Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran.
Iran Biomed J. 2023 Jul 1;27(4):191-8. doi: 10.61186/ibj.3874.
T-cell immunoglobulin domain and mucin domain-3 (TIM-3) is an inhibitory receptor expressed in a variety of cells, including dendritic cells, T-helper 1 lymphocytes, and natural killer cells. Binding of this protein to its ligand, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), causes T-cell exhaustion, a specific condition in which effector T cells lose their ability to proliferate and produce cytokines. Blocking this inhibitory receptor is known to be an effective strategy for treating cancer and other related diseases. Therefore, in this study, in order to block the inhibitory receptor of TIM-3, we designed and produced recombinantly a protein with a high binding affinity to this receptor.
The extracellular domain of CEACAM1 involved in binding to TIM-3 was mutated using R script to obtain a variant with the increased binding affinity to TIM-3. The binding energy of the mutant protein was calculated using the FoldX module. Finally, after recombinant production of the most appropriate CEACAM1 variant (variant 39) in E. coli, its secondary structure was determined by CD spectroscopy.
The binding free energy between variant 39 and TIM-3 decreased from -5.63 to -14.49 kcal/mol, indicating an increased binding affinity to the receptor. Analysis of the secondary structure of this variant also showed that the mutation did not significantly alter the structure of the protein.
Our findings suggest that variant 39 could bind to TIM-3 with a higher binding affinity than the wild-type, making it a proper therapeutic candidate for blocking TIM-3.
T 细胞免疫球蛋白域和粘蛋白域-3（TIM-3）是一种在多种细胞中表达的抑制性受体，包括树突状细胞、辅助性 T 细胞 1 和自然杀伤细胞。该蛋白与它的配体，癌胚抗原相关细胞粘附分子 1（CEACAM1）结合，导致 T 细胞耗竭，这是效应 T 细胞失去增殖和产生细胞因子能力的一种特定状态。阻断这种抑制性受体被认为是治疗癌症和其他相关疾病的有效策略。因此，在这项研究中，为了阻断 TIM-3 的抑制性受体，我们设计并生产了一种与该受体具有高结合亲和力的重组蛋白。
使用 R 脚本对涉及与 TIM-3 结合的 CEACAM1 的胞外结构域进行突变，以获得与 TIM-3 结合亲和力增加的变体。使用 FoldX 模块计算突变蛋白的结合能。最后，在大肠杆菌中重组生产最合适的 CEACAM1 变体（变体 39）后，通过 CD 光谱学确定其二级结构。
变体 39 与 TIM-3 之间的结合自由能从-5.63 降低到-14.49 kcal/mol，表明与受体的结合亲和力增加。对该变体的二级结构的分析也表明，突变没有显著改变蛋白质的结构。
我们的研究结果表明，变体 39 与 TIM-3 的结合亲和力比野生型更高，因此可能是阻断 TIM-3 的合适治疗候选物。
