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Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells

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 target...

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Main Authors: Teo, Michelle Yee Mun, Ng, Jeremy Jeack Ceen, Jung, Yin Fong, Jung, Shan Hwang, Song, Adelene Ai Lian, Lim, Renee Lay Hong, In, Lionel Lian Aun
Format: Article
Language:English
Published: PeerJ Publishing 2021
Online Access:http://psasir.upm.edu.my/id/eprint/96690/1/ABSTRACT.pdf
http://psasir.upm.edu.my/id/eprint/96690/
https://peerj.com/articles/11063/
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spelling my.upm.eprints.966902022-12-14T13:16:09Z http://psasir.upm.edu.my/id/eprint/96690/ Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells Teo, Michelle Yee Mun Ng, Jeremy Jeack Ceen Jung, Yin Fong Jung, Shan Hwang Song, Adelene Ai Lian Lim, Renee Lay Hong In, Lionel Lian Aun 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. PeerJ Publishing 2021 Article PeerReviewed text en http://psasir.upm.edu.my/id/eprint/96690/1/ABSTRACT.pdf Teo, Michelle Yee Mun and Ng, Jeremy Jeack Ceen and Jung, Yin Fong and Jung, Shan Hwang and Song, Adelene Ai Lian and Lim, Renee Lay Hong and In, Lionel Lian Aun (2021) Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells. PeerJ, 9. art. no. 11063. pp. 1-26. ISSN 2689-7733 https://peerj.com/articles/11063/ 10.7717/peerj.11063
institution Universiti Putra Malaysia
building UPM Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Putra Malaysia
content_source UPM Institutional Repository
url_provider http://psasir.upm.edu.my/
language English
description 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.
format Article
author Teo, Michelle Yee Mun
Ng, Jeremy Jeack Ceen
Jung, Yin Fong
Jung, Shan Hwang
Song, Adelene Ai Lian
Lim, Renee Lay Hong
In, Lionel Lian Aun
spellingShingle Teo, Michelle Yee Mun
Ng, Jeremy Jeack Ceen
Jung, Yin Fong
Jung, Shan Hwang
Song, Adelene Ai Lian
Lim, Renee Lay Hong
In, Lionel Lian Aun
Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
author_facet Teo, Michelle Yee Mun
Ng, Jeremy Jeack Ceen
Jung, Yin Fong
Jung, Shan Hwang
Song, Adelene Ai Lian
Lim, Renee Lay Hong
In, Lionel Lian Aun
author_sort Teo, Michelle Yee Mun
title Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
title_short Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
title_full Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
title_fullStr Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
title_full_unstemmed Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS colorectal cancer cells
title_sort development of a single-chain fragment variable fused-mutant halt-1 recombinant immunotoxin against g12v mutated kras colorectal cancer cells
publisher PeerJ Publishing
publishDate 2021
url http://psasir.upm.edu.my/id/eprint/96690/1/ABSTRACT.pdf
http://psasir.upm.edu.my/id/eprint/96690/
https://peerj.com/articles/11063/
_version_ 1753789936390111232
score 13.211869

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