BLOCKING NEGATIVE SIGNALS TO NK CELLS TO TREAT LEUKEMIA

阻断 NK 细胞的负信号来治疗白血病

• 批准号: 6633105
• 负责人: Michael Bennett
• 金额: $ 21.06万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6633105
• 关键词: NOD mouse; SCID mouse; antireceptor antibody; athymic mouse; biological signal transduction; bone marrow transplantation; cytotoxicity; disease /disorder model; human tissue; method development; monoclonal antibody; myelogenous leukemia; natural killer cells; neoplasm /cancer immunotherapy

DESCRIPTION: (Adapted from the investigator's abstract) NK cells are cytolytic for tumor cells but clinical use of autologous NK cell treatment has been relatively unsuccessful. A major potential cause for this is that NK cells have receptors for 'self' class I transplantation antigens, and the majority of these receptors respond by sending negative signals that prevent NK cell lysis. This explains why NK cells preferentially lyse HLA or H2 allogeneic, or class I deficient target cells. During the first 3 years of this project, the investigators have obtained evidence that blocking negative signals for two inhibitory receptors, Ly-49I and C, with F(ab')2 5E6 MAbs enhanced survival of B6 mice infused with syngeneic C1498 myeloid leukemia cells. Use of T and B cell deficient mice indicated that NK cells were the effectors. The same treatment did not inhibit growth of syngeneic BMC in irradiated B6 mice (a safety concern) but did enhance the ability of mice to reject allogeneic BMC grafts. A new 8H7 anti-Ly-49I MAb at low doses blocks negative signals without depleting NK cells and can be used as a reagent with a longer half-life than MAb fragment. The F(ab')2 reagent is limited in function due to short half-life in serum (<18h). This renewal application has 5 specific aims: Aim 1. Generate more effective MAb reagents to block negative signals to NK cells without depleting them - the investigators have mutated the Fc region of 5E6 MAb to remove a critical N-carbohydrate attachment site that is required for the MAbs to deplete cells in vivo. Generate similar reagents against Ly-49G2, an inhibitory receptor expressed on a large fraction of murine NK cells.Aim 2. Develop rapid assays for growth assessment of leukemia cells in vivo to quicken the pace of developing new reagents, e.g., infusion of leukemic cells i.v. into irradiated hosts, and assessing proliferation 5 days later by measuring DNA synthesis, an assay for proliferating cells. Use 123I-iododeoxyuridine to label proliferating cells that can be used for imaging of growing tumors and for labeling leukemia cells that are infused so that survival can be determined by whole body counting. Aim 3. Test the reagents for the ability to 'purge' leukemia cells from syngeneic marrow cells 'spiked' with different numbers of leukemia cells.Aim 4. Expand the clinical treatment protocol to include supplemental treatment of mice with IL-2 after the infusion of syngeneic or allogeneic IL-2 activated NK cells coated with anti-5E5 and/or anti-Ly49G2 F(ab')2 MAbs. Aim 5. Extend the studies to the use of human myeloid leukemia cells, human NK cells, and immunodeficient SCID mice pretreated with asialo GM1 or SCID-NOD mice, which accept grafts of human leukemia cells. Non-depleting MAbs or fragments to human negative signaling receptors for class I antigens expressed on the leukemia cells will be tested for anti-leukemia effects. Success with these studies will hopefully determine if this approach has potential for clinical application.

