Humanization of mouse anti-human IL-8 antibody and development of anti-inflammatory agent against cytokine regulatory factor, NFkB

小鼠抗人IL-8抗体的人源化和针对细胞因子调节因子NFkB的抗炎剂的开发

• 批准号: 07557031
• 负责人: MATSUSHIMA Kouji
• 金额: $ 7.36万
• 依托单位: University of Tokyo (1996-1997)Kanazawa University (1995)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557031/
• 关键词: interleukin-8; anti-inflammatory agent; acute inflammation; anti-interleukin-8 antibody; humanization; NF-kB; IkB-bound kinase; phosphorylation; ケモカイン; 白血球; 転写因子; NFkB; IkBα; 単球走化活性化因子; 抗体

IL-8 is essentially involved in neutrophil-dependent tissue damage in acute inflammatory reactions. We established the humanized mouse anti-human IL-8 by mean of complementarity determining region grafting and succeeded to purify the large amount of this antibody. For the application of this antibody on various clinical condition, we established various acute inflammatory ananimal models. Especially, we have established preclinical condition animal models such as acute respiratory distress syndrom-like lung injury and brain reperfusion injury. In these models, we showed that anti-IL-8 antibody treatment almostly prevented these injury. These results strongly suggest the possibility of clinical application of humarized anti-human IL-8 antibody against acute inflammatory dieases.We have identified a kinase in cell extracts from the LPS-stimulated human monocytic cell line, THP-1, that specifically bind and phosphorylates IkBa. LPS-stimulation transiently enhanced the IkBa-bound kinase activity in THP-1 cells. Mutation analysis of IkBa and competition experiments with the synthetic peptides identified major phosphorylation site by the bound kinase as Ser and Thr residues in the C-terminal acidic domain of IkBa. Moreover, this IkBa-boundkinase is novel kinase but not Ikka, b which are related to signal pathway of TNF-a and IL-1. So that we try to purify the kinase SDS-PAGE analysis showed that the kinase. is 40 Kd. We are now analyzing the amino acid squence of the sample and trying to clone the gene.

IL-8基本上参与急性炎症反应中嗜中性粒细胞依赖性组织损伤。我们通过互补性确定区域移植，并成功净化了大量抗体。为了在各种临床状况上应用这种抗体，我们建立了各种急性炎症性非征模模型。特别是，我们已经建立了临床前疾病模型，例如急性呼吸窘迫综合症样肺损伤和脑再灌注损伤。在这些模型中，我们表明抗IL-8抗体治疗几乎阻止了这些损伤。这些结果强烈表明，临床应用造成型抗人IL-8抗体针对急性炎症性DIEASE，我们已经从LPS刺激的人类单核细胞系THP-1（特异性结合和磷酸化ikba）中鉴定出了细胞提取物中的激酶。 LPS刺激瞬时增强了THP-1细胞中IKBA结合的激酶活性。与合成肽的IKBA和竞争实验的突变分析通过结合的激酶确定了主要的磷酸化位点为IKBA的C末端酸性结构域中的Ser和THR残基。此外，这种IKBA结合激酶是新颖的激酶，但不是IKKA，B，与TNF-A和IL-1的信号途径有关。因此，我们尝试纯化激酶SDS-PAGE分析表明激酶。是40 kd。现在，我们正在分析样品的氨基酸挤压并试图克隆基因。

项目成果

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Mukaida,N.: "In Leukocyte Typing V" Oxford University Press, 3 (1995)

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Mukaida,N.et al.: "Cytokine Growth Fact.Rev." Interleukin-8 and momocyte chemotactic and activating factor (MCAF/MCP-i),chenokines essentially involved in inflemmatory and immone reactions. (in press), (1997)

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Zhang,Y.: "TNFa as a physiological regulator of hematopoietic progenitor cells: Increase of early hematopoietic progenitor cells in TNF-R55-1-mice in vivo and potent inhibition of progenitor cell proliferation by TNFa in vitro." Blood. 6. 2930-2937 (1995)

张，Y.：“TNFa 作为造血祖细胞的生理调节剂：体内 TNF-R55-1-小鼠的早期造血祖细胞增加，体外 TNFa 有效抑制祖细胞增殖。”