Production of Single-chain Fragment Variable and Its Application in Tumor Diagnosis and Treatment

doi:10.13523/j.cb.2301026

China Biotechnology

2023, Vol. 43

Issue (6): 76-86 DOI: 10.13523/j.cb.2301026

Production of Single-chain Fragment Variable and Its Application in Tumor Diagnosis and Treatment

LIN Peng^1,²,QIAN Jing^1,^2,³,FENG Qiang²,LEI Jing²,JIANG Ya²,YANG Ju-lun^2,^***(

)

1 School of Medicine, Kunming University of Science and Technology, Kunming 650500, China
2 Department of Pathology, 920th Hospital of the Joint Logistics Support Force of PLA, Kunming 650032, China
3 School of Life Sciences, Kunming University of Science and Technology, Kunming 650032, China

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Abstract

Single-chain fragment variable (scFv) is a small molecule recombinant antibody consisting of a variable heavy chain (VH) and a variable light chain (VL) linked together by a flexible peptide junction. The mRNA of single chain antibodies is isolated mainly from hybridomas and reverse transcribed into cDNA as a template for scFv gene amplification, resulting in a gene library containing a large number of different VH and VL fragments of scFv. Screening and identification of the affinity and specificity of the scFv is accomplished using different display techniques, and the resulting scFv can be used to successfully express its proteins through various expression systems. Despite its small molecular weight, scFv contains the antigen-binding domain of intact antibodies and has high specificity and affinity for antigens, as well as low immunogenicity, and also has a strong ability to penetrate and spread through tumor tissue. Therefore, scFv has become a hot research topic in the development of oncology therapeutic approaches. This review details the methods and problems in the preparation of scFv and focuses on the research progress of scFv in tumor diagnosis and treatment, with a view to providing a theoretical basis for the preparation of scFv and its application in diagnosis and treatment of tumors.

Key words： Single-chain fragment variable (scFv) Recombinant antibody Tumor diagnosis Tumor therapy

Received: 18 January 2023 Published: 04 July 2023

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Cite this article:

LIN Peng, QIAN Jing, FENG Qiang, LEI Jing, JIANG Ya, YANG Ju-lun. Production of Single-chain Fragment Variable and Its Application in Tumor Diagnosis and Treatment. China Biotechnology, 2023, 43(6): 76-86.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2301026 OR https://manu60.magtech.com.cn/biotech/Y2023/V43/I6/76

Fig.1 IgG antibody molecule and its different fragments and scFv

Fig.2 Phage display technique for screening scFv

Fig.3 Functions of scFv applications in tumor diagnosis and treatment

Table 1 scFvs applied in the diagnosis and treatment of tumours

Fig.4 A simple schematic diagram of the composition of CAR-T(a) and BiTE(b)


[1]	Chiu M L, Goulet D R, Teplyakov A, et al. Antibody structure and function: the basis for engineering therapeutics. Antibodies, 2019, 8(4): 55. doi: 10.3390/antib8040055

[2]	Altshuler E P, Serebryanaya D V, Katrukha A G. Generation of recombinant antibodies and means for increasing their affinity. Biochemistry (Moscow), 2010, 75(13): 1584-1605. doi: 10.1134/S0006297910130067

[3]	Lyu X C, Zhao Q C, Hui J L, et al. The global landscape of approved antibody therapies. Antibody Therapeutics, 2022, 5(4): 233-257. doi: 10.1093/abt/tbac021 pmid: 36213257

[4]	Ahmad Z A, Yeap S K, Ali A M, et al. scFv antibody: principles and clinical application. Clinical & Developmental Immunology, 2012, 2012: 980250.

[5]	Alfthan K, Takkinen K, Sizmann D, et al. Properties of a single-chain antibody containing different linker peptides. Protein Engineering, Design and Selection, 1995, 8(7): 725-731. doi: 10.1093/protein/8.7.725

[6]	Wilson I A, Stanfield R L. Antibody-antigen interactions: new structures and new conformational changes. Current Opinion in Structural Biology, 1994, 4(6): 857-867. pmid: 7536111

[7]	Hussack G, MacKenzie C R, Tanha J. Characterization of single-domain antibodies with an engineered disulfide bond. Single domain antibodies, Totowa, NJ: Humana Press, 2012: 417-429.

