Lintuzumab-Ac225 Exerts Mutation Agnostic Antileukemic Activity in Preclinical Models of AML
Authors: Chin Amanda , Jiao Rubin , Allen Kevin J.H. , Li Jason , Chen Mary , Vusirikala Madhuri , Desai Avinash , Xu Le-Cun , Brodin Patrik , Roy Monideepa , Malo Mackenzie , Beckford-Vera Denis , Dadachova Ekaterina , Kotanides Helen 
Published: December 2024

Introduction
Acute myeloid leukemia (AML) is a highly heterogeneous hematologic malignancy with a poor survival prognosis. Genetic aberrations are drivers of leukemogenesis and can negatively impact treatment efficacy and durability of responses in patients, with most relapsing following treatment with small molecule inhibitors and/or standard-of-care chemotherapy. We have shown that myeloid leukemic cells are broadly targeted by lintuzumab-Ac225, a CD33-directed monoclonal antibody conjugated with the alpha particle-emitting radionuclide actinium-225. In clinical trials, lintuzumab-Ac225 has demonstrated promising therapeutic responses when added to CLAG-M chemotherapy in heavily pretreated relapsed/refractory AML patients, including high risk populations with venetoclax failures and TP53 mutations. Since alpha radiation directly causes lethal DNA double strand breaks within targeted cells, the aim of these studies is to determine that lintuzumab-Ac225 is mutation-agnostic agent to commonly found genetic abnormalities especially in AML, including FMS-like tyrosine kinase-3 (FLT3), tumor protein p53 (TP53), isocitrate dehydrogenases 1/2 (IDH1/2), nucleophosmin 1 (NPM1) and lysine methyltransferase 2A (KMT2A). We investigate lintuzumab-Ac225 anti-leukemic effects in preclinical AML models harboring these mutations either as a single agent or in combinations, to determine how alpha radiation may enhance response of targeted therapies for AML.
Methods
Lintuzumab-Ac225 was generated by conjugating lintuzumab with p-SCN-Bn-DOTA and subsequently radiolabeled with Ac-225. A viability assay was performed in vitro with test drugs using flow cytometry to assess the response across genetically altered human AML cell lines (mutant FLT3, KMT2A: MV-4-11, MOLM-13 cells, mutant NPM1: OCI-AML3, and mutant TP53: KASUMI-1, HL-60 cells). Lintuzumab-Ac225, FLT3 inhibitors (gilteritinib, quizartinib) and KMT2A inhibitors (revumenib, ziftomenib) were evaluated as single agents and combinations. The modulation of DNA damage markers (phosphorylated H2A.X, p21) in response to lintuzumab-Ac225 was examined by western blot. The effect of lintuzumab-Ac225 on AML growth in vivo was investigated in subcutaneous MV-4-11 leukemia xenograft models in nude mice.
Results
Treatment with lintuzumab-Ac225 caused a potent dose-dependent reduction of leukemia cell viability in vitro relative to cold lintuzumab across all AML models irrespective of FLT3, KMT2A, NPM1 or TP53 mutations. All evaluated cell lines showed a response, with IC50 activity at 48 hours that ranged between 0.5 and 5 nCi/mL, indicating absence of resistance to lintuzumab-Ac225 radiation induced cell death. Consistent with these data, markers of DNA damage and proapoptotic pathways including phosphorylated H2A.X and p21, showed rapid activation by exposure of the mutant cells to lintuzumab-Ac225. Although the single agent cytotoxicity of FLT3 inhibitors (gilteritinib, quizartinib) and KMT2A inhibitors (revumenib, ziftomenib) was observed with varied sensitivity in both MV-4-11 and MOLM-13 FLT3/KMT2A mutation-carrying cells, the combinations with lintuzumab-Ac225 were additive at all studied dose levels (p&lt;0.01). In the MV-4-11 in vivo AML efficacy model, monotherapy of FLT3 and KMT2A inhibitors delayed tumor growth as single agents. Lintuzumab-Ac225 in combination with each of these inhibitors demonstrated statistically significant enhanced tumor control (p&lt;0.002).
Conclusions
Our findings demonstrate broad single agent anti-leukemic activity of lintuzumab-Ac225 agnostic to the genomic mutational landscape of AML especially FLT3, KMT2A, NPM1 and TP53. CD33-targeted radiation-induced damage improves AML control in adverse-risk settings, and further potentiates response durability when added to standard-of-care, including molecularly targeted therapy. Collectively, these data support the backbone therapy potential of lintuzumab-Ac225 in a mutation agnostic manner, warranting further clinical evaluation in difficult to treat relapsed/refractory AML.