Simkins Stephen G, Knapp Steven L, Brough George H, Lenz Karen L, Barley-Maloney Lise, Baker Jeffrey P, Dekking Liesbeth, Wai Hobert, Dixon Eric P
BD Diagnostics, Women's Health and Cancer, Durham, North Carolina, USA.
Hybridoma (Larchmt). 2011 Oct;30(5):433-43. doi: 10.1089/hyb.2011.0037.
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.
急性髓系白血病中常见的染色体易位t(8;21)会产生致癌融合蛋白AML1-ETO。这种嵌合致癌蛋白会破坏野生型AML1的功能，并使对正常骨髓生成至关重要的基因失调。能够捕获和检测AML1-ETO融合蛋白的单克隆抗体将有助于急性髓系白血病的早期诊断和治疗预后。我们报告了针对由AML1或ETO编码的表位特异性结合的鼠单克隆抗体（MAb）的研发情况。由于与人类ETO蛋白比对显示与小鼠直系同源物几乎100%同源，因此需要一种策略来指导小鼠的体液免疫聚焦并响应自身表位。我们开发捕获/检测试剂的策略包括产生能与未融合的骨髓蛋白（即AML1或ETO）内表位结合的单克隆抗体。这包括一个选择抗体的过程，这些抗体还需具备识别易位染色体AML1-ETO融合蛋白的能力，并鉴定互补的捕获/检测抗体对。构建肽半抗原-载体复合物并使用快速免疫方案产生了IgM-IgG ETO特异性单克隆抗体。这些单克隆抗体特异性结合在HEK中表达的重组形式的AML1-ETO融合蛋白以及在Kasumi-1中表达的内源性AML1-ETO融合蛋白形式。我们报告了源自用肽“自身表位”免疫的鼠杂交瘤单克隆抗体的研发情况。我们的研究结果提供了一种潜在策略，可指导小鼠的体液免疫聚焦并响应自身表位。该策略已通过参与急性髓系白血病的致癌融合蛋白AML1-ETO得到验证。
