Top

Published in:

Open Access 01-12-2024 | Colorectal Cancer | Research

RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP

Authors: Peng Lin, Jing Qian, Cheng-Cheng Huang, Wen-Mang Xu, Yuan-Yuan Wang, Zi-Ran Gao, Shi-Qi Zheng, Peng Wang, Da-Qi Jia, Qiang Feng, Ju-Lun Yang

Published in: BMC Cancer | Issue 1/2024

Abstract

Background

Ras gene mutation and/or overexpression are drivers in the progression of cancers, including colorectal cancer. Blocking the Ras signaling has become a significant strategy for cancer therapy. Previously, we constructed a recombinant scFv, RGD-p21Ras-scFv by linking RGD membrane-penetrating peptide gene with the anti-p21Ras scFv gene. Here, we expressed prokaryotically RGD-p21Ras-scFv on a pilot scale, then investigated the anti-tumor effect and the mechanism of blocking Ras signaling.

Methods

The E. coli bacteria which could highly express RGD-p21Ras-scFv was screened and grown in 100 L fermentation tank to produce RGD-p21Ras-scFv on optimized induced expression conditions. The scFv was purified from E. coli bacteria using His Ni-NTA column. ELISA was adopted to test the immunoreactivity of RGD-p21Ras-scFv against p21Ras proteins, and the IC50 of RGD-p21Ras-scFv was analyzed by CCK-8. Immunofluorescence colocalization and pull-down assays were used to determine the localization and binding between RGD-p21Ras-scFv and p21Ras. The interaction forces between RGD-p21Ras-scFv and p21Ras after binding were analyzed by molecular docking, and the stability after binding was determined by molecular dynamics simulations. p21Ras-GTP interaction was detected by Ras pull-down. Changes in the MEK-ERK /PI3K-AKT signaling paths downstream of Ras were detected by WB assays. The anti-tumor activity of RGD-p21Ras-scFv was investigated by nude mouse xenograft models.

Results

The technique of RGD-p21Ras-scFv expression on a pilot scale was established. The wet weight of the harvested bacteria was 31.064 g/L, and 31.6 mg RGD-p21Ras-scFv was obtained from 1 L of bacterial medium. The purity of the recombinant antibody was above 85%, we found that the prepared on a pilot scale RGD-p21Ras-scFv could penetrate the cell membrane of colon cancer cells and bind to p21Ras, then led to reduce of p21Ras-GTP (active p21Ras). The phosphorylation of downstream effectors MEK-ERK /PI3K-AKT was downregulated. In vivo antitumor activity assays showed that the RGD-p21Ras-scFv inhibited the proliferation of colorectal cancer cell lines.

Conclusion

RGD-p21Ras-scFv prokaryotic expressed on pilot-scale could inhibited Ras-driven colorectal cancer growth by partially blocking p21Ras-GTP and might be able to be a hidden therapeutic antibody for treating RAS-driven tumors.

Available only for authorised users

Parada LF, Tabin CJ, Shih C, Weinberg RA. Human EJ bladder carcinoma oncogene is homologue of Harvey sarcoma virus ras gene. Nature. 1982;297(5866):474–8.CrossRefPubMed

Emel’ianova MA, Amosenko FA, Chudinov AV, Surzhikov SA, Kazubskaia TP, Liubchenko LN, Nasedkina TV. [Detection of KRAS mutations in Tumor cells using biochips]. Mol Biol (Mosk). 2011;45(5):863–70.PubMed

Zinatizadeh MR, Momeni SA, Zarandi PK, Chalbatani GM, Dana H, Mirzaei HR, Akbari ME, Miri SR. The role and function of ras-association domain family in Cancer: a review. Genes Dis. 2019;6(4):378–84.CrossRefPubMedPubMedCentral

Drosten M, Barbacid M. Targeting the MAPK pathway in KRAS-Driven tumors. Cancer Cell. 2020;37(4):543–50.CrossRefPubMed

Bai S, Feng Q, Pan XY, Zou H, Chen HB, Wang P, Zhou XL, Hong YL, Song SL, Yang JL. Overexpression of wild-type p21Ras plays a prominent role in Colorectal cancer. Int J Mol Med. 2017;39(4):861–8.CrossRefPubMedPubMedCentral

Hofmann MH, Gmachl M, Ramharter J, Savarese F, Gerlach D, Marszalek JR, Sanderson MP, Kessler D, Trapani F, Arnhof H, et al. BI-3406, a potent and selective SOS1-KRAS Interaction inhibitor, is effective in KRAS-Driven cancers through combined MEK Inhibition. Cancer Discov. 2021;11(1):142–57.CrossRefPubMed

Thein KZ, Biter AB, Hong DS. Therapeutics targeting mutant KRAS. Annu Rev Med. 2021;72:349–64.CrossRefPubMed

Canon J, Rex K, Saiki AY, Mohr C, Cooke K, Bagal D, Gaida K, Holt T, Knutson CG, Koppada N, et al. The clinical KRAS(G12C) inhibitor AMG 510 drives anti-tumour immunity. Nature. 2019;575(7781):217–23.CrossRefPubMed

Dueling, KRAS(G12C). Inhibitors achieve responses. Cancer Discov. 2020;10(1):10.CrossRef

10.

