Development of a single-chain fragment variable fused-mutant HALT-1 recombinant immunotoxin against G12V mutated KRAS c olorectal 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 targeted therapies such as immunotoxins.
Methods: This study aims to develop a recombinant anti-m KRAS 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 (V _H ) and light chain (V _L ) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-m KRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific m KRAS 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-m KRAS 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 V _H and V _L 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-m KRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 10 ⁶ and 2.9 × 10 ⁶ individual clones, respectively. After three rounds of bio-panning, the anti-m KRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-m KRAS 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 IC ₅₀ 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.
Competing Interests: The authors declare that they have no competing interests.
(© 2021 Teo et al.)