Enever C, Pupecka-Swider M, Sepp A
Biopharm Innovation, RD Biopharm R&D, GlaxoSmithKline Plc., 315 Cambridge Science Park, Cambridge CB4 0WG, UK
Biopharm Innovation, RD Biopharm R&D, GlaxoSmithKline Plc., 315 Cambridge Science Park, Cambridge CB4 0WG, UK.
Protein Eng Des Sel. 2015 Mar;28(3):59-66. doi: 10.1093/protein/gzu057. Epub 2015 Feb 4.
In addition to the desired specificity and affinity for their respective therapeutic targets, antibody-based drugs must also demonstrate an ability to be manufactured and formulated at the concentrations needed for therapeutic application and to remain resistant to aggregation during storage to reduce the risk of induced immunogenicity. Improvements to the thermodynamic stability of the folded state of the protein are considered to be critical for decreasing the aggregation propensity of the protein. In this work, we have improved the biophysical properties of a number of human domain antibodies (dAbs) by identifying mutations which decrease the propensity for dAb self-aggregation without compromising the affinity for their respective target antigen. The mutations were identified by subjecting phage-displayed error-prone PCR-generated libraries to a variety of generic environmental conditions (temperature, pH and protease) followed by antigen capture, facilitating selection for improved thermodynamic stability of the protein. The results indicate that sufficient sequence diversity usually exists within the complementarity determining regions of dAbs to allow for mutations that lead to improvements to biophysical properties with full retention of parent lead biochemical and biological properties. Improved biophysical properties were often accompanied by higher apparent melting temperature values, while alternative selection pressures often identified similar features, suggesting generic nature of these mutations.
除了对各自治疗靶点具有所需的特异性和亲和力外，基于抗体的药物还必须能够按照治疗应用所需的浓度进行生产和配制，并在储存期间保持抗聚集能力，以降低诱导免疫原性的风险。蛋白质折叠态的热力学稳定性的提高被认为对于降低蛋白质的聚集倾向至关重要。在这项工作中，我们通过鉴定能够降低结构域抗体（dAb）自聚集倾向而又不损害其对各自靶抗原亲和力的突变，改善了多种人源结构域抗体的生物物理性质。通过使噬菌体展示的易错PCR生成的文库经受各种一般环境条件（温度、pH值和蛋白酶），然后进行抗原捕获，来鉴定这些突变，从而有助于选择提高蛋白质的热力学稳定性。结果表明，在结构域抗体的互补决定区内通常存在足够的序列多样性，以允许发生导致生物物理性质改善且完全保留亲本先导生化和生物学性质的突变。改善的生物物理性质通常伴随着更高的表观解链温度值，而其他选择压力通常也能鉴定出类似特征，这表明这些突变具有一般性。
