Ataee Mohammad Hossein, Mirhosseini Seyed Ali, Mirnejad Reza, Rezaie Ehsan, Hosseini Hamideh Mahmoodzadeh, Amani Jafar
Student Research Committee, Baqiyatallah University of Medical Sciences, Tehran, I.R. Iran.
Applied Microbiology Research Center, Systems Biology and Poisonings Institute, Baqiyatallah University of Medical Sciences, Tehran, I.R. Iran.
Res Pharm Sci. 2022 Jul 14;17(4):428-444. doi: 10.4103/1735-5362.350243. eCollection 2022 Aug.
The lack of a new effective treatment for small cell lung cancer (SCLC) is an unresolved problem. Due to the new identification of delta-like ligand 3 (DLL3) and its high expression in SCLC patients, the use of DLL3 in target therapy can be effective. The use of bacterial toxins belonging to the ADP-ribosyl transferase toxins family and human enzymes to remove cancerous cells has been effective in the structure of immunotoxins. In this study, single-chain fragment variable of rovalpituzumab antibody fused to granzyme B (Rova-GrB) and PltA of typhoid toxin (Rova-Typh) as immunotoxins were designed, and bioinformatics analysis was done.
analysis including the physicochemical properties, evaluation of the secondary and tertiary structure, refinement and validation of 3D models, and docking were performed. Immunotoxin genes were cloned and expressed in the BL21 (DE3) host, purified, subsequently confirmed by western blotting and their secondary structure was evaluated by the circular dichroism method.
FINDINGS/RESULTS: The bioinformatics analysis showed that Rova-GrB and Rova-Typh had hydrophilic properties, their codon optimization parameters were standard, validation parameters were improved after immunotoxin refinement, and docking analysis showed that the binding domain of immunotoxins could bind the N-terminal region of DLL3. immunotoxins had high expression and after purification under denaturing condition by Ni-NTA column, the immunotoxins were dialyzed against PBS buffer.
The immunotoxins had the right structure and can be produced in a prokaryotic host. The recombinant immunotoxins against DLL3 can be promising therapeutic agents for SCLC cancer.
小细胞肺癌（SCLC）缺乏新的有效治疗方法是一个尚未解决的问题。由于新发现了δ样配体3（DLL3）及其在SCLC患者中的高表达，将DLL3用于靶向治疗可能有效。在免疫毒素结构中，使用属于ADP-核糖基转移酶毒素家族的细菌毒素和人类酶来清除癌细胞已被证明是有效的。在本研究中，设计了与颗粒酶B融合的罗伐单抗单链可变片段（Rova-GrB）和伤寒毒素的PltA（Rova-Typh）作为免疫毒素，并进行了生物信息学分析。
进行了包括理化性质分析、二级和三级结构评估、3D模型的优化和验证以及对接分析。免疫毒素基因在BL21（DE3）宿主中克隆和表达，纯化后通过蛋白质印迹法进行确认，并通过圆二色法评估其二级结构。
生物信息学分析表明，Rova-GrB和Rova-Typh具有亲水性，其密码子优化参数符合标准，免疫毒素优化后的验证参数有所改善，对接分析表明免疫毒素的结合域可与DLL3的N端区域结合。免疫毒素表达量高，在变性条件下通过Ni-NTA柱纯化后，将免疫毒素用PBS缓冲液透析。
免疫毒素具有正确的结构，可在原核宿主中产生。针对DLL3的重组免疫毒素有望成为SCLC癌症的治疗药物。
