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  1. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M
    Clin. Dev. Immunol., 2012;2012:980250.
    PMID: 22474489 DOI: 10.1155/2012/980250
    To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
    Matched MeSH terms: Immunotoxins/immunology; Immunotoxins/therapeutic use*
  2. Jiemy WF, Hiew LF, Sha HX, In LLA, Hwang JS
    BMC Biotechnol, 2020 Jun 17;20(1):31.
    PMID: 32552895 DOI: 10.1186/s12896-020-00628-9
    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 from Hydra 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 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.

    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.

    Matched MeSH terms: Immunotoxins
  3. Ng TJ, Teo MYM, Liew DS, Effiong PE, Hwang JS, Lim CSY, et al.
    PeerJ, 2019;7:e6639.
    PMID: 31106043 DOI: 10.7717/peerj.6639
    Background: Hydra actinoporin like toxin -1 (HALT-1), is a small 18.5 kDa pore forming toxin derived from Hydra magnipapillata which has been shown to elicit strong haemolytic and cytolytic activity when in contact with cell membranes. Due to its cytotoxic potency, HALT-1 was further investigated for its potential as a toxin moiety candidate in immunotoxin developmental efforts, ideally as a form of targeted therapy against cancer.

    Methods: In this study, wtHALT-1 (wild type) and its Y110A mutated binding domain counterpart (mHALT-1) were produced and evaluated for their cytotoxic and apoptotic effects on various cancer cell lines. A total of seven different tumour and non-tumour cell lines including HeLa, HepG2, SW-620, MCF-7, CCD841CoN, NHDF and HCT116 were used. Immunofluorescence assays were used to observe membrane binding and localization changes between both HALT-1 recombinant proteins based on 6xHis-tag detection.

    Result: Based on MTT data, mHALT-1 demonstrated a significant reduction of 82% ±  12.21% in cytotoxic activity across all cell lines after the membrane recognition domain had been mutated in comparison to the wtHALT-1. Annexin V FITC/PI assay data also indicated that HeLa, HepG2 and MCF-7 demonstrated an apoptosis-mediated cell death after being treated with wtHALT-1. Additionally, a notable difference between wtHALT-1 and mHALT-1 binding affinity was clearly observed where emission of green fluorescence along the cell membrane was observed only in wtHALT-1 treated cells.

    Discussion: These results suggest that mHALT-1 (Y110A) can be potentially developed as a toxin-moiety candidate for the development of future immunotoxins against various human cell-based diseases.

    Matched MeSH terms: Immunotoxins
  4. Teo MYM, Ng JJC, Fong JY, Hwang JS, Song AA, Lim RLH, et al.
    PeerJ, 2021;9:e11063.
    PMID: 33959410 DOI: 10.7717/peerj.11063
    Background: KRAS oncogenes harboring codon G12 and G13 substitutions are considered gatekeeper mutations which drive oncogenesis in many cancers. To date, there are still no target-specific vaccines or drugs available against this genotype, thus reinforcing the need towards the development of targeted therapies such as immunotoxins.

    Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.

    Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.

    Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.

    Matched MeSH terms: Immunotoxins
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