Furth ME, Davis LJ, Fleurdelys B, Scolnick EM. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982;43(1):294–304.CrossRefPubMedPubMedCentral

11.

Yoshida K, Hamatani K, Koide H, Ikeda H, Nakamura N, Akiyama M, Tsuchiyama H, Nakayama E, Shiku H. Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers. Cancer Res. 1988;48(19):5503–9.PubMed

12.

Yang JL, Liu DX, Zhen SJ, Zhou YG, Zhang DJ, Yang LY, Chen HB, Feng Q. A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues. BMC Cancer. 2016;16:131.CrossRefPubMedPubMedCentral

13.

Yang JL, Pan XY, Zhao WX, Hu QC, Ding F, Feng Q, Li GY, Luo Y. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo. Int J Oncol. 2016;48(3):1218–28.CrossRefPubMed

14.

Pan XY, Liu XJ, Li J, Zhen SJ, Liu DX, Feng Q, Zhao WX, Luo Y, Zhang YL, Li HW, et al. The antitumor efficacy of anti-p21Ras scFv mediated by the dual-promoter-regulated recombinant adenovirus KGHV300. Gene Ther. 2017;24(1):40–8.CrossRefPubMed

15.

Liu FR, Bai S, Feng Q, Pan XY, Song SL, Fang H, Cui J, Yang JL. Anti-colorectal cancer effects of anti-p21Ras scFv delivered by the recombinant adenovirus KGHV500 and cytokine-induced killer cells. BMC Cancer. 2018;18(1):1087.CrossRefPubMedPubMedCentral

16.

Wang M, Hong Y, Feng Q, Pan X, Song S, Cui J, Lei J, Fang H, Yang J. Recombinant adenovirus KGHV500 and CIK cells Codeliver Anti-p21-Ras scFv for the treatment of gastric Cancer with Wild-Type Ras Overexpression. Mol Ther Oncolytics. 2018;11:90–101.CrossRefPubMedPubMedCentral

17.

Lin XR, Zhou XL, Feng Q, Pan XY, Song SL, Fang H, Lei J, Yang JL. CIK cell-based delivery of recombinant adenovirus KGHV500 carrying the anti-p21Ras scFv gene enhances the anti-tumor effect and safety in Lung cancer. J Cancer Res Clin Oncol. 2019;145(5):1123–32.CrossRefPubMed

18.

Dai F, Zhang PB, Feng Q, Pan XY, Song SL, Cui J, Yang JL. Cytokine-induced killer cells carrying recombinant oncolytic adenovirus expressing p21Ras scFv inhibited Liver cancer. J Cancer. 2021;12(9):2768–76.CrossRefPubMedPubMedCentral

19.

Qian J, Yang M, Feng Q, Pan XY, Yang LL, Yang JL. Inhibition of glioma by adenovirus KGHV500 encoding anti-p21Ras scFv and carried by cytokine-induced killer cells. Exp Biol Med (Maywood). 2021;246(10):1228–38.CrossRefPubMed

20.

Huang CC, Liu FR, Feng Q, Pan XY, Song SL, Yang JL. RGD4C peptide mediates anti-p21Ras scFv entry into Tumor cells and produces an inhibitory effect on the human colon Cancer cell line SW480. BMC Cancer. 2021;21(1):321.CrossRefPubMedPubMedCentral

21.

Yu T, Shi Y, Pan X, Feng Q, Wang P, Song S, Yang L, Yang J. BR2 cell penetrating peptide effectively delivers anti-p21Ras scFv to Tumor cells with ganglioside expression for therapy of ras-driven Tumor. PLoS ONE. 2022;17(6):e0269084.CrossRefPubMedPubMedCentral

22.

Du Y, Lin X, Feng Q, Pan X, Song S, Yang J. Inhibition of human Lung cancer cells by anti-p21Ras scFv mediated by the activatable cell-penetrating peptide. Anticancer Drugs. 2022;33(1):e562–72.CrossRefPubMed

23.

Chen J, Yang H, Feng Y, Shi Q, Li Z, Tao Z, Fan J, Jin Y, Li S, Cheng J, et al. A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNFα in the E. Coli M15-pQE30 system by improving gene transcription. Appl Microbiol Biotechnol. 2021;105(4):1447–60.CrossRefPubMedPubMedCentral

24.

Studier FW. Protein production by auto-induction in high density shaking cultures. Protein Expr Purif. 2005;41(1):207–34.CrossRefPubMed

25.

Brenke R, Hall DR, Chuang GY, Comeau SR, Bohnuud T, Beglov D, Schueler-Furman O, Vajda S, Kozakov D. Application of asymmetric statistical potentials to antibody-protein docking. Bioinformatics. 2012;28(20):2608–14.CrossRefPubMedPubMedCentral

26.

Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, Lindahl E. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25.CrossRef

27.

Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem. 2005;26(16):1701–18.CrossRefPubMed

28.

