PROTEINASE 3 SPECIFIC IMMUNOTHERAPY OF LEUKEMIA

同种异体骨髓移植（BMT）后，由供体淋巴细胞介导的一种显著的移植物抗白血病（GVL）免疫反应有助于40- 50%的髓系白血病成年患者的治愈。然而，由于患者的年龄和供体的可用性，同种异体骨髓移植仅适用于25%的患者。因此，本提案的总体目标是制定策略，以产生抗原特异性抗白血病免疫反应，用于治疗髓系白血病，可应用于更多患者。蛋白酶3 （Pr3）是一种26kd的骨髓限制性氮性粒细胞颗粒蛋白，通常仅在发育中的早幼粒细胞中最大表达，是韦格纳肉芽肿患者抗中性粒细胞胞浆抗体（ANCA）的抗原。Pr3在75%的慢性髓性白血病（CML）和50%的急性髓性白血病（AML）细胞中过表达3 - 6倍。从Pr3衍生的hla - a2限制性9氨基酸肽PR1已被证明是细胞毒性淋巴细胞（CTL）的靶表位，CTL在体外优先溶解髓系白血病而不是正常骨髓祖细胞。CTL裂解的量与靶细胞中Pr3的过表达有关。在正常供体和一些髓性白血病患者中检测到对PR1的免疫，强烈暗示患者自身的白血病细胞可以作为免疫蛋白的来源，并回避了对PR1存在的免疫反应是否在减缓白血病进展中起作用以及增强免疫反应是否可以提供任何治疗益处的问题。目前的NEW研究者提案将评估对PR1肽的免疫应答的临床重要性，评估髓性白血病患者中基于PR1肽的肿瘤疫苗，并且由于PR1是第一个确定的组织限制性表位，将研究其他独特的hla - a2相关Pr3肽作为CTL的白血病特异性表位。为了研究PR1免疫应答的临床意义，将在CML、AML患者及其正常骨髓供者BMT前后使用限制性稀释分析和使用荧光标记的PR1-四聚体直接标记PR1特异性CTL来测定抗PR1 CTL前体（CTLP）频率。我们将检测白血病中Pr3的表达、白血病表面表型和pr1特异性CTL与CTLP频率和临床结果的相关性。为了研究是否可以增强对PR1的免疫应答，一项I/II期研究（NCI研究编号T98-0017， Molldrem P.I.）将在CML、AML和骨髓增生异常综合征患者中进行不完全弗氏佐剂（IFA） PR1肽基疫苗的研究。德克萨斯大学安德森癌症中心已批准该研究，NCI将提供GMP质量的PR1肽和IFA。对疫苗的免疫反应将通过CTLP的LDA和用荧光pr1四聚体分析pr1特异性CTL的数量来确定，并将与临床反应和骨髓中白血病母细胞的百分比相关。最后，研究人员将研究从Pr3合成的其他hla - a2相关肽在体外诱导供体PBMC中肽特异性CTL的潜力，然后测试其裂解髓性白血病的能力。

A significant graft versus leukemia (GVL) immune response, mediated by donor lymphocytes after allogeneic bone marrow transplantation (BMT), contributes to the cure of 40-50 percent of adult patients with myeloid leukemias. However, allogeneic BMT is only available for 25 percent of all patients due to patient age and donor availability. Therefore, the overall objective of this proposal is to develop strategies for the generation of antigen-specific antileukemia immune responses for the treatment of myeloid leukemias that can be applied to more patients. Proteinase 3 (Pr3) is a 26kd myeloid-restricted azurophil granule protein that is normally maximally expressed only in developing promyelocytes and is an antigen for the antineutrophil cytoplasmic antibody (ANCA) in patients with Wegener's granulomatosis. Pr3 is overexpressed by 3 to 6 fold in 75 percent of chronic myeloid leukemia (CML) and 50 percent of acute myeloid leukemia (AML) cells. An HLA-A2-restricted 9 amino acid peptide derived from Pr3, PR1, has been shown to be a target epitope of cytotoxic lymphocytes (CTL) that preferentially lyse myeloid leukemia over normal bone marrow progenitors in vitro. The amount of CTL lysis correlates with Pr3 overexpression in the target cells. Detection of immunity to PR1 in normal donors and some patients with myeloid leukemia strongly implies that patient's own leukemia cells can act as a source of immunizing protein and begs the issue of whether existent immune responses to PR1 play any role in slowing leukemia progression and whether boosting of immune responses can offer any therapeutic benefit. The current NEW INVESTIGATOR proposal will assess the clinical importance of immune responses to PR1 peptide, evaluate a PR1 peptide-based tumor vaccine in patients with myeloid leukemia, and since Pr1 is the first tissue-restricted epitope identified, will investigate other unique HLA-A2-associated Pr3 peptides as leukemic-specific epitopes for CTL. To investigate the clinical significance of immune responses to PR1, CTL precursor (CTLP) frequency against PR1 will be determined in patients with CML, AML and their normal marrow donors before and after BMT using limiting dilution analysis and by using a fluorescence-tagged PR1-tetramer to directly label PR1-specific CTL. Pr3 expression in leukemia and the surface phenotype of leukemia and PR1-specific CTL will be examined and correlated to CTLP frequency and clinical outcomes. To investigate whether an immune response to PR1 can be boosted, a phase I/II study (NCI study number T98-0017, Molldrem P.I.) of PR1 peptide-based vaccine with incomplete Freund's adjuvant (IFA) will be conducted in patients with CML, AML, and myelodysplastic syndrome. The University of Texas M. D. Anderson Cancer Center has approved the investigation, and NCI will supply GMP quality PR1 peptide and IFA. Immune responses to the vaccine will be determined by LDA of CTLP and by analyzing the number of PR1-specific CTL with the fluorescent PR1-tetramer and will be correlated to clinical response and percent of leukemia blasts in bone marrow. Lastly, other synthetic HLA-A2-associated peptides from Pr3 will be investigated for their potential to elicit peptide-specific CTL from donor PBMC in vitro and then tested for ability to lyse myeloid leukemia.