Background: 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 fromHydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin.
Results: 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 inEscherichia 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(50)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.
Conclusions: 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.
- alpha-Pore forming toxin
- Single chain fragment variable
- PORE-FORMING TOXIN
- ACUTE MYELOID-LEUKEMIA
- CUTANEOUS INFLAMMATION
- TARGETING CD64