The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo

Yang, Ju-Lun; Pan, Xinyan; Zhao, Wanying; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; GuiYun, Li; Luo, Ying

doi:10.3892/ijo.2016.3334

Cited by 14 publications

(18 citation statements)

References 37 publications

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“…ScFvs play an important role in the diagnosis and treatment of diseases (Wang et al 2016b ). They can be used as powerful tools that directly target specific antigens to exert strong antitumoral effects and can also be used to visualize tumor masses in vivo (Duranti et al 2018 ; Yang et al 2016 ). Therefore, passive immunization with scFvs directed against PEDV may be an effective strategy for controlling PEDV infection.…”

Section: Introductionmentioning

confidence: 99%

Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus

Wang

Zhang

et al. 2022

Appl Microbiol Biotechnol

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Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, which can cause death in suckling piglets. Vaccines confer only partial protection against new mutant strains, whereas antibodies targeting virus-encoded proteins may be effective prophylactics. In this study, we constructed a recombinant single chain variable fragment (scFv) library from the spleens of two pigs immunized with a recombinant PEDV nucleocapsid (N) protein. Among the positive clones directed against PEDV N protein isolated from the library, four scFvs that showed higher affinity for N were functionally analyzed. These scFvs specifically bound to the PEDV N protein, but not to the transmissible gastroenteritis virus (TGEV) N protein. Their framework regions were highly conserved, whereas their complementarity-determining regions displayed clear diversity. An immunofluorescence assay showed the co-localization of the four scFvs with PEDV N protein in cells. They significantly suppressed PEDV replication, detected with reverse transcription (RT)-quantitative PCR (qPCR; P < 0.01). Two of them significantly reduced the viral titer at 48 hpi and 72 hpi ( P < 0.05). In addition, they observably suppressed the production of viral protein at 72 hpi. The expression of interferons, interferon regulatory factor 3 (IRF3), and IRF7 was assessed with RT-qPCR, which indicated that PEDV dramatically suppressed the transcription of interferon-λ1 and IRF7 and that the scFvs significantly upregulated their expression ( P < 0.05). These findings facilitated the investigation of the mechanism by which PEDV evaded the host immune response and suggested that these porcine scFvs were potential candidate agents for the prevention and treatment of porcine diarrhea caused by PEDV. Key points • Four scFvs targeting PEDV N protein were generated from porcine spleens • These scFvs co-localized with PEDV N protein and suppressed PEDV replication • These scFvs significantly upregulated IFN-λ1 expression Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11722-z.

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Section: Introductionmentioning

confidence: 99%

Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus

Wang

Zhang

et al. 2022

Appl Microbiol Biotechnol

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“…However, no targeted drugs that target against p21Ras directly have been exploited. Recently, we prepared a novel anti-p21Ras Mab, KGH-R1, which can recognize and react with three type of p21Ras, including H-p21Ras, N-p21Ras and K-p21Ras, and the single chain antibody derived from this Mab could regress p21Ras-overexpressing tumors in vitro and in vivo (26,42). In this study, the Mab was employed to examine p21Ras expression in normal colorectal epithelium, inflammatory polyps, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and invasive CRC.…”

