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主要参与急性炎症反应中中性粒细胞依赖的组织损伤。我们通过互补决定区嫁接的方法建立了人源化的小鼠抗人IL-8抗体，并成功地大量纯化了该抗体。为了在不同的临床条件下应用该抗体，我们建立了各种急性炎症动物模型。特别是建立了急性呼吸窘迫综合征样肺损伤、脑再灌注损伤等临床前状态动物模型。在这些模型中，我们表明抗IL-8抗体治疗几乎可以防止这些损伤。这些结果有力地表明了抗人IL-8抗体的临床应用的可能性。我们已经在脂多糖刺激的人单核细胞系THP-1的细胞提取物中发现了一种能特异性结合和磷酸化IkBA的激酶。内毒素刺激可瞬时增强THP-1细胞中IkBA结合的激酶活性。IkBA的突变分析和与合成肽的竞争实验表明，IkBA的C-末端酸性结构域上的结合激酶主要是丝氨酸和苏氨酸残基。此外，该IkBA型结合蛋白是一种新的激酶，而不是与肿瘤坏死因子-a和白介素1的信号通路相关的Ikka，b。使我们试着提纯得到的蛋白激酶进行了SDS-PAGE分析，结果表明该蛋白具有较高的活性。是40kD。我们现在正在分析样本的氨基酸序列，并试图克隆基因。

项目成果

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Matsumoto,T.et al.：“通过白细胞介素 8 抗体预防再灌注损伤中的脑水肿和梗塞。”

Matsumoto,T.et al.: "Pivotal role of interleukin-8 (IL-8) in acute inflammation (Invited Review)" J.Leukocyte Biol.62, 7(581-587) (1997)

Matsumoto,T.et al.：“白细胞介素 8 (IL-8) 在急性炎症中的关键作用（特邀评论）”J.Leukcyto Biol.62, 7(581-587) (1997)