[8]	Roberts C J. Therapeutic protein aggregation: mechanisms, design, and control. Trends in Biotechnology, 2014, 32(7): 372-380. doi: 10.1016/j.tibtech.2014.05.005 pmid: 24908382

[9]	Galeffi P, Lombardi A, Pietraforte I, et al. Functional expression of a single-chain antibody to ErbB-2 in plants and cell-free systems. Journal of Translational Medicine, 2006, 4(1): 1-13. doi: 10.1186/1479-5876-4-1

[10]	Finlay W J J, Shaw I, Reilly J P, et al. Generation of high-affinity chicken single-chain fv antibody fragments for measurement of the Pseudonitzschia pungens Toxin domoic acid. Applied and Environmental Microbiology, 2006, 72(5): 3343-3349. doi: 10.1128/AEM.72.5.3343-3349.2006

[11]	Zhang J L, Gou J J, Zhang Z Y, et al. Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen. Hepatobiliary Pancreat Dis Int, 2006, 5: 237-241.

[12]	Zhang X T, Dong S, Huang Y Y, et al. A scFv phage targeting the C. albicans cell wall screened from a bacteriophage-based library of induced immune protection in mice. Infection, Genetics and Evolution, 2022, 102: 105303. doi: 10.1016/j.meegid.2022.105303

[13]	Li L, Wu S M si Y, et al. Single-chain fragment variable produced by phage display technology: construction, selection, mutation, expression, and recent applications in food safety. Comprehensive Reviews in Food Science and Food Safety, 2022, 21(5): 4354-4377. doi: 10.1111/1541-4337.13018 pmid: 35904244

[14]	Ahangarzadeh S, Bandehpour M, Kazemi B. Selection of single-chain variable fragments specific for Mycobacterium tuberculosis ESAT-6 antigen using ribosome display. Iran J Basic Med Sci, 2017, 20: 327-333. doi: 10.22038/ijbms.2017.8363 pmid: 28392906

[15]	Ban B, Blake R C II, Blake D A. Yeast surface display platform for rapid selection of an antibody library via sequential counter antigen flow cytometry. Antibodies, 2022, 11(4): 61. doi: 10.3390/antib11040061

[16]	Sandomenico A, Sivaccumar J P, Ruvo M. Evolution of Escherichia coli expression system in producing antibody recombinant fragments. International Journal of Molecular Sciences, 2020, 21(17): 6324. doi: 10.3390/ijms21176324

[17]	Yaghoobizadeh F, Ardakani M R, Ranjbar M M, et al. Expression, purification, and study on the efficiency of a new potent recombinant scFv antibody against the SARS-CoV-2 spike RBD in E. coli BL21. Protein Expression and Purification, 2023, 203: 106210. doi: 10.1016/j.pep.2022.106210

[18]	Dolgikh V V, Senderskiy I V, Timofeev S A, et al. Construction of scFv antibodies against the outer loops of the Microsporidium Nosema bombycis ATP/ADP-transporters and selection of the fragment efficiently inhibiting parasite growth. International Journal of Molecular Sciences, 2022, 23(23): 15307. doi: 10.3390/ijms232315307

[19]	Kee O H, Nguyen Ngan T B, Bi J W, et al. Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells. Antibody Therapeutics, 2022, 5(4): 288-300. doi: 10.1093/abt/tbac025 pmid: 36518226

[20]	Satheeshkumar P K. Expression of single chain variable fragment (scFv) molecules in plants: a comprehensive update. Molecular Biotechnology, 2020, 62(3): 151-167. doi: 10.1007/s12033-020-00241-3 pmid: 32036549

[21]	Lo K M, Leger O, Hock B. Antibody engineering. Microbiology Spectrum, 2014, 2(1). DOI: 10.1128/microbiolspec.AID-0007-12. doi: 10.1128/microbiolspec.AID-0007-12

