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 IC50 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.

中文翻译：

---

评价Hydra HALT-1作为重组免疫毒素的毒素部分。

免疫毒素是由毒素部分组成的杂合蛋白，该毒素部分与靶向部分相连，目的是特异性消除靶细胞。传统的免疫毒素中使用的毒素实际上很难大量生产，组织渗透性差，内部化过程复杂。我们假设较小的HALT-1（一种来自非洲乳头蛇菌的细胞溶血素）可以用作重组免疫毒素构建中的毒素部分。在这项研究中，由于炎性巨噬细胞在多种炎性和自身免疫性疾病中起主要作用，因此将其选择为靶细胞。我们旨在通过在两个方向上将HALT-1与抗CD64-scFv结合来构建靶向巨噬细胞的重组免疫毒素，并评估在分子融合后是否可以保留它们的细胞毒活性和结合能力。重组免疫毒素HALT-1-scFv和scFv-HALT-1已成功构建并在大肠杆菌（E. coli）中表达。我们的数据表明，与抗CD64 scFv融合后，HALT-1仍对CD64 +和CD64-细胞系表现出明显的细胞毒性，尽管与单独的HALT-1相比，其半数具有细胞毒性。由于将HALT-1定位在N端或C端不影响其效力，因此这两种构建体显示出可比的细胞毒活性，其中CD64 +细胞系的IC50低于CD64-细胞系。相反，靶向部分抗CD64 scFv在C末端的位置对于维持scFv结合能力至关重要。HALT-1可以通过易变性多肽接头与抗CD64-scFv融合。成功生产此重组HALT-1 scFv融合蛋白后，HALT-1被证明可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在治疗性细胞靶向中具有作为毒素部分的潜力。