Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach
Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach
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DOI：
10.1080/19420862.2015.1088618
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发表时间：
2015-11-02
期刊：
影响因子：
5.3
通讯作者：
Robinson, Matthew K.
中科院分区：
文献类型：
--
作者：
Lehmann, Andreas;Wixted, Josephine H. F.;Robinson, Matthew K.
噬菌体展示技术有助于从大型重组文库中快速选择抗原特异性单链可变片段（scFv）抗体。由V-H和V-L结构域组成的ScFv抗体易于工程化为多聚体形式以用于诊断和靶向疗法的开发。然而，选择策略的重组性质可能导致V-H和V-L结构域具有次优的生物物理性质，例如降低的热力学稳定性和增强的聚集倾向，这导致生产差和应用受限。我们发现C10抗表皮生长因子受体（EGFR）scFv及其亲和突变体P2224在E.杆菌有趣的是，这些scFv包含了一个融合的LVDa 3和LVDa 1V区（LV 3和LV 1）基因，最有可能的结果，在文库构建过程中的PCR畸变。为了增强这些scFv的生物物理性质，我们利用基于结构的方法来替换和重新设计V-L结构域的预先存在的框架，使其与现有的V-H最佳配对。我们描述了一种方法来交换lambda序列与一个更稳定的kappa 3框架（KV 3）内的V-L域，其中包括原始的lambda DE-环。所得到的scFv C10KV3_LV1DE和P2224KV3_LV1DE在生物学上更稳定，并且更容易从细菌培养物中产生。此外，C10KV3_LV1DE和P2224KV3_LV1DE保留了对EGFR的结合亲和力，这表明这种剧烈的框架交换不会显着影响scFv结合。我们在此提供了一种新的策略，用于重新设计有问题的scFv的轻链，以增强其稳定性和治疗适用性。
Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a V-H and V-L domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in V-H and V-L domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the V-L domain to one that best pairs with the existing V-H. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the V-L domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.