BLOCKING NEGATIVE SIGNALS TO NK CELLS TO TREAT LEUKEMIA

描述：（改编自研究者摘要）NK细胞具有细胞溶解性 但自体NK细胞治疗的临床应用已经 相对不成功。其主要潜在原因是NK细胞具有 “自身”I类移植抗原的受体，以及大多数 这些受体通过发送阻止NK细胞溶解的负信号来响应。 这解释了为什么NK细胞优先溶解HLA或H2同种异体，或I类 缺陷的靶细胞。在该项目的前三年， 调查人员已经获得证据， 抑制性受体Ly-49 I和C与F（ab '）2 5E 6单抗联合使用， 输注同系C1498髓系白血病细胞的B6小鼠。T和B的使用 细胞缺陷小鼠表明NK细胞是效应细胞。相同的 治疗没有抑制辐射B6小鼠中同基因BMC的生长（a 但确实增强了小鼠排斥同种异体BMC的能力 移植物一种新的8H 7抗Ly-49 I单抗在低剂量下阻断阴性信号， 消耗NK细胞，并且可以用作具有比 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 h）。本更新申请有5个具体目标：目标1。生成 更有效的单克隆抗体试剂阻断NK细胞的阴性信号， 耗尽它们-研究人员已经突变了5E 6单抗的Fc区， 去除MAb所需的关键N-碳水化合物连接位点 来消耗体内的细胞。生成针对Ly-49 G2的类似试剂， 目的1.在小鼠NK细胞上大部分表达抑制性受体。 开发快速检测白血病细胞体内生长的方法， 开发新试剂的速度，例如，将白血病细胞静脉内输注到 照射宿主，并在5天后通过测量DNA 合成，用于增殖细胞的测定。用123 I-碘脱氧尿苷标记 增殖细胞，其可用于生长肿瘤的成像， 标记输注的白血病细胞， 全身计数。目标3。测试试剂的“清除”能力 白血病细胞从同源骨髓细胞“掺入”不同数量的 目的4.扩大临床治疗方案， 用IL-2补充治疗小鼠，在输注同基因或 用抗5E 5和/或抗Ly 49 G2包被的同种异体IL-2活化的NK细胞 F（ab '）2MAb。目标5。将研究扩展到人类骨髓性白血病 细胞、人NK细胞和用去唾液酸GM 1预处理的免疫缺陷SCID小鼠 或SCID-NOD小鼠，接受人类白血病细胞移植。非消耗 针对I类抗原的人负信号受体的MAb或片段 将测试在白血病细胞上表达的抗白血病作用。 这些研究的成功将有望确定这种方法是否具有 临床应用潜力。

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.