PROTEINASE 3 SPECIFIC IMMUNOTHERAPY OF LEUKEMIA

异基因骨髓移植(BMT)后供者淋巴细胞介导的移植物抗白血病(GVL)免疫反应有助于40%-50%的成人髓系白血病患者的治愈。然而，由于患者年龄和供者可获得性的原因，异基因骨髓移植只适用于25%的患者。因此，这项建议的总体目标是开发产生抗原特异性抗白血病免疫反应的策略，用于治疗髓系白血病，并可应用于更多的患者。蛋白水解酶3(PR3)是一种26kD的髓系限制性天青颗粒蛋白，正常情况下仅在发育中的早幼粒细胞中大量表达，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%的慢性髓系白血病(CML)和50%的急性髓系白血病(AML)细胞中过度表达3-6倍。人类白细胞抗原A2限制性9氨基酸肽来源于PR3，PR1，已被证明是细胞毒性淋巴细胞(CTL)的靶表位，在体外优先溶解髓系白血病而不是正常骨髓前体细胞。CTL裂解量与靶细胞中PR3的过度表达有关。在正常供者和一些髓系白血病患者中检测到对PR1的免疫，强烈表明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的现有免疫反应是否在减缓白血病进展中发挥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。目前的新研究方案将评估对PR1多肽的免疫反应的临床重要性，评估基于PR1多肽的肿瘤疫苗在髓系白血病患者中的应用，由于PR1是第一个被发现的组织限制性表位，因此将研究其他独特的与HLA-A2相关的PR3多肽作为白血病CTL的特异性表位。为探讨PR1免疫应答的临床意义，采用限制性稀释法和荧光标记的PR1四聚体直接标记PR1特异性CTL，检测了CML、AML患者及其正常骨髓供者骨髓移植前后抗PR1的CTL前体细胞(CTLP)频率。将检测白血病中PR3的表达、白血病的表面表型和PR1特异性CTL，并将其与CTLP频率和临床结果相关联。为了研究是否可以增强对PR1的免疫反应，一项I/II期研究(NCI研究号T98-0017，Molldrem P.I.)将对CML、AML和骨髓增生异常综合征患者进行带有不完全弗氏佐剂(IFA)的PR1多肽疫苗的研究。德克萨斯大学安德森癌症中心已经批准了这项研究，NCI将提供GMP质量的PR1多肽和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细胞的百分比相关。最后，我们将研究从PR3中合成的其他人类白细胞抗原-A2相关多肽在体外从供者PBMC中诱生多肽特异性CTL的潜力，然后测试它们溶解髓系白血病的能力。

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.