Abstract:

Conventional antibody screening is based on physiological pH conditions for affinity evaluation and screening， which may ignore the influence of acidic characteristics of tumor microenvironment on antibody binding. The purpose of this study is to establish a multi-pH gradient screening strategy to screen 5T4 antibody clones with high affinity and stability under acidic pH conditions by simulating tumor microenvironment conditions， so as to improve the clinical transformation success rate of antibody development. The 5T4 hybridoma clones obtained in the previous screening were selected. After the antibody concentration in the supernatant was quantified， a multi-pH gradient （7.4， 7.0， 6.6，6.0， 5.5） experimental system was constructed by Biacore T200 system， and the dynamic changes of antibody affinity were monitored in real time by surface plasmon resonance technology. The results showed that the affinity of high affinity antibody 5T4-15 screened under normal physiological conditions decreased gradually with the decrease of pH in acidic environment， and it lost its binding ability to antigen when pH was 5.5. In contrast， the antibody 5T4-45 obtained by pressure screening at pH 5.5 can maintain stable high affinity in the tumor microenvironment related range of pH 7.4 to 6.0， showing excellent adaptability to acidic environment. The introduction of acid stress test （pH 5.5） in the initial antibody screening stage could effectively identify and screen tumor microenvironment-adaptive antibodies， break through the limitation of screening high-affinity antibodies at conventional physiological pH， improve the screening efficiency of high-quality candidate molecules， and provide a new strategy for developing therapeutic antibodies for solid tumors with higher clinical transformation potential.

Key words: tumor microenvironment, antibody screening, pH gradient, Biacore

摘要：

常规抗体筛选主要基于生理pH条件进行亲和力评估和筛选，可能忽略了肿瘤微环境酸性特征对抗体结合作用的影响。研究旨在建立多pH梯度筛选策略，通过模拟肿瘤微环境条件，筛选出酸性pH条件下保持高亲和力与稳定性的5T4抗体克隆，提升抗体开发的临床转化成功率。选取前期筛选得到的5T4杂交瘤克隆，上清抗体经浓度定量后，利用Biacore T200系统构建多pH梯度（7.4、7.0、6.6、6.0、5.5）实验体系，采用表面等离子共振技术实时监测抗体亲和力动态变化。结果发现，常规生理条件下筛选的高亲和力抗体5T4-15，在酸性环境中亲和力随pH降低呈逐步下降趋势，至pH 5.5时丧失与抗原的结合能力。相比之下，经pH 5.5压力筛选获得的抗体5T4-45，在pH 7.4~6.0的肿瘤微环境相关区间内均能保持稳定的高亲和力，展现出卓越的酸性环境适应性。在抗体初筛阶段引入酸性压力测试（pH 5.5），可有效鉴别并筛选出肿瘤微环境适应性抗体，突破常规生理pH筛选高亲和力抗体的局限性，并提升优质候选分子的筛选效率，为开发临床转化潜力更高的实体瘤治疗性抗体提供新策略。

关键词: 肿瘤微环境, 抗体筛选, pH梯度, Biacore