Department of Biotechnology, Faculty of Applied Sciences, UCSI University, 56000, Kuala Lumpur, Malaysia.
Department of Biological Sciences, School of Science and Technology, Sunway University, No. 5, Jalan Universiti, Bandar Sunway, 47500 Selangor Darul Ehsan, Malaysia.
BMC Biotechnol. 2020 Jun 17;20(1):31. doi: 10.1186/s12896-020-00628-9.
Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin.
In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64 and CD64 cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC lower in CD64 cell line than in CD64 cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability.
HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.
免疫毒素是一种由毒素部分与靶向部分连接而成的杂合蛋白，旨在特异性消除靶细胞。传统免疫毒素中使用的毒素在大量生产时实际上很难，组织穿透性差，内化过程复杂。我们假设较小的 HALT-1，一种来源于Hydra magnipapillata 的细胞溶解素，可以用作构建重组免疫毒素的毒素部分。
在这项研究中，由于促炎巨噬细胞在许多炎症和自身免疫性疾病中起着重要作用，因此选择其作为靶细胞。我们旨在通过将 HALT-1 与抗 CD64-scFv 以两种方向结合，构建巨噬细胞靶向的重组免疫毒素，并评估其在分子融合后是否保留其细胞毒性和结合能力。重组免疫毒素 HALT-1-scFv 和 scFv-HALT-1 成功构建并在大肠杆菌（E. coli）中表达。我们的数据表明，HALT-1 与抗 CD64-scFv 融合后仍对 CD64 和 CD64 细胞系表现出显著的细胞毒性，尽管其细胞毒性比单独的 HALT-1 低一半。由于将 HALT-1 置于 N-或 C-末端不会影响其效力，因此两种构建体在 CD64 细胞系中的 IC 低于 CD64 细胞系，表现出相当的细胞毒性。相比之下，靶向部分抗 CD64-scFv 位于 C 末端的位置对于保持 scFv 的结合能力至关重要。
HALT-1 可以通过一个灵活的多肽接头与抗 CD64-scFv 融合。在成功生产这种重组 HALT-1 scFv 融合蛋白后，证明 HALT-1 能够有效杀死两种人类细胞系。因此，这项初步研究强烈表明 HALT-1 有可能成为治疗性细胞靶向的毒素部分。
