ABSTRACT
The chromosomal translocation t(8;21) often found in acute myeloid leukemia generates an oncogenic fusion protein AML1-ETO. This chimeric oncoprotein disrupts wild-type AML1 function and dysregulates genes important for normal myelopoiesis. Monoclonal antibodies that can capture and detect the AML1-ETO fusion protein would help with early diagnosis and treatment prognosis of acute myeloid leukemia. We report the development of murine monoclonal antibodies (MAbs) that specifically bind epitopes encoded by either AML1 or ETO. Since alignment to the human ETO protein indicated almost 100% homology to the mouse ortholog, a strategy was needed to instruct humoral immunity in mice to focus and respond to self-epitopes. Our strategy to develop capture/detector reagents involved producing MAbs that would bind to epitopes within the non-fused myelopic protein (i.e., either AML1 or ETO). This included a process to select antibodies for their ability to also recognize the translocated chromosomal AML1-ETO fusion protein and to identify complementary capture/detector antibody pairs. Construction of a peptide hapten-carrier complex and use of a rapid immunization protocol resulted in IgM-IgG ETO specific MAbs. These MAbs bound specifically to a recombinant form of AML1-ETO fusion protein expressed in HEK and to an endogenous AML1-ETO form of the fusion protein expressed in Kasumi-1. We report the development of murine hybridoma MAbs derived from immunizations with a peptide "self-epitope." Our findings provide a potential strategy to instruct humoral immunity in mice to focus and respond to self-epitopes. This strategy has been validated with the oncogenic fusion protein AML1-ETO involved in acute myeloid leukemia.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/biosynthesis , Core Binding Factor Alpha 2 Subunit/immunology , Proto-Oncogene Proteins/immunology , Recombinant Fusion Proteins/immunology , Transcription Factors/immunology , Amino Acid Sequence , Animals , Antibody Affinity , Antibody Specificity , Cloning, Molecular , Core Binding Factor Alpha 2 Subunit/isolation & purification , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , Humans , Hybridomas/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Proto-Oncogene Proteins/isolation & purification , RUNX1 Translocation Partner 1 Protein , Recombinant Fusion Proteins/isolation & purification , Sequence Homology, Amino Acid , Transcription Factors/isolation & purificationABSTRACT
Tyrosine kinase inhibitors have profoundly modified the treatment and prognosis of chronic myeloid leukemia and Ph(+) acute lymphoblastic leukemia. A rapid and accurate detection of the BCR-ABL fusion protein is paramount today for an optimal management of Ph(+) acute lymphoblastic leukemia. We have utilized a recently described and commercialized immunoassay that identifies qualitatively the presence of the BCR-ABL protein in leukemic cell lysates. The BCR-ABL fusion protein is captured and detected by a cytometric bead assay and analyzed by flow cytometry. The assay was applied to 101 primary patient samples (94 acute leukemias and 7 chronic myeloid leukemia blast crisis) and the results of the immunoassay were concordant with those obtained by conventional molecular techniques. The method proved reliable, reproducible, of simple execution and it was successfully completed within four hours. This flow cytometric immunoassay has important implications for perfecting the management of Ph(+) acute lymphoblastic leukemia patients worldwide.
