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: 2390914
• 负责人: Michael Bennett
• 金额: $ 18.3万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-06-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2390914
• 关键词: MHC class I antigen; antireceptor antibody; biological signal transduction; bone marrow transplantation; chimeric proteins; disease /disorder model; flow cytometry; human genetic material tag; hybridomas; immunoglobulin G; interferons; interleukin 2; laboratory mouse; leukemia; lymphoma; melanoma; method development; molecular cloning; natural killer cells; neoplasm /cancer immunotherapy; tissue /cell culture

Natural killer (NK) cells are cytolytic for tumor cells, and stimulation of NK cells with low doses of interleukin-2 (IL2) have produced dramatic treatment results, especially in patients receiving T cell depleted allogeneic marrow transplants. Less success has been obtained by infusing autologous NK cells and giving high doses of IL2. NK cells are also involved in the rejection of incompatible bone marrow cell (BMC) grafts in rodents. The immunogenetics of BMC transplantation is unusual in that parental strain BMC are often rejected by F1 hybrid mice (hybrid resistance). Apparently NK cells will lyse target cells that fail to send negative signals to NK cells through certain receptors. These receptors are coded for by genes in the Ly49 NK gene family on mouse chromosome 6. One of these receptors, 5E6(Ly49C), receives negative signals from H2+ class I Ags. This explains why the 5E6 subset of Nk cells is responsible for the rejection of parental or allogeneic H2d but not H2b BMC grafts. A new in vitro assay mimics BMC grafts in vivo, which involves the lysis of lymphoblasts by NK cells. The inability of 5E6+ NK cells to lyse H2b target cells can be reversed by adding F(ab')2 anti-5E6 mAb, blocking the negative signals. In this assay syngeneic target cells are not lysed; even 5E6+ H2d NK cells fail to lyse syngeneic blasts unless F(ab'2 anti-5E6 is added. The knowledge that presence or absence of negative signals from class I Ags to NK cell receptors regulates lysis of target cells provides a new approach to utilize NK cells to treat syngeneic or autologous leukemia/lymphoma and other neoplasias. We have developed two specific aims for this proposal. In Aim 1, we will generate F(ab')2 anti5E6 by enzymatic digestion of whole mAbs and a recombinant anti-E6 mAb in which the murine Fc portion is replaced by human IgGI (Hgamma1)Fc fragment. To create this, the VDJ fragment of the anti-5E6 hybridoma IgG2a heavy chain gene will be spliced to the Hgamma1 gene in one vector, and the anti-5E6 kappa light chain gene and a neomycin resistance gene will be created in a second vector. These two vectors will be cotransfected into SP2/O hybridoma cells. The anti-5E6-Hgamma1 should not fix complement or cause antibody dependent cellular cytotoxicity (ADCC). We will also generate a recombinant 5E6 (extracellular domain)-Hgamma1 construct 5E6-Hgamma1 to be transfected into SP2/O cells. In Aim 2, we will first determine if the administration of F(ab')2 anti5E6, anti-5E6-Hgamma1 or 5E6-Hgamma1 to irradiated mice infused with syngeneic BMC can cause resistance to engraftment, presumably by blocking negative signals in a large fraction of 5E6+NK cells. We will then proceed to treat EL4 lymphomas and B16.F10 melanomas [B6 mice], L1210 lymphoma cells [DBA/2 and C.B-17 severe combined immune deficient (SCID) mice], and Friend virus leukemia [BALB/c mice]. Some mice will receive iL2 or interferon alpha/beta to boost NK cell activity. The methods will be to initially inject the reagents intraperitoneally (i.p.), and later the hybridomas secreting the anti-5E6- Hgamma1 or 5E6-Hgamma1 will be put in diffusion chambers i.p. to create a continuous supply of the reagents. Thus, this is a model of cancer therapy.

自然杀伤（NK）细胞对肿瘤细胞具有细胞溶解性，并且刺激肿瘤细胞的生长。 NK细胞与低剂量的白细胞介素-2（IL-2）产生了显着的 治疗结果，特别是在接受T细胞耗竭的患者中 同种异体骨髓移植 通过注入 自体NK细胞和给予高剂量的IL 2。 NK细胞也是 参与不相容骨髓细胞（BMC）移植物的排斥反应， 啮齿动物 BMC移植的免疫遗传学是不寻常的， 亲本品系BMC经常被F1杂交小鼠（杂交 电阻）。 显然，NK细胞会溶解那些不能向细胞内 通过某些受体向NK细胞传递负信号。 这些受体 由小鼠6号染色体上Ly49 NK基因家族中的基因编码。 这些受体之一，5E6（Ly49C），接收来自H2+的负信号 I类Ags。 这解释了为什么5E6亚群的NK细胞是负责 对于亲本或同种异体H2d但不是H2b BMC移植物的排斥。 一 一种新的体外试验模拟了体内BMC移植物， 淋巴母细胞的NK细胞。 5E6 + NK细胞不能裂解H2b 靶细胞可以通过加入F（ab '）2抗5E6 mAb来逆转， 负面信号 在该测定中，同源靶细胞不裂解;甚至 5E6 + H2d NK细胞不能裂解同系母细胞，除非F（ab ′ 2抗5E6）被 补充说 如果有负面信号， 针对NK细胞受体的I类Ag调节靶细胞的溶解， 一种利用NK细胞治疗同基因或自体 白血病/淋巴瘤和其它瘤形成。 我们开发了两个具体的 针对这一提议。 在目标1中，我们将通过以下步骤产生F（ab '）2抗5E6： 酶消化完整mAb和重组抗E6 mAb，其中 鼠Fc部分被人IgGI（Hgamma1）Fc片段取代。 到 产生抗5E6杂交瘤IgG2a重链的VDJ片段 基因将在一个载体中剪接到Hgamma1基因上，而抗5E6 κ轻链基因和新霉素抗性基因将在 第二个矢量 将这两种载体共转染入SP2/O 杂交瘤细胞 抗5E6-Hgamma1不应固定补体或引起 抗体依赖性细胞毒性（ADCC）。 我们还将生成一个 重组5E6（细胞外结构域）-Hgamma1构建体5E6-Hgamma1将被 转染SP2/O细胞。 在目标2中，我们将首先确定 施用F（ab '）2抗5E6、抗5E6-Hgamma1或5E6-Hgamma1至 注入同基因BMC的辐射小鼠可导致对 移植，大概是通过阻断大部分的负信号， 5E6 + NK细胞。 然后，我们将继续治疗EL4淋巴瘤和B16.F10 黑色素瘤[B6小鼠]、L1210淋巴瘤细胞[DBA/2和C. B-17重度组合 免疫缺陷（SCID）小鼠]和Friend病毒白血病[BALB/c小鼠]。 一些小鼠将接受iL2或干扰素α/β以增强NK细胞 活动 方法是首先注入试剂 腹膜内（i.p.），然后是分泌抗5E6抗体的杂交瘤 Hgamma1或5E6-Hgamma1将被置于扩散室中，腹膜内，以产生 试剂的连续供应。 因此，这是一个癌症模型 疗法