulation in mice bearing U937 tumors as demonstrated by PET. Phagocytosis of CD33 positive cells was significantly enhanced by the ²²⁵Ac-anti-CD33 and anti-CD47 combination compared to each agent alone. The ²²⁵Ac-anti-CD33 and anti-CD47 combination therapy demonstrated a reproducible increase in survival and reduced toxicity in a human AML preclinical model.

Conclusions: This study suggests that the combination of CD47 (“don’t eat me” signal) blockade and CRT (“eat me” signal) upregulation by ²²⁵Ac-anti-CD33 can synergize to increase innate immune response and significantly improve survival in a preclinical model of AML. Our findings warrant further investigation of the potential for this combination to enhance responses in hematological cancers.