Mikolajczyk S D, Meyer D L, Fagnani R, Hagan M S, Law K L, Starling J J
Hybritech Inc., San Diego, California 92196-9006, USA.
Bioconjug Chem. 1996 Jan-Feb;7(1):150-8. doi: 10.1021/bc950088j.
The physical and pharmacological properties of proteins can be altered by chemical modification with polymers. Preliminary studies showed that attachment of oxidized dextran to the bacterial protein, beta-lactamase (beta L) effectively reduced in vivo immunogenicity in mice with no loss of enzymatic activity. This report describes a general method for differentially dextran modifying the Fab' component of a Fab'--beta-lactamase conjugate by the use of amine-blocking reagents. Methyl acetimidate (MeAcm) and the N-succinimidyl derivative of (methylsulfonyl)ethyl carbonate (NHS-Msc), reagents which can reversibly block primary amines, were used in model studies to modulate the level of available reactive amines on the F(ab')2 fragments of both the anti-carcinoembryonic antigen antibody, ZCE025, and the antitumor-associated glycoprotein-72 antibody, CC49. MeAcm had little or no effect on immunoreactivity and was maximally effective in modulating dextran attachment, while NHS-Msc was much less effective. A comparison of NHS-Msc and MeAcm is described. Treatment of F(ab')2 with 5-300 mM MeAcm prior to dextran treatment showed a proportional decline in the level of dextran attachment as well as intramolecular cross-linking of the protein by the dextran polymers (6 kDa or 33-mer). A conjugate of beta L coupled to MeAcm-treated ZCE025 Fab' [reduced F(ab')2] was constructed under standard conditions using sulfosuccinimidyl N-[(4-carboxycyclohexyl)methyl]maleimide. After dextran modification, this conjugate maintained good immunoreactivity and enzymatic activity. Biodistribution studies in tumor-bearing nude mice of dextranated and nondextranated conjugate showed comparable overall distribution profiles except that the clearance of the dextranated conjugate from both blood and tumor was delayed about 48-72 h.
蛋白质的物理和药理性质可通过与聚合物进行化学修饰而改变。初步研究表明，将氧化葡聚糖连接到细菌蛋白β-内酰胺酶（βL）上可有效降低小鼠体内的免疫原性，且酶活性无损失。本报告描述了一种通过使用胺阻断试剂对Fab'-β-内酰胺酶偶联物的Fab'成分进行差异葡聚糖修饰的通用方法。在模型研究中使用了甲基乙酰亚胺酯（MeAcm）和（甲基磺酰基）乙基碳酸酯的N-琥珀酰亚胺衍生物（NHS-Msc），这两种试剂可可逆地阻断伯胺，用于调节抗癌胚抗原抗体ZCE025和抗肿瘤相关糖蛋白-72抗体CC49的F(ab')2片段上可用反应性胺的水平。MeAcm对免疫反应性影响很小或没有影响，在调节葡聚糖连接方面效果最佳，而NHS-Msc的效果则差得多。本文描述了NHS-Msc和MeAcm的比较。在葡聚糖处理之前，用5-300 mM MeAcm处理F(ab')2，结果显示葡聚糖连接水平以及葡聚糖聚合物（6 kDa或33聚体）对蛋白质的分子内交联呈比例下降。在标准条件下，使用磺基琥珀酰亚胺N-[(4-羧基环己基)甲基]马来酰亚胺构建了与经MeAcm处理的ZCE025 Fab'[还原F(ab')2]偶联的βL偶联物。葡聚糖修饰后，该偶联物保持了良好的免疫反应性和酶活性。对荷瘤裸鼠进行的生物分布研究表明，经葡聚糖修饰和未经葡聚糖修饰的偶联物的总体分布情况相当，只是经葡聚糖修饰的偶联物从血液和肿瘤中的清除延迟了约48-72小时。
