Structural and biologic characterization of pegylated recombinant IFN-alpha2b.
Structural and biologic characterization of pegylated recombinant IFN-alpha2b.
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发表时间：
2001
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通讯作者：
M. Grace;S. Youngster;G. Gitlin;W. Sydor;L. Xie;L. Westreich;S. Jacobs;D. Brassard;J. Bausch;R. Bordens
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作者：
M. Grace;S. Youngster;G. Gitlin;W. Sydor;L. Xie;L. Westreich;S. Jacobs;D. Brassard;J. Bausch;R. Bordens
I型干扰素-α（IFN-α）家族是具有临床重要的抗感染和抗肿瘤活性的天然小蛋白家族。我们通过将12，000-Da单甲氧基聚乙二醇（PEG-12000）聚合物连接到蛋白质上，开发了IFN-α 2b（内含子A）的半合成蛋白质-聚合物缀合物。PEG缀合被认为增加血清半衰期，从而延长患者对IFN-α 2b的暴露，而不改变蛋白质的生物学效力。基质辅助激光解吸电离/质谱法（MALDI-MS），高效分子排阻色谱法（HPSEC），圆二色性（CD）分析和胰蛋白酶消化肽分析的PEG内含子表明，IFN-α 2b蛋白质约95%的PEG化，一级，二级和三级结构不变。聚乙二醇化不影响用于内含子A定量的抗体的表位识别。对聚乙二醇化位置异构体的广泛分析显示，约50%的PEG内含子在IFN-α 2b蛋白的His（34）残基上被双乙二醇化。PEG内含子的聚乙二醇化位置异构体的最高抗病毒活性与His（34）聚乙二醇化异构体相关。在抗病毒细胞病变保护试验中，PEG内含子的比活性相对于内含子A为28%。然而，PEG内含子的效力（定义为与蛋白浓度无关的生物活性）在一系列体外试验中在分子和细胞水平上与内含子A相当。在Molt 4细胞中，PEG内含子和内含子A的等效单位对于几个关键的IFN诱导基因的诱导是不可区分的，包括2 '，5'-寡腺苷酸合成酶（2 '，5'-OAS）和蛋白激酶R（PKR）。抗病毒剂量-反应曲线显示PEG内含子和内含子A之间无显著差异。这表明，在抗病毒测定中，引入与PEG内含子的等效单位给药相关的更多IFN-α 2b蛋白不会产生任何拮抗作用或激动作用。在免疫应答的测定中，PEG内含子和内含子A显示出对自然杀伤（NK）和淋巴因子激活的杀伤（LAK）细胞的细胞溶解活性以及对I类主要组织相容性蛋白的诱导的相当的效力。这些结果表明，PEG内含子保持了与内含子A高度相当的抗病毒和免疫活性的体外生物学效力特征。
The type I interferon-alpha (IFN-alpha) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-alpha2b (Intron A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-alpha2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-alpha2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.