Evaluation ofHydraHALT-1 as a toxin moiety for recombinant immunotoxin

William F. Jiemy, Lih Fhung Hiew, Hong Xi Sha, Lionel L. A. In, Jung Shan Hwang*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

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Abstract

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.

Original languageEnglish
Article number31
Number of pages13
JournalBMC Biotechnology
Volume20
Issue number1
DOIs
Publication statusPublished - 17-Jun-2020
Externally publishedYes

Keywords

  • Actinoporin
  • Cnidaria
  • alpha-Pore forming toxin
  • Proinflammatory
  • Single chain fragment variable
  • Immunotoxin
  • PORE-FORMING TOXIN
  • ACUTE MYELOID-LEUKEMIA
  • CUTANEOUS INFLAMMATION
  • SEA-ANEMONE
  • MONOCLONAL-ANTIBODY
  • TARGETING CD64
  • EQUINATOXIN-II
  • IN-VITRO
  • MACROPHAGES
  • ELIMINATION

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