Section: Discussionmentioning

confidence: 99%

Overexpression of wild-type p21Ras plays a prominent role in colorectal cancer

Bai

Feng

Pan

et al. 2017

International Journal of Molecular Medicine

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Colorectal cancer (CRC) is the most common gastrointestinal type of cancer. The overexpression of Ras proteins, particularly p21Ras, are involved in the development of CRC. However, the subtypes of the p21Ras proteins that are overexpressed and the mutation status remain unknown restricting the development of therapeutic antibodies targeting p21Ras proteins. The present study aimed to investigate the mutation status of ras genes associated with Ras proteins that are overexpressed in CRC and explore whether or not wild-type p21Ras could be a target for CRC therapy. p21Ras expression was examined immunohistochemically in normal colorectal epithelium, benign lesions and malignant colorectal tumor tissues by monoclonal antibody (Mab) KGH-R1 which is able to react with three types of p21Ras proteins: H-p21Ras, N-p21Ras and K-p21Ras. Then, the expression levels of p21Ras subtypes were determined in CRC by a specific Mab for each p21Ras subtype. Mutation status of ras genes in p21Ras-overexpressing CRC was detected by DNA sequencing. There was rare p21Ras expression in normal colorectal epithelium but a high level of p21Ras expression in CRC, with a significant increase from normal colorectal epithelium to inflammatory polyps, low-grade intraepithelial neoplasia, high-grade intraepithelial neoplasia and invasive colorectal adenocarcinoma, respectively. Overexpression of K-p21Ras was found in all CRC tissues tested, overexpression of N-p21Ras was found in 85.7% of the CRC tissues, while H-p21Ras expression was not found in any CRC tissue. DNA sequencing showed that there were no K-ras mutations in 60% of the K-p21Ras-overexpressing CRC, while 40% of the CRC tissues harbored K-ras mutations. N-ras mutations were not found in any N-p21Ras-overexpressing CRC. Our findings indicate that overexpression of wild-type p21Ras may play a prominent role in the development of CRC in addition to ras mutations and could be a promising target for CRC therapy.

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“…Subsequently, the membranes were washed and incubated with a goat anti-mouse/rabbit IgG antibody and horseradish peroxidase (HRP) (ZSGB-Bio, ZB-5305, China) at 37°C for 45 min. After washing with TBST, the protein bands were visualized with a 3,3'-diaminobenzidine chemiluminescence system (ZSGB-Bio) [29].…”

Section: Detection Of the Immunoreactivity Of Rgd4c-p21ras Scfvmentioning

confidence: 99%

“…Mutated p21Ras proteins become key drivers in the development of cancers [26,27]. Moreover, wild-type p21Ras overexpression is an important cause of colorectal cancer [28], and the expression rate of p21Ras has been found to reach 29% -76% [29]. p21Ras could become a promising therapeutic target for colorectal cancer treatment.…”

Section: Introductionmentioning

confidence: 99%

RGD4C Peptide Mediates anti-p21Ras scFv Entry Into Tumor Cells and Produces an Inhibitory Effect on the Human Colon Cancer Cell Line SW480

Huang

Liu

Feng

et al. 2020

Preprint

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BackgroundAn anti-p21Ras scFv can specifically bind with mutant and wild-type p21Ras but cannot penetrate the cell membrane, which prevents it from binding to p21Ras in the cytoplasm. Here, the RGD4C peptide was used to mediate scFv penetration into tumor cells and produce an inhibitory effect.MethodsRGD4C-linker-EGFP and RGD4C-p21Ras-scFv recombinant expression plasmids were constructed, and the fusion proteins were expressed in E. coli and purified with HisPur Ni-NTA. RGD4C-linker-EFGP was used to test the factors affecting RGD4C penetration of the tumor cell membrane. The immunoreactivity of RGD4C-p21Ras-scFv toward p21Ras was identified by ELISA and western blotting. Moreover, immunocytochemistry was used to detect the ability of RGD4C-p21Ras-scFv to penetrate SW480 cells. Cell migration, colony formation, cell killing, and apoptosis assays were used to assess the inhibitory effect of RGD4C-p21Ras-scFv on SW480 cells in vitro.ResultsThe RGD4C peptide could target tumor cells, but endocytosis inhibitors and a low temperature inhibited RGD4C peptide endocytosis into cells, and tumor cell entry was time and concentration dependent. Additionally, a change in the cell membrane potential did not affect penetrability. We found that RGD4C-p21Ras-scFv could penetrate SW480 cells; effectively inhibit the growth, proliferation and migration of SW480 cells; and promote apoptosis in SW480 cells. ConclusionThe RGD4C peptide can mediate anti-p21Ras scFv entry into SW480 cells and produce an inhibitory effect, which indicates that RGD4C-p21Ras-scFv may be a potential therapeutic antibody for the treatment of RAS-mutant colorectal cancer.

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The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo

Cited by 14 publications

References 37 publications

Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus

Generation and functional analysis of single chain variable fragments (scFvs) targeting the nucleocapsid protein of Porcine epidemic diarrhea virus

Overexpression of wild-type p21Ras plays a prominent role in colorectal cancer

RGD4C Peptide Mediates anti-p21Ras scFv Entry Into Tumor Cells and Produces an Inhibitory Effect on the Human Colon Cancer Cell Line SW480

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