ABSTRACT
Targeted immunotherapy in acute myeloid leukemia (AML) is challenged by the lack of AML-specific target antigens and clonal heterogeneity, leading to unwanted on-target off-leukemia toxicity and risk of relapse from minor clones. We hypothesize that combinatorial targeting of AML cells can enhance therapeutic efficacy without increasing toxicity. To identify target antigen combinations specific for AML and leukemic stem cells, we generated a detailed protein expression profile based on flow cytometry of primary AML (n = 356) and normal bone marrow samples (n = 34), and a recently reported integrated normal tissue proteomic data set. We analyzed antigen expression levels of CD33, CD123, CLL1, TIM3, CD244 and CD7 on AML bulk and leukemic stem cells at initial diagnosis (n = 302) and relapse (n = 54). CD33, CD123, CLL1, TIM3 and CD244 were ubiquitously expressed on AML bulk cells at initial diagnosis and relapse, irrespective of genetic characteristics. For each analyzed target, we found additional expression in different populations of normal hematopoiesis. Analyzing the coexpression of our six targets in all dual combinations (n = 15), we found CD33/TIM3 and CLL1/TIM3 to be highly positive in AML compared with normal hematopoiesis and non-hematopoietic tissues. Our findings indicate that combinatorial targeting of CD33/TIM3 or CLL1/TIM3 may enhance therapeutic efficacy without aggravating toxicity in immunotherapy of AML.
Subject(s)
Antigens, Neoplasm/metabolism , Antineoplastic Agents/therapeutic use , Bone Marrow/metabolism , Leukemia, Myeloid, Acute/metabolism , Molecular Targeted Therapy , Neoplastic Stem Cells/metabolism , Proteome/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Bone Marrow/drug effects , Bone Marrow/pathology , Case-Control Studies , Cells, Cultured , Cohort Studies , Female , Follow-Up Studies , Humans , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/pathology , Male , Middle Aged , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/pathology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Prognosis , Young AdultABSTRACT
The fms-related tyrosine kinase 3 (FLT3) receptor has been extensively studied over the past two decades with regard to oncogenic alterations that do not only serve as prognostic markers but also as therapeutic targets in acute myeloid leukemia (AML). Internal tandem duplications (ITDs) became of special interest in this setting as they are associated with unfavorable prognosis. Because of sequence-dependent protein conformational changes FLT3-ITD tends to autophosphorylate and displays a constitutive intracellular localization. Here, we analyzed the effect of tyrosine kinase inhibitors (TKIs) on the localization of the FLT3 receptor and its mutants. TKI treatment increased the surface expression through upregulation of FLT3 and glycosylation of FLT3-ITD and FLT3-D835Y mutants. In T cell-mediated cytotoxicity (TCMC) assays, using a bispecific FLT3 × CD3 antibody construct, the combination with TKI treatment increased TCMC in the FLT3-ITD-positive AML cell lines MOLM-13 and MV4-11, patient-derived xenograft cells and primary patient samples. Our findings provide the basis for rational combination of TKI and FLT3-directed immunotherapy with potential benefit for FLT3-ITD-positive AML patients.
Subject(s)
Leukemia, Myeloid, Acute/therapy , Protein Kinase Inhibitors/pharmacology , Protein-Tyrosine Kinases/metabolism , Tandem Repeat Sequences/drug effects , fms-Like Tyrosine Kinase 3/metabolism , Biomarkers, Tumor/metabolism , Cell Line, Tumor , Humans , Immunotherapy/methods , Leukemia, Myeloid, Acute/metabolism , Mutation/drug effects , Mutation/genetics , Prognosis , Up-Regulation/drug effects , Up-Regulation/geneticsABSTRACT
Bispecific T-cell engagers (BiTEs) are very effective in recruiting and activating T cells. We tested the cytotoxicity of the CD33/CD3 BiTE antibody construct AMG 330 on primary acute myeloid leukemia (AML) cells ex vivo and characterized parameters contributing to antileukemic cytolytic activity. The E:T ratio and the CD33 expression level significantly influenced lysis kinetics in long-term cultures of primary AML cells (n=38). AMG 330 induced T-cell-mediated proinflammatory conditions, favoring the upregulation of immune checkpoints on target and effector cells. Although not constitutively expressed at the time of primary diagnosis (n=123), PD-L1 was strongly upregulated on primary AML cells upon AMG 330 addition to ex vivo cultures (n=27, P<0.0001). This phenomenon was cytokine-driven as the sole addition of interferon (IFN)-γ and tumor necrosis factor-α also induced expression. Through blockade of the PD-1/PD-L1 interaction, AMG 330-mediated lysis (n=9, P=0.03), T-cell proliferation (n=9, P=0.01) and IFN-γ secretion (n=8, P=0.008) were significantly enhanced. The combinatorial approach was most beneficial in settings of protracted AML cell lysis. Taken together, we have characterized a critical resistance mechanism employed by primary AML cells under AMG 330-mediated proinflammatory conditions. Our results support the evaluation of checkpoint molecules in upcoming clinical trials with AMG 330 to enhance BiTE antibody construct-mediated cytotoxicity.
