
不同细胞消化方式对靶向CD3L1的抗体结合肿瘤细胞的影响
The impact of different cell digestion methods on the binding of an anti-CD3L1 antibody to tumor cells
目的: 探讨不同的细胞消化方式对靶向CD3L1的人源化单克隆抗体(代号:5H)结合肿瘤细胞的影响。方法: 使用胰蛋白酶溶液或胶原酶与中性蛋白酶混合液,在室温和37 ℃两种消化温度下对不同贴壁肿瘤细胞系进行离解、传代和收集。随后,通过流式细胞术和共聚焦显微镜成像,比较不同消化条件下肿瘤细胞与5H抗体的结合差异。结果: 使用胰蛋白酶溶液在室温离解和传代的细胞仅表现出与5H抗体的微弱结合,而相同的细胞系用胶原酶与中性蛋白酶混合液在室温消化后,则显示出与5H抗体的明显结合,两组之间抗体结合信号及抗体染色阳性细胞比例的差异有统计学意义(P < 0.01)。胶原酶与中性蛋白酶混合液处理的细胞与抗体的结合信号呈现出抗体浓度依赖性,可用于受体占位分析。相比室温,37 ℃的条件下消化后略微削弱了靶细胞与5H抗体的结合,但该差异无统计学意义。结论: 胰蛋白酶溶液消化方式显著削弱了抗CD3L1抗体与肿瘤细胞的结合,而胶原酶与中性蛋白酶混合液的消化方式能有效保护该结合。在选择适宜的消化酶处理细胞的前提下,消化温度的影响相对次要。在研究抗体药物与细胞表面抗原相互作用时,应采用合理且经过优化的细胞消化方案。
Objective: To investigate the impact of different cell digestion methods on the binding of a humanized monoclonal antibody targeting CD3L1 (designated as 5H) to tumor cells. Methods: Trypsin solution and a collagenase/neutral protease cocktail, combined with two digestion temperatures (room temperature and 37℃), were used to dissociate, passage, and collect various adherent tumor cell lines. Flow cytometry and confocal microscopy were then employed to compare differences in 5H antibody binding under different cell digestion conditions. Results: Cells dissociated and passaged with trypsin solution at room temperature exhibited only weak binding to the 5H antibody, whereas those treated with the collagenase/neutral protease cocktail at room temperature showed significantly stronger binding. There were notable differences in the binding signal and the proportion of antibody-stained positive cells between the two groups (P < 0.01). Additionally, cells treated with the collagenase/neutral protease cocktail exhibited antibody binding signals that were dependent on the concentration of the 5H antibody, enabling receptor occupancy analysis. Compared to digestion at room temperature, treatment at 37℃ resulted in a slight reduction in the binding of target cells to the 5H antibody, however, this difference was not statistically significant. Conclusion: Trypsin digestion significantly reduces the binding of the anti-CD3L1 antibody to tumor cells, whereas the collagenase/neutral protease cocktail effectively preserves it. With an appropriate choice of digestion enzymes, the impact of digestion temperature is relatively minor. These findings suggest that optimized cell digestion protocols should be employed when studying interactions between antibody drugs and cell surface antigens.
CD3L1 / 胞膜蛋白 / 细胞消化 / 抗体-抗原结合 / 肿瘤免疫治疗 {{custom_keyword}} /
CD3L1 / cell membrane proteins / cell digestion / antibody-antigen binding / cancer immunotherapy {{custom_keyword}} /
图 1 细胞消化方式影响肿瘤细胞与5H抗体的结合信号Fig 1 Cell digestion methods influence the binding signal of tumor cells with the 5H antibody A: Representative images showing the fluorescence signal shift of various tumor cell lines stained with the 5H antibody versus those stained with the control-IgG1 antibody under different digestion methods. B: Comparing the fold change in GMFI of cells stained with the 5H antibody relative to cells stained with the control-IgG1 antibody under different digestion methods. Trypsin digestion significantly weakened the binding between cells and the 5H antibody, whereas the effect of digestion temperature on this binding signal was minimal (n=3 for each group, One-way ANOVA, (1)P < 0.05, (2)P < 0.01, (3)P < 0.001, (4)P < 0.000 1). Ab: Antibody; Accutase: The collagenase/neutral protease cocktail; GMFI: Geometric mean fluorescence intensity; ns: Not significant; RT: Room temperature. |
图 2 细胞消化方式影响5H抗体染色阳性RD细胞的占比Fig 2 Cell digestion methods affect the percentage of 5H-stained positive RD cells A: Representative images showing the binding between RD cells and the 5H antibody under different cell digestion conditions. Blue fluorescence indicates the cell nuclei, with green fluorescence representing the 5H antibody and red fluorescence marking the early endosomes. There was no colocalization between the green and red fluorescence. B: Comparison of the percentage of 5H-stained positive RD cells in each field of view under various digestion conditions. Trypsin digestion significantly reduced this percentage, while the impact of digestion temperature was insignificant (n=5 views for each group, One-way ANOVA, (1)P < 0.000 1). Accutase: The collagenase/neutral protease cocktail; ns: Not significant; RT: Room temperature. |
图 3 胶原酶与中性蛋白酶混合液结合室温消化细胞辅助受体占位分析Fig 3 Receptor occupancy analysis of cells digested with the collagenase/neutral protease cocktail at room temperature The receptor occupancy curve of RD and RKO cells digested with the collagenase/neutral protease cocktail at room temperature when incubated with serially diluted 5H (n=3 for each antibody concentration). RO: Receptor occupancy. |
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作者贡献声明 张远 实验实施,数据采集和整理,论文撰写。邓守言 实验指导。许杰 实验设计和指导,论文修订。
利益冲突声明 所有作者均声明不存在利益冲突。
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