Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.
Cytokine refacing effect reduces granulocyte macrophage colony-stimulating factor susceptibility to antibody neutralization.
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DOI：
10.1093/protein/gzv019
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发表时间：
2015-10
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影响因子：
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通讯作者：
Pete Heinzelman;S. Carlson;G. N. Cox
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文献类型：
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作者：
Pete Heinzelman;S. Carlson;G. N. Cox
克罗恩病（CD）困扰着50多万美国人，每年的经济影响超过100亿美元。粒细胞巨噬细胞集落刺激因子（GM-CSF）可以增加患者对促进CD的肠道微生物的免疫反应，并在临床试验中对一些患者有效。我们在开发GM-CSF变体方面取得了重要进展，这些变体可能是更有效的CD治疗剂，因为它们不太容易被CD患者中高度表达的内源性GM-CSF自身抗体中和。酵母展示工程揭示了在表面等离子体共振实验中使GM-CSF变体对GM-CSF受体α和β亚基两者的结合亲和力增加高达0.33倍的突变。在常规体外人白细胞增殖试验中，结合亲和力增加不会降低GM-CSF半数有效浓度（EC 50）值。然而，增强亲和力的突变确实促进了“表面重塑效应”，该效应赋予所有五种评估的GM-CSF变体在GM-CSF中和多克隆抗血清存在下增加的体外生物活性。改进最多的变体H15 L/R23 L的活性是野生型GM-CSF的6倍。引入额外的已知亲和力增加突变可以增强本文所述的表面重塑效应和伴随的生物活性改善。
Crohn's Disease (CD) afflicts over half a million Americans with an annual economic impact exceeding $10 billion. Granulocyte macrophage colony-stimulating factor (GM-CSF) can increase patient immune responses against intestinal microbes that promote CD and has been effective for some patients in clinical trials. We have made important progress toward developing GM-CSF variants that could be more effective CD therapeutics by virtue of being less prone to neutralization by the endogenous GM-CSF autoantibodies that are highly expressed in CD patients. Yeast display engineering revealed mutations that increase GM-CSF variant binding affinity by up to ∼3-fold toward both GM-CSF receptor alpha and beta subunits in surface plasmon resonance experiments. Increased binding affinity did not reduce GM-CSF half-maximum effective concentration (EC50) values in conventional in vitro human leukocyte proliferation assays. Affinity-enhancing mutations did, however, promote a 'refacing effect' that imparted all five evaluated GM-CSF variants with increased in vitro bioactivity in the presence of GM-CSF-neutralizing polyclonal antisera. The most improved variant, H15L/R23L, was 6-fold more active than wild-type GM-CSF. Incorporation of additional known affinity-increasing mutations could augment the refacing effect and concomitant bioactivity improvements described here.