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：（改编自研究者的摘要）NK 细胞具有溶细胞作用 对于肿瘤细胞，但自体 NK 细胞治疗的临床应用已 比较不成功。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，并且大多数 这些受体通过发送阻止 NK 细胞裂解的负信号来做出反应。 这解释了为什么 NK 细胞优先裂解 HLA 或 H2 同种异体或 I 类 靶细胞缺陷。在该项目的前 3 年中， 调查人员已获得证据，可以阻止两个人的负面信号 抑制性受体 Ly-49I 和 C 与 F(ab')2 5E6 单克隆抗体可增强 B6 小鼠输注同基因 C1498 髓系白血病细胞。 T 和 B 的使用 细胞缺陷小鼠表明 NK 细胞是效应细胞。相同 治疗不会抑制受辐射的 B6 小鼠中同基因 BMC 的生长（a 安全问题）但确实增强了小鼠排斥同种异体 BMC 的能力 移植物。低剂量的新型 8H7 抗 Ly-49I 单克隆抗体可阻断负信号，且无需 消耗 NK 细胞，可用作半衰期比 NK 细胞更长的试剂 单克隆抗体片段。由于半衰期短，F(ab')2 试剂的功能受到限制 血清中（<18小时）。此续订应用程序有 5 个具体目标： 目标 1. 生成 更有效的 MAb 试剂可阻断 NK 细胞的负信号，而无需 耗尽它们 - 研究人员已将 5E6 MAb 的 Fc 区突变为 去除 MAb 所需的关键 N-碳水化合物附着位点 以消耗体内的细胞。生成针对 Ly-49G2 的类似试剂， 抑制性受体在大部分小鼠 NK 细胞上表达。目标 2。 开发用于体内白血病细胞生长评估的快速检测方法，以加快 开发新试剂的步伐，例如静脉注射白血病细胞进入 受辐射的宿主，并在 5 天后通过测量 DNA 来评估增殖情况 合成，增殖细胞的测定。使用123I-碘脱氧尿苷标记 增殖细胞可用于生长肿瘤的成像和 标记输注的白血病细胞，以便可以通过以下方式确定存活率 全身计数。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“掺入”了不同数量的 目标 4. 扩大临床治疗方案以包括 在输注同基因或IL-2后对小鼠进行补充治疗 涂有抗 5E5 和/或抗 Ly49G2 的同种异体 IL-2 激活的 NK 细胞 F(ab')2 单克隆抗体。目标 5. 将研究扩展到人类髓系白血病的使用 用 asialo GM1 预处理的细胞、人类 NK 细胞和免疫缺陷 SCID 小鼠 或 SCID-NOD 小鼠，接受人类白血病细胞的移植物。不消耗 针对 I 类抗原的人类负信号受体的单克隆抗体或片段 将测试在白血病细胞上表达的抗白血病作用。 这些研究的成功有望确定这种方法是否有效 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