Pitti R M, Marsters S A, Haak-Frendscho M, Osaka G C, Mordenti J, Chamow S M, Ashkenazi A
Department of Molecular Biology, Genentech, Inc., South San Francisco, CA 94080.
Mol Immunol. 1994 Dec;31(17):1345-51. doi: 10.1016/0161-5890(94)90053-1.
Overproduction of the cytokine interleukin 1 (IL-1) is an important factor in the pathogenesis of several autoimmune and inflammatory disease. To develop a recombinant inhibitor of IL-1 with an extended pharmacologic half-life, we constructed an IL-1 receptor immunoadhesin (IL-1R-IgG), by fusing the extracellular domain of the type IL-1 receptor with the hinge and Fc regions of human IgG1 heavy chain. Transfected human 293 cells express IL-1R-IgG as a secreted, disulfide-bonded homodimer. The secreted protein contains an intact antibody Fc region, as indicated by immunoblotting, and a functional IL-1 receptor region, as indicated by ligand-blotting. Saturation binding analysis indicates an equilibrium dissociation constant (KD) of 350 pM for the binding of IL-1R-IgG to its ligand, IL-1 beta. Kinetic analysis of the binding reveals an off rate of 0.1 min-1 and an on rate of 1.5 x 10(8) min-1 M-1, yielding a calculated KD of 770 pM. These binding properties are similar to those of cell-surface type I IL-1 receptor. IL-1R-IgG is capable of inhibiting the biological activity of IL-1 beta in vitro, as evidenced in a thymocyte proliferation assay. Pharmacokinetic analysis in mice indicates that IL-1R-IgG has a terminal half-life of 91 hr in the blood circulation. This half-life is markedly longer than the values reported for other recombinant inhibitors of IL-1 such as the IL-1 receptor antagonist or soluble IL-1 receptor. Thus, IL-1R-IgG may be useful for investigating the interaction of IL-1 with its receptor and the role of IL-1 in disease, as well as for potential intervention in pathological situations involving overproduction of IL-1.
细胞因子白细胞介素1（IL-1）的过度产生是几种自身免疫性和炎症性疾病发病机制中的一个重要因素。为了开发一种具有延长药理半衰期的IL-1重组抑制剂，我们通过将I型IL-1受体的细胞外结构域与人IgG1重链的铰链区和Fc区融合，构建了一种IL-1受体免疫粘附素（IL-1R-IgG）。转染的人293细胞将IL-1R-IgG表达为一种分泌型、通过二硫键连接的同二聚体。免疫印迹显示，分泌的蛋白质含有完整的抗体Fc区，配体印迹显示含有功能性的IL-1受体区。饱和结合分析表明，IL-1R-IgG与其配体IL-1β结合的平衡解离常数（KD）为350 pM。结合动力学分析显示，解离速率为0.1 min-1，结合速率为1.5×108 min-1 M-1，计算得出的KD为770 pM。这些结合特性与细胞表面I型IL-1受体的特性相似。在胸腺细胞增殖试验中证实，IL-1R-IgG能够在体外抑制IL-1β的生物活性。小鼠药代动力学分析表明，IL-1R-IgG在血液循环中的终末半衰期为91小时。这个半衰期明显长于其他IL-1重组抑制剂（如IL-1受体拮抗剂或可溶性IL-1受体）报道的值。因此，IL-1R-IgG可能有助于研究IL-1与其受体的相互作用以及IL-1在疾病中的作用，也可能用于对涉及IL-1过度产生的病理情况进行潜在干预。
