Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, Thailand.
Center of Research Excellence on Therapeutic Proteins and Antibody Engineering, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
PLoS One. 2023 Apr 20;18(4):e0284708. doi: 10.1371/journal.pone.0284708. eCollection 2023.
Hodgkin's lymphoma and anaplastic large cell lymphoma, especially relapsed or refractory diseases, could recently be cured by CD30-targeted immunotherapy. However, the CD30 antigen releases the soluble ectodomain of CD30, which might obscure the targeted therapy. Therefore, the membrane epitope of CD30 (mCD30), left on the cancer cells, might be a prospective target for lymphoma treatment. The discovery of novel mCD30 monoclonal antibodies (mAbs) using phage technology yielded 59 potential human single-chain variable fragments (HuscFvs). Ten candidate HuscFv clones have been selected based on various methods, i.e., direct PCR, ELISA and western blot assays, and nucleotide sequencing techniques. Fortunately, only one potential HuscFv clone, clone #A4, was determined by the prediction of HuscFv-peptide molecular docking and the binding affinity test using isothermal titration calorimetry. Finally, we proved that the HuscFv #A4, which had a binding affinity (Kd) of 421e-9 ± 2.76e-6 M, might be the novel mCD30 mAb. We generated chimeric antigen receptor-modified T lymphocytes using HuscFv #A4 as an antigen detection part (anti-mCD30-H4CART). The cytotoxicity assay of anti-mCD30-H4CART cells showed significant eradication of the CD30-expressing cell line, K562 (p = 0.0378). We found a novel mCD30 HuscFv using human phage technology. We systematically examined and proved that our HuscFv #A4 could specifically eradicate CD30-expressing cancers.
霍奇金淋巴瘤和间变大细胞淋巴瘤，尤其是复发或难治性疾病，最近可以通过 CD30 靶向免疫疗法治愈。然而，CD30 抗原释放 CD30 的可溶性细胞外结构域，这可能会使靶向治疗变得模糊。因此，留在癌细胞上的 CD30 膜表位（mCD30）可能成为淋巴瘤治疗的一个有前景的靶点。使用噬菌体技术发现新型 mCD30 单克隆抗体（mAb）产生了 59 个潜在的人源单链可变片段（HuscFvs）。基于各种方法，即直接 PCR、ELISA 和 Western blot 测定以及核苷酸测序技术，已经选择了 10 个候选 HuscFv 克隆。幸运的是，只有一个潜在的 HuscFv 克隆，克隆#A4，通过 HuscFv-肽分子对接的预测和使用等温滴定量热法的结合亲和力测试来确定。最后，我们证明了 HuscFv#A4 的结合亲和力（Kd）为 421e-9±2.76e-6M，可能是新型 mCD30 mAb。我们使用 HuscFv#A4 作为抗原检测部分（抗-mCD30-H4CART）生成嵌合抗原受体修饰的 T 淋巴细胞。抗-mCD30-H4CART 细胞的细胞毒性测定显示出对表达 CD30 的细胞系 K562 的显著清除（p=0.0378）。我们使用人类噬菌体技术发现了一种新型 mCD30 HuscFv。我们系统地检查并证明了我们的 HuscFv#A4 可以特异性地清除表达 CD30 的癌症。
