Libentech Co. LTD, Daejeon, Republic of Korea.
Graduate School of Medical Science, College of medicine, Yonsei University, Seoul, Republic of Korea.
Virol J. 2024 Jan 4;21(1):7. doi: 10.1186/s12985-023-02276-9.
Oncolytic viruses are being studied and developed as novel cancer treatments. Using directed evolution technology, structural modification of the viral surface protein increases the specificity of the oncolytic virus for a particular cancer cell. Newcastle disease virus (NDV) does not show specificity for certain types of cancer cells during infection; therefore, it has low cancer cell specificity. Hemagglutinin is an NDV receptor-binding protein on the cell surface that determines host cell tropism. NDV selectivity for specific cancer cells can be increased by artificial amino acid changes in hemagglutinin neuraminidase HN proteins via directed evolution, leading to improved therapeutic effects.
Sialic acid-binding sites (H domains) of the HN protein mutant library were generated using error-prone PCR. Variants of the H domain protein were screened by enzyme-linked immunosorbent assay using HCT 116 cancer cell surface molecules. The mutant S519G H domain protein showed the highest affinity for the surface protein of HCT 116 cells compared to that of different types of cancer cells. This showed that the S519G mutant H domain protein gene replaced the same part of the original HN protein gene, and S519G mutant recombinant NDV (rNDV) was constructed and recovered. S519G rNDV cancer cell killing effects were tested using the MTT assay with various cancer cell types, and the tumor suppression effect of the S519G mutant rNDV was tested in a xenograft mouse model implanted with cancer cells, including HCT 116 cells.
S519G rNDV showed increased specificity and enhanced killing ability of HCT 116 cells among various cancer cells and a stronger suppressive effect on tumor growth than the original recombinant NDV. Directed evolution using an artificial amino acid change in the NDV HN (S519G mutant) protein increased its specificity and oncolytic effect in colorectal cancer without changing its virulence.
These results provide a new methodology for the use of directed evolution technology for more effective oncolytic virus development.
溶瘤病毒正在被研究并开发为新型癌症治疗方法。通过定向进化技术，对病毒表面蛋白进行结构修饰可以提高溶瘤病毒对特定癌细胞的特异性。在感染过程中，新城疫病毒（NDV）对某些类型的癌细胞没有特异性；因此，它对癌细胞的特异性较低。血凝素是细胞表面上的 NDV 受体结合蛋白，决定着宿主细胞的嗜性。通过定向进化，在血凝素神经氨酸酶 HN 蛋白的人工氨基酸变化，可以增加 NDV 对特定癌细胞的选择性，从而提高治疗效果。
使用易错 PCR 生成 HN 蛋白突变文库的唾液酸结合位点（H 结构域）。通过酶联免疫吸附试验筛选 HCT116 癌细胞表面分子的 H 结构域蛋白变体。与不同类型的癌细胞相比，突变的 S519G H 结构域蛋白对 HCT116 细胞表面蛋白表现出最高的亲和力。这表明 S519G 突变 H 结构域蛋白基因取代了原始 HN 蛋白基因的相同部分，并构建和恢复了 S519G 突变重组 NDV（rNDV）。用 MTT 法检测 S519G rNDV 对各种癌细胞的杀伤作用，并在植入癌细胞的异种移植小鼠模型中测试 S519G 突变 rNDV 的肿瘤抑制作用，包括 HCT116 细胞。
S519G rNDV 对各种癌细胞中 HCT116 细胞的特异性和杀伤能力增强，对肿瘤生长的抑制作用强于原始重组 NDV。在 NDV HN（S519G 突变体）蛋白中使用人工氨基酸变化进行定向进化，提高了其在结直肠癌中的特异性和溶瘤作用，而不改变其毒力。
这些结果为使用定向进化技术开发更有效的溶瘤病毒提供了一种新方法。