[22]	Lu Q, Hou Y Y, Liu X X, et al. Construction, expression and functional analysis of anti-clenbuterol codon-optimized scFv recombinant antibody. Food and Chemical Toxicology, 2020, 135: 110973. doi: 10.1016/j.fct.2019.110973 pmid: 31738983

[23]	Li L, Hou R, Shen W, et al. Development of a monoclonal-based ic-ELISA for the determination of kitasamycin in animal tissues and simulation studying its molecular recognition mechanism. Food Chemistry, 2021, 363: 129465. doi: 10.1016/j.foodchem.2021.129465

[24]	Godino A, Amaranto M, Manassero A, et al. His-tagged lactate oxidase production for industrial applications using fed-batch fermentation. Journal of Biotechnology, 2023, 363: 1-7. doi: 10.1016/j.jbiotec.2022.12.011 pmid: 36608873

[25]	Koo C W, Hershewe J M, Jewett M C, et al. Cell-free protein synthesis of particulate methane monooxygenase into nanodiscs. ACS Synthetic Biology, 2022, 11(12): 4009-4017. doi: 10.1021/acssynbio.2c00366 pmid: 36417751

[26]	刘丽琴, 陈婷婷, 李少伟, 等. 大肠杆菌表达系统在基因工程疫苗研发中的应用与策略优化. 中国新药杂志, 2020, 29(21):2434-2442.

[26]	Liu L Q, Chen T T, Li S W, et al. Escherichia coli expression system applied in the development of recombinant human vaccines and its potential improvement. Chinese Journal of New Drugs, 2020, 29(21):2434-2442.

[27]	Salavatifar M, Amin S D, Jahromi Z M, et al. Green fluorescent-conjugated anti-CEA single chain antibody for the detection of CEA-positive cancer cells. Hybridoma, 2011, 30(3): 229-238. doi: 10.1089/hyb.2011.0009 pmid: 21707357

[28]	van der Steen S C H A, van Tilborg A A G, Vallen M J E, et al. Prognostic significance of highly sulfated chondroitin sulfates in ovarian cancer defined by the single chain antibody GD3A11. Gynecologic Oncology, 2016, 140(3): 527-536. doi: 10.1016/j.ygyno.2015.12.024 pmid: 26731725

[29]	Eyvazi S, Kazemi B, Bandehpour M, et al. Identification of a novel single chain fragment variable antibody targeting CD24-expressing cancer cells. Immunology Letters, 2017, 190: 240-246. doi: S0165-2478(17)30200-6 pmid: 28866120

[30]	Verachi F, Percario Z, Di Bonito P, et al. Purification and characterization of antibodies in single-chain format against the E6 oncoprotein of human papillomavirus type 16. BioMed Research International, 2018, 2018: 1-9.

[31]	Jalilzadeh-Razin S, Mantegi M, Tohidkia M R, et al. Phage antibody library screening for the selection of novel high-affinity human single-chain variable fragment against gastrin receptor: an in silico and in vitro study. DARU Journal of Pharmaceutical Sciences, 2019, 27(1): 21-34. doi: 10.1007/s40199-018-0233-1

[32]	Fogaça R L, Alvarenga L M, Woiski T D, et al. Biomolecular engineering of antidehydroepiandrosterone antibodies: a new perspective in cancer diagnosis and treatment using single-chain antibody variable fragment. Nanomedicine, 2019, 14(6): 689-705. doi: 10.2217/nnm-2018-0230

[33]	Banisadr A, Safdari Y, Kianmehr A, et al. Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells. Human Vaccines & Immunotherapeutics, 2018, 14(4): 856-863.

