BLOCKING NEGATIVE SIGNALS TO NK CELLS TO TREAT LEUKEMIA

自然杀伤(NK)细胞对肿瘤细胞具有杀伤作用，而刺激 低剂量白介素2(IL2)的NK细胞产生了戏剧性的 治疗结果，特别是在接受T细胞耗尽的患者 异基因骨髓移植。通过输液获得的成功较少 自体NK细胞和给予大剂量IL-2。NK细胞也是 参与血型不合的骨髓细胞(BMC)移植的排斥反应 啮齿动物。骨髓细胞移植的免疫遗传学的不同寻常之处在于 亲本品系BMC经常被F1(杂交)小鼠排斥 抵抗)。显然，NK细胞将溶解未能发送给 通过某些受体向NK细胞发出负信号。这些受体 是由小鼠6号染色体上Ly49 NK基因家族的基因编码的。 其中一个受体，5E6(Ly49C)，从H2+接收负信号 I类AGS。这就解释了为什么NK细胞的5E6亚群 对亲本或同种异体H_2d移植有排斥作用，但对H_2B-BMC移植物无排斥作用。一个 一种新的体外模拟体内BMC移植的方法，它涉及到裂解 NK细胞诱导的淋巴母细胞。5E6+NK细胞不能裂解H_2B 加入F(ab‘)2抗5E6单抗可逆转靶细胞，阻断 负面信号。在这个实验中，同基因的靶细胞不被裂解；甚至 5E6+H2d NK细胞不能溶解同基因母细胞，除非F(ab‘2抗5E6是 添加了。从负面信号的存在或不存在 类抗原结合NK细胞受体调节靶细胞裂解提供 利用NK细胞治疗同基因或自体移植的新途径 白血病/淋巴瘤和其他肿瘤。我们已经开发出两种特定的 旨在提出这项建议。在目标1中，我们将通过以下方式生成F(ab‘)2抗5E6 全长单抗和重组抗E6单抗的酶消化 用人Iggi(HGamma1)Fc片段取代鼠Fc部分。至 创造这个，抗5E6杂交瘤IgG2a重链的VDJ片段 将基因与HGamma1基因在一个载体中拼接，并将抗5E6 Kappa轻链基因和新霉素抗性基因将在 第二个矢量。将这两个载体共转染入SP2/O 杂交瘤细胞。抗5E6-HGamma1不应修复补体或引起 抗体依赖性细胞毒性(ADCC)。我们还将生成一个 重组5E6(胞外区)-HGamma1构建5E6-HGamma1 转染SP2/O细胞。在目标2中，我们将首先确定是否 F(ab‘)2抗5E6、抗5E6-HGamma1或5E6-HGamma1给药 注射同基因骨髓细胞的受照小鼠可产生抗药性 嫁接，想必是通过阻断很大一部分的负面信号 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严重联合 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来增强NK细胞 活动。这些方法将首先将试剂注入 (Ip)，然后是分泌抗5E6的杂交瘤- HGamma1或5E6-HGamma1将被放入扩散室I.P.要创建 试剂的持续供应。因此，这是一种癌症模型 心理治疗。

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.