Biologics Research, Centocor R&D, 145 King of Prussia Radnor, PA 19087-4557, USA.
Protein Eng Des Sel. 2010 Aug;23(8):643-51. doi: 10.1093/protein/gzq037. Epub 2010 Jun 11.
Protein aggregation is of great concern to pharmaceutical formulations and has been implicated in several diseases. We engineered an anti-IL-13 monoclonal antibody CNTO607 for improved solubility. Three structure-based engineering approaches were employed in this study: (i) modifying the isoelectric point (pI), (ii) decreasing the overall surface hydrophobicity and (iii) re-introducing an N-linked carbohydrate moiety within a complementarity-determining region (CDR) sequence. A mutant was identified with a modified pI that had a 2-fold improvement in solubility while retaining the binding affinity to IL-13. Several mutants with decreased overall surface hydrophobicity also showed moderately improved solubility while maintaining a similar antigen affinity. Structural studies combined with mutagenesis data identified an aggregation 'hot spot' in heavy-chain CDR3 (H-CDR3) that contains three residues ((99)FHW(100a)). The same residues, however, were found to be essential for high affinity binding to IL-13. On the basis of the spatial proximity and germline sequence, we reintroduced the consensus N-glycosylation site in H-CDR2 which was found in the original antibody, anticipating that the carbohydrate moiety would shield the aggregation 'hot spot' in H-CDR3 while not interfering with antigen binding. Peptide mapping and mass spectrometric analysis revealed that the N-glycosylation site was generally occupied. This variant showed greatly improved solubility and bound to IL-13 with affinity similar to CNTO607 without the N-linked carbohydrate. All three engineering approaches led to improved solubility and adding an N-linked carbohydrate to the CDR was the most effective route for enhancing the solubility of CNTO607.
蛋白质聚集是药物制剂中非常关注的问题，并且与几种疾病有关。我们设计了一种抗 IL-13 单克隆抗体 CNTO607，以提高其溶解度。本研究采用了三种基于结构的工程方法：（i）修饰等电点（pI），（ii）降低整体表面疏水性，（iii）在互补决定区（CDR）序列内重新引入 N-连接的碳水化合物部分。鉴定出一种具有修饰 pI 的突变体，其溶解度提高了 2 倍，同时保留了与 IL-13 的结合亲和力。几个整体表面疏水性降低的突变体也表现出适度改善的溶解度，同时保持类似的抗原亲和力。结构研究与突变数据相结合，确定了重链 CDR3（H-CDR3）中存在一个聚集“热点”，其中包含三个残基（（99）FHW（100a））。然而，相同的残基对于与 IL-13 的高亲和力结合是必不可少的。基于空间接近性和种系序列，我们在 H-CDR2 中重新引入了原始抗体中存在的共识 N-糖基化位点，预计碳水化合物部分将屏蔽 H-CDR3 中的聚集“热点”，同时不干扰抗原结合。肽图谱和质谱分析显示，N-糖基化位点通常被占据。该变体显示出大大提高的溶解度，并且与 IL-13 的亲和力与没有 N-连接碳水化合物的 CNTO607 相似。这三种工程方法都提高了溶解度，并且在 CDR 中添加 N-连接的碳水化合物是提高 CNTO607 溶解度的最有效途径。