Tian XP, Cai J, Ma SY, Fang Y, Huang HQ, Lin TY, Rao HL, Li M, Xia ZJ, Kang TB, et al. BRD2 induces drug resistance through activation of the RasGRP1/Ras/ERK signaling pathway in adult T-cell lymphoblastic Lymphoma. Cancer Commun (Lond). 2020;40(6):245–59.CrossRefPubMed

29.

Keam B, Im SA, Lee KH, Han SW, Oh DY, Kim JH, Lee SH, Han W, Kim DW, Kim TY, et al. Ki-67 can be used for further classification of triple negative Breast cancer into two subtypes with different response and prognosis. Breast Cancer Res. 2011;13(2):R22.CrossRefPubMedPubMedCentral

30.

Curnis F, Gasparri A, Sacchi A, Longhi R, Corti A. Coupling Tumor necrosis factor-alpha with alphaV integrin ligands improves its antineoplastic activity. Cancer Res. 2004;64(2):565–71.CrossRefPubMed

31.

Li X, Zhou S, Wang Y, Lian H, Zuo A, Zhou K, Tong L, Zhou Z, Gao J. The pilot-scale preparation of the SA-hGM-CSF bi-functional fusion protein. Bioengineered. 2019;10(1):108–20.CrossRefPubMedPubMedCentral

32.

Zhang H, Wu J, Zhang Y, Fu N, Wang J, Zhao S. Optimized procedure for expression and renaturation of recombinant human bone morphogenetic protein-2 at high protein concentrations. Mol Biol Rep. 2010;37(7):3089–95.CrossRefPubMed

33.

Zhang X, Xie J, Sun Y, Xu H, Du T, Liu Z, Chen J, Zheng Z, Liu K, Zhang J, et al. High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein. Appl Microbiol Biotechnol. 2014;98(12):5499–506.CrossRefPubMed

34.

Zhang S, Huang J, Zhang L, Gu J, Song Q, Cai Y, Zhong J, Zhong H, Deng Y, Zhu W, et al. Fermentation, purification, and Tumor Inhibition of a disulfide-stabilized Diabody Against Fibroblast Growth Factor-2. Front Oncol. 2021;11:585457.CrossRefPubMedPubMedCentral

35.

Tsubaki M, Takeda T, Noguchi M, Jinushi M, Seki S, Morii Y, Shimomura K, Imano M, Satou T, Nishida S. Overactivation of akt contributes to MEK inhibitor primary and Acquired Resistance in Colorectal Cancer cells. Cancers (Basel) 2019, 11(12).

36.

Roberts PJ, Der CJ. Targeting the Raf-MEK-ERK mitogen-activated protein kinase cascade for the treatment of cancer. Oncogene. 2007;26(22):3291–310.CrossRefPubMed

37.

Zips D, Thames HD, Baumann M. New anticancer agents: in vitro and in vivo evaluation. In Vivo. 2005;19(1):1–7.PubMed

Title: RGD-p21Ras-scFv expressed prokaryotically on a pilot scale inhibits ras-driven colorectal cancer growth by blocking p21Ras-GTP
Authors: Peng Lin
Jing Qian
Cheng-Cheng Huang
Wen-Mang Xu
Yuan-Yuan Wang
Zi-Ran Gao
Shi-Qi Zheng
Peng Wang
Da-Qi Jia
Qiang Feng
Ju-Lun Yang
Publication date: 01-12-2024
Publisher: BioMed Central
Keywords: Colorectal Cancer
Colorectal Cancer
Published in: BMC Cancer / Issue 1/2024
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-023-11686-5

Webinar | 19-02-2024 | 17:30 (CET)

Keynote webinar | Spotlight on antibody–drug conjugates in cancer

Watch now

Antibody–drug conjugates (ADCs) are novel agents that have shown promise across multiple tumor types. Explore the current landscape of ADCs in breast and lung cancer with our experts, and gain insights into the mechanism of action, key clinical trials data, existing challenges, and future directions.

Dr. Véronique Diéras

Prof. Fabrice Barlesi

Developed by: Springer Medicine

Keynote webinar | Spotlight on medication adherence

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

Please log in to get access to this content

Other articles of this Issue 1/2024

Retraction Note: Curcumin inhibits proliferation, migration, invasion and promotes apoptosis of retinoblastoma cell lines through modulation of miR-99a and JAK/STAT pathway

The effects of oxaliplatin-based adjuvant chemotherapy in high-risk stage II colon cancer with mismatch repair-deficient: a retrospective study

Comparison of sexual function after robot-assisted radical prostatectomy and carbon-ion radiotherapy for Japanese prostate cancer patients using propensity score matching

Anticipated time to seek medical advice for possible lung cancer symptoms and barriers to timely presentation in Palestine: a national cross-sectional study

Cardiovascular health assessment in routine cancer follow-up in community settings: survivor risk awareness and perspectives

Automated evaluation of masseter muscle volume: deep learning prognostic approach in oral cancer

Keynote webinar | Spotlight on antibody–drug conjugates in cancer