描述：(改编自研究人员的摘要)NK细胞具有细胞溶解功能 但临床上使用自体NK细胞治疗肿瘤细胞已 相对不成功。造成这种情况的一个主要潜在原因是NK细胞 自身I类移植抗原的受体，以及大多数 这些受体通过发出阻止NK细胞裂解的负面信号来做出反应。 这就解释了为什么NK细胞优先溶解人类白细胞抗原或H2同种异体，或I类 目标细胞不足。在这个项目的头3年里， 调查人员已获得证据，阻断两种负性信号 抑制性受体Ly-49I和C与F(ab‘)25E6单抗联合应用提高小鼠存活率 B6小鼠输注同基因C1498髓系白血病细胞。T和B的用法 细胞缺陷小鼠提示NK细胞是效应细胞。相同 治疗不能抑制B6小鼠(A)同基因BMC的生长 安全性考虑)，但确实增强了小鼠排斥异基因BMC的能力 嫁接。一种新的8H7抗Ly-49I单抗在低剂量时阻断负信号而不 消耗NK细胞，可用作半衰期长于 单抗片段。F(ab‘)2试剂由于半衰期短而功能受限 在血清中(&lt；18h)。此续订申请有5个具体目标：目标1.生成 更有效的单抗试剂可以阻断NK细胞的负面信号，而无需 耗尽它们--研究人员已经将5E6单抗的Fc区突变为 去除单抗所需的关键N-碳水化合物结合部位 消耗体内的细胞。产生针对Ly-49G2的类似试剂，以及 抑制性受体在小鼠NK细胞上的大量表达。 建立白血病细胞体内生长的快速检测方法 开发新试剂的速度，例如，静脉输注白血病细胞。vt.进入，进入 照射宿主，并在5天后通过测量DNA来评估增殖 合成，一种细胞增殖的检测方法。用~(123)I-碘脱氧尿苷标记 增殖细胞，可用于正在生长的肿瘤的成像和 标记注入的白血病细胞，以便通过以下方式确定存活率 整个身体都在数。目标3.测试试剂的“净化”能力 来自同基因骨髓细胞的白血病细胞被不同数量的 白血病细胞。目标4.扩大临床治疗方案以包括 小鼠输注同基因或异体血后补充IL-2的实验研究 抗5E5和/或抗Ly49G2包被的同种异体IL-2激活的NK细胞 F(ab‘)2个单抗。目的5.将研究扩展到人类髓系白血病的用途 Asialo GM1预处理的细胞、人NK细胞和免疫缺陷的SCID小鼠 或者接受人类白血病细胞移植的SCID-NOD小鼠。不会耗尽 抗人I类抗原负性信号受体的单抗或片段 在白血病细胞上表达的蛋白将被检测为抗白血病效果。 这些研究的成功将有望确定这种方法是否 具有临床应用潜力。

项目成果

B6 strain Ly49I inhibitory gene expression on T cells in FVB.Ly49IB6 transgenic mice fails to prevent normal T cell functions.

B6株Ly49I抑制FVB.Ly49IB6转基因小鼠T细胞上的基因表达，不能阻止正常T细胞功能。

• DOI: 10.4049/jimmunol.169.7.3661
• 发表时间: 2002
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Morris,MargaretA;Liu,Jingxuan;Arora,Veera;George,ThaddeusC;Klem,Jennifer;Schatzle,JohnD;Kumar,Vinay;Bennett,Michael
• 通讯作者: Bennett,Michael

NK inhibitory-receptor blockade for purging of leukemia: effects on hematopoietic reconstitution.

NK 抑制性受体阻断清除白血病：对造血重建的影响。

• DOI: 10.1053/bbmt.2002.v8.pm11846352
• 发表时间: 2002
• 期刊: Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation
• 作者: Koh,CrystalY;Raziuddin,Arati;Welniak,LisbethA;Blazar,BruceR;Bennett,Michael;Murphy,WilliamJ
• 通讯作者: Murphy,WilliamJ

Inhibition of the death receptor pathway by cFLIP confers partial engraftment of MHC class I-deficient stem cells and reduces tumor clearance in perforin-deficient mice.

cFLIP 对死亡受体途径的抑制使 MHC I 类缺陷干细胞部分植入，并减少穿孔素缺陷小鼠的肿瘤清除率。

• DOI: 10.4049/jimmunol.167.8.4230
• 发表时间: 2001
• 期刊: Journal of immunology (Baltimore, Md. : 1950)
• 作者: Taylor,MA;Chaudhary,PM;Klem,J;Kumar,V;Schatzle,JD;Bennett,M
• 通讯作者: Bennett,M

Definition of additional functional ligands for Ly49I(B6) using FVBLy49I(B6) transgenic mice and B6 natural killer cell effectors.

使用 FVBLy49I(B6) 转基因小鼠和 B6 自然杀伤细胞效应器定义 Ly49I(B6) 的其他功能配体。

• DOI: 10.1097/00007890-200211270-00018
• 发表时间: 2002
• 期刊: Transplantation
• 影响因子: 6.2
• 作者: Morris,MargaretA;Koulich,Elena;Liu,Jingxuan;Arora,Veera;George,ThaddeusC;Schatzle,JohnD;Kumar,Vinay;Bennett,Michael
• 通讯作者: Bennett,Michael