Stem Cell Transplantation and Cellular Therapy, Surgical Oncology, and Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.
Clin Cancer Res. 2013 Jan 1;19(1):247-57. doi: 10.1158/1078-0432.CCR-12-2753. Epub 2012 Nov 12.
Immunotherapy targeting aberrantly expressed leukemia-associated antigens has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy.
We used Immune Epitope Database and in vitro binding assays to identify immunogenic epitopes derived from CG. Flow cytometry, immunoblotting, and confocal microscopy were used to characterize the expression and processing of CG in AML patient samples, leukemia stem cells, and normal neutrophils. Cytotoxicity assays determined the susceptibility of AML to CG-specific cytotoxic T lymphocytes (CTL). Dextramer staining and cytokine flow cytometry were conducted to characterize the immune response to CG in patients.
CG was highly expressed and ubiquitinated in AML blasts, and was localized outside granules in compartments that facilitate antigen presentation. We identified five HLA-A0201 binding nonameric peptides (CG1-CG5) derived from CG, and showed immunogenicity of the highest HLA-A0201 binding peptide, CG1. We showed killing of primary AML by CG1-CTL, but not normal bone marrow. Blocking HLA-A0201 abrogated CG1-CTL-mediated cytotoxicity, further confirming HLA-A0201-dependent killing. Finally, we showed functional CG1-CTLs in peripheral blood from AML patients following allogeneic stem cell transplantation.
CG is aberrantly expressed and processed in AML and is a novel immunotherapeutic target that warrants further development.
针对异常表达的白血病相关抗原的免疫疗法在急性髓细胞白血病（AML）的治疗中显示出了一定的前景。然而，由于髓性白血病的异质性和克隆进化是其特征之一，针对单一肽表位的靶向治疗取得的成功有限，这突显了对新型抗原发现的需求。在这项研究中，我们研究了髓系嗜天青颗粒蛋白酶组织蛋白酶 G（CG）作为 AML 免疫治疗的新靶点的作用。
我们使用免疫表位数据库和体外结合实验来鉴定源自 CG 的免疫原性表位。流式细胞术、免疫印迹和共聚焦显微镜用于分析 AML 患者样本、白血病干细胞和正常中性粒细胞中 CG 的表达和加工。细胞毒性实验确定了 AML 对 CG 特异性细胞毒性 T 淋巴细胞（CTL）的敏感性。通过 dextramer 染色和细胞因子流式细胞术分析来研究患者对 CG 的免疫反应。
CG 在 AML 原始细胞中高度表达和泛素化，并定位于有助于抗原呈递的颗粒外的隔室中。我们鉴定了源自 CG 的五个 HLA-A0201 结合的非肽 CG1-CG5，并且显示了最高 HLA-A0201 结合肽 CG1 的免疫原性。我们表明 CG1-CTL 可杀伤原代 AML，但不能杀伤正常骨髓。阻断 HLA-A0201 可消除 CG1-CTL 介导的细胞毒性，进一步证实了 HLA-A0201 依赖性杀伤。最后，我们在异基因干细胞移植后 AML 患者的外周血中观察到了功能性的 CG1-CTL。
CG 在 AML 中异常表达和加工，是一种新型免疫治疗靶标，值得进一步开发。
