Lee S C, Parthasarathy R, Botwin K, Kunneman D, Rowold E, Lange G, Klover J, Abegg A, Zobel J, Beck T, Miller T, Hood W, Monahan J, McKearn J P, Jansson R, Voliva C F
Department of Cellular and Molecular Biochemistry, and the Dorothy M. Davis Heart and Lung Research Institute, 473 W. 12th Avenue, Ohio State University, Columbus, OH 43210, USA.
Biomed Microdevices. 2004 Sep;6(3):191-202. doi: 10.1023/B:BMMD.0000042048.18186.ff.
Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these results show that they can be used successfully in conjugates to bioactive proteins.
在此，我们描述了SC - 63032的一种翻译后修饰，SC - 63032是物种受限的多谱系造血因子人白细胞介素 - 3（hIL - 3）的一个变体。我们制备了两种新型树枝状聚合物（聚酰胺胺或第5代PAMAM树枝状大分子）与SC - 63032的生物共轭物。使用两种不同的化学方法（其中一种是本研究新采用的），我们实现了与连接到树枝状大分子上的蛋白质中的氨基酸进行位点特异性共轭。在这两种生物共轭物中，共轭细胞因子保持了其结合hIL - 3α受体亚基的能力，但在诱导hIL - 3依赖的体外细胞增殖方面，其效力显著降低（约10倍），低于游离细胞因子。体内数据表明，共轭作用适度降低了共轭细胞因子的免疫原性。在不存在与生物共轭物相关的药代动力学或生物分布效应从而增加其体内效力的情况下（由于hIL - 3及其衍生物的物种限制，这只能在高等灵长类动物中进行测试），这些免疫减轻效应可能太小而无治疗意义。尽管未修饰的PAMAM树枝状大分子不会在小鼠中引发抗体反应，但蛋白质与树枝状大分子共轭会使其成为半抗原，并产生针对树枝状大分子的特异性抗体反应。总体而言，本文中树枝状大分子 - 细胞因子生物共轭物的主要局限性在于其体外受体亲和力和效力降低。如果生物共轭物的体内效力与本文报道的共轭物的体外效力相当，那么相对于母体SC - 63032分子，特定的树枝状大分子生物共轭物可能无法证明其更高的商品成本是合理的；不过共轭物中保留了SC - 63032的生物活性，这表明其他细胞因子 - 树枝状大分子生物共轭物可能具有生物活性。这对纳米技术领域来说是个好消息，因为PAMAM树枝状大分子属于现有的单分散聚合物纳米材料，这些结果表明它们可以成功用于与生物活性蛋白的共轭物中。
