SPECIFIC T CELL RESPONSES TO BCR-ABL CHIMERIC PROTEIN

慢性粒细胞白血病(CML)的标志是易位 从9号染色体到特定断裂点的c-abl癌基因 22号染色体上bcr基因的簇区(Bcr)。换位 结果形成了一种新的融合bcr-abl基因，它编码一种 210kD的嵌合蛋白具有异常的酪氨酸激酶活性。C- ABL和BCR基因由正常细胞表达，因此编码的 蛋白质大概是非免疫原性的。然而，加入地区 P210嵌合蛋白的片段由独特的序列组成 C-ABL氨基酸连接到BCR氨基酸连接到BCR氨基酸 它们只在恶性细胞中表达。初步研究表明 结果表明，bcr-abl蛋白连接区对T细胞具有免疫原性。 细胞。Bcr-abl蛋白是吸引人的候选靶点 免疫治疗的几个原因包括：(A)bcr-abl蛋白是一种 许多恶性肿瘤患者共有的抗原-在 超过95%的慢性粒细胞白血病患者和13%的急性白血病患者 淋巴细胞白血病；(B)bcr-abl蛋白与 恶变与恶变的维护 表型。因此，抗原阴性的变种是不可能的；(C)T细胞 能够识别并响应BCR/ABL的接合区域片段 仅由恶性细胞表达的蛋白质。因此，治疗将会 有选择性，毒性最小；(D)正常骨髓 前体通常与恶性克隆共存于骨髓中 对慢性粒细胞白血病患者，从而选择性地摧毁恶性克隆 将保持正常的造血功能；(E)慢性粒细胞白血病的T细胞治疗 异基因骨髓移植(BMT)的形式已经 已经被证明是有效的。发生移植物抗宿主病的慢性粒细胞白血病患者 人类白细胞抗原相合骨髓移植后的疾病(GVHD)很少复发，这意味着 识别次要组织相容性抗原的供体T细胞能够 根除慢性粒细胞白血病细胞。免疫BCR-ABL的T细胞可能作为T细胞发挥作用 对次要组织相容性抗原免疫但没有 GVHD的毒性；(F)T细胞耐受性和 对恶性细胞表达的蛋白质无反应很容易 通过使用人类白细胞抗原相合的骨髓捐赠者作为来源 免疫T细胞用于特定的过继治疗--一种已经展示的程序 有效地转移病毒特异性免疫。 目前提案的具体目标是：(1)制定方法，以 用bcr-abl特异性的CD4+T细胞治疗小鼠肿瘤模型； (2)开发生成和使用BCR-ABL特定语言的方法 小鼠模型中I类MHC限制性CD8+T细胞；(3)确定 慢性粒细胞白血病患者是否存在p210 bcr-T细胞 ABL蛋白；(4)判断CML患者是否存在 CD8+T细胞与p210 bcr-abl结合；(5)确定T细胞 可以检测和获得与p210 bcr-abl蛋白有反应的蛋白 来自正常人，特别是人类白细胞抗原相合的兄弟姐妹 作为免疫T细胞的来源，作为骨髓的一部分 移植程序；(6)确定慢性粒细胞白血病患者是否 对p210 bcr-abl蛋白产生特异性体液反应；(7) 在小鼠身上开发和评估适合于 最终在人类身上使用。

The hallmark of chronic myelogenous leukemia (CML) is the transposition of the c-abl oncogene from chromosome 9 to the specific breakpoint cluster region (bcr) of the bcr gene on chromosome 22. The transposition results in the formation of a new fusion bcr-abl gene which encodes a 210 kD chimeric protein with abnormal tyrosine kinase activity. The c- abl and bcr genes are expressed by normal cells and thus the encoded proteins are presumably non-immunogenic. However, the joining region segment of the p210 chimeric protein is composed of unique sequences of c-abl amino acids joined to bcr amino acids joined to bcr amino acids which are expressed only by malignant cells. Preliminary studies have shown that the joining region of bcr-abl protein is immunogenic to T cells. Bcr-abl proteins are appealing candidate targets for immunotherapy for several reasons including: (a) bcr-abl protein is an antigen shared by many individuals with malignancy-being detected in greater than 95% of patients with CML and 13% of patients with acute lymphocytic leukemia; (b) bcr-abl protein is intimately associated with malignant transformation as well as maintenance of the malignant phenotype. Thus, antigen-negative variants are not likely; (c) T cells can recognize and respond to the joining region segment of bcr/abl protein which is expressed only by malignant cells. Thus therapy would be selective with minimal potential for toxicity; (d) normal bone marrow precursors commonly co-exist with the malignant clone in the bone marrow of CML patients, so that selective destruction of the malignant clone would leave normal hematopoiesis intact; (e) T cell therapy of CML in the form of allogeneic bone marrow transplantation (BMT) has already been shown to be effective. Patients with CML who develop graft-vs-host disease (GVHD) following HLA-identical BMT rarely relapse, implying that donor T cells recognizing minor histocompatibility antigens are capable of eradicating CML cells. T cells immune to bcr-abl might function as T cells immune to minor histocompatibility antigens but without the toxicity of GVHD; (f) the potential problems of T cell tolerance and anergy to proteins expressed by malignant cells can be easily circumvented by using an HLA-identical bone marrow donor as a source of immune T cells for specific adoptive therapy- a procedure already shown to be effective for transferring virus-specific immunity. The specific aims of the current proposal are: (1) tp develop methods to use bcr-abl specific CD4+ T cells for tumor therapy in murine models; (2) to develop methods for generating and utilizing bcr-abl specific class I MHC-restricted CD8+ T cells in murine models; (3) to determine whether patients with CML have existent CD4+ T cells primed to p210 bcr- abl protein; (4) to determine whether patients with CML have existent CD8+ T cells primed to p210 bcr-abl; (5) to determine whether T cells with reactivity to p210 bcr-abl protein can be detected and procured from normal individuals, particularly HLA-identical siblings who might serve as a source of immune T cells to be used as part of a bone marrow transplantation procedure; (6) to determine whether patients with CML develop specific humoral responses to p210 bcr-abl protein; (7) to develop and evaluate bcr-abl vaccines in mice that are appropriate for eventual use in humans.