Luo Ruiqi, Qu Baole, An Lili, Zhao Yun, Cao Yang, Ren Peng, Hang Haiying
Key Laboratory for Protein and Peptide Pharmaceuticals, National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Bioengineering (Basel). 2022 Aug 2;9(8):360. doi: 10.3390/bioengineering9080360.
Antibody stability and affinity are two important features of its applications in therapy and diagnosis. Antibody display technologies such as yeast and bacterial displays have been successfully used for improving both affinity and stability. Although mammalian cell display has also been utilized for maturing antibody affinity, it has not been applied for improving antibody stability. Previously, we developed a Chinese hamster ovary (CHO) cell display platform in which activation-induced cytidine deaminase (AID) was used to induce antibody mutation, and antibody affinity was successfully matured using the platform. In the current study, we developed thermo-resistant (TR) CHO cells for the purpose of maturing both antibody stability and affinity. We cultured TR CHO cells displaying an antibody mutant library and labeled them at temperatures above 41 °C, enriching cells that displayed antibody mutants with both the highest affinities and the highest display levels. To evaluate our system, we chose three antibodies to improve their affinities and stabilities. We succeeded in simultaneously improving both affinities and stabilities of all three antibodies. Of note, we obtained an anti-TNFα antibody mutant with a Tm (dissolution temperature) value 12 °C higher and affinity 160-fold greater than the parent antibody after two rounds of cell proliferation and flow cytometric sorting. By using CHO cells with its advantages in protein folding, post-translational modifications, and code usage, this procedure is likely to be widely used in maturing antibodies and other proteins in the future.
抗体稳定性和亲和力是其在治疗和诊断应用中的两个重要特性。酵母展示和细菌展示等抗体展示技术已成功用于提高亲和力和稳定性。尽管哺乳动物细胞展示也已用于优化抗体亲和力，但尚未应用于提高抗体稳定性。此前，我们开发了一种中国仓鼠卵巢（CHO）细胞展示平台，其中使用激活诱导的胞苷脱氨酶（AID）诱导抗体突变，并使用该平台成功优化了抗体亲和力。在本研究中，我们开发了耐热（TR）CHO细胞，以同时优化抗体稳定性和亲和力。我们培养展示抗体突变文库的TR CHO细胞，并在41°C以上的温度下对其进行标记，富集展示具有最高亲和力和最高展示水平的抗体突变体的细胞。为了评估我们的系统，我们选择了三种抗体来提高它们的亲和力和稳定性。我们成功地同时提高了所有三种抗体的亲和力和稳定性。值得注意的是，经过两轮细胞增殖和流式细胞术分选后，我们获得了一种抗TNFα抗体突变体，其熔解温度（Tm）值比亲本抗体高12°C，亲和力比亲本抗体高160倍。通过利用CHO细胞在蛋白质折叠、翻译后修饰和密码子使用方面的优势，该方法未来可能会广泛应用于优化抗体和其他蛋白质。