[34]	Mahgoub E, Bolad A. Construction, expression and characterisation of a single chain variable fragment in the Escherichia coli periplasmic that recognise MCF-7 breast cancer cell line. Journal of Cancer Research and Therapeutics, 2014, 10(2): 265. doi: 10.4103/0973-1482.136551 pmid: 25022376

[35]	Gur D, Liu S L, Shukla A, et al. Identification of single chain antibodies to breast cancer stem cells using phage display. Biotechnology Progress, 2009, 25(6): 1780-1787. doi: 10.1002/btpr.285 pmid: 19899107

[36]	Khantasup K, Saiviroonporn P, Jarussophon S, et al. Anti-EpCAM scFv gadolinium chelate: a novel targeted MRI contrast agent for imaging of colorectal cancer. Magnetic Resonance Materials in Physics, Biology and Medicine, 2018, 31(5): 633-644. doi: 10.1007/s10334-018-0687-7

[37]	Abe K, Shoji M, Chen J, et al. Regulation of vascular endothelial growth factor production and angiogenesis by the cytoplasmic tail of tissue factor. Proceedings of the National Academy of Sciences of the United States of America, 1999, 96(15): 8663-8668.

[38]	Liu Q, Pang H, Hu X L, et al. Construction of human single-chain variable fragment antibodies of medullary thyroid carcinoma and single photon emission computed tomography/computed tomography imaging in tumor-bearing nude mice. Oncology Reports, 2016, 35(1): 171-178. doi: 10.3892/or.2015.4345 pmid: 26498224

[39]	Yakushiji H, Kobayashi K, Takenaka F, et al. Novel single-chain variant of antibody against mesothelin established by phage library. Cancer Science, 2019, 110(9): 2722-2733. doi: 10.1111/cas.14150 pmid: 31461572

[40]	Zhang X, Liu C B, Hu F, et al. PET imaging of VCAM-1 expression and monitoring therapy response in tumor with a 68Ga-labeled single chain variable fragment. Molecular Pharmaceutics, 2018, 15(2): 609-618. doi: 10.1021/acs.molpharmaceut.7b00961 pmid: 29308904

[41]	Chowdhury P S, Viner J L, Beers R, et al. Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(2): 669-674.

[42]	Seyed-Alireza E, Foroogh N, Amirhossein S. Inhibition of intercellular communication between prostate cancer cells by A specific anti-STEAP-1 single chain antibody. Anti-Cancer Agents in Medicinal Chemistry, 2018, 18(12): 1674-1679. doi: 10.2174/1871520618666171208092115 pmid: 29219059

[43]	Ji X N, Shen Y L, Sun H, et al. A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG 2 cells as a single agent or in combination with paclitaxel. Tumor Biology, 2016, 37(8): 10085-10096. doi: 10.1007/s13277-016-4803-x

[44]	Liu J N, Yi B, Zhang Z, et al. CD176 single-chain variable antibody fragment inhibits the adhesion of cancer cells to endothelial cells and hepatocytes. Frontiers of Medicine, 2016, 10(2): 204-211. doi: 10.1007/s11684-016-0443-1 pmid: 27090911

[45]	Veisi K, Farajnia S, Zarghami N, et al. Development and evaluation of a cetuximab-based humanized single chain antibody against EGFR-overexpressing tumors. Drug Research, 2014, 65(12): 624-628. doi: 10.1055/s-00023610

[46]	Nickho H, Younesi V, Aghebati-Maleki L, et al. Developing and characterization of single chain variable fragment (scFv) antibody against frizzled 7 (Fzd7) receptor. Bioengineered, 2017, 8(5): 501-510. doi: 10.1080/21655979.2016.1255383 pmid: 27849134

[47]	Crépin R, Goenaga A L, Jullienne B, et al. Development of human single-chain antibodies to the transferrin receptor that effectively antagonize the growth of leukemias and lymphomas. Cancer Research, 2010, 70(13): 5497-5506. doi: 10.1158/0008-5472.CAN-10-0938 pmid: 20530676

[48]	Wu Y, Tang W W, Cao Y H, et al. A cyclin D1-specific single-chain variable fragment antibody that inhibits HepG 2 cell growth and proliferation. Biotechnology Journal, 2020, 15(8): 1900430. doi: 10.1002/biot.v15.8

[49]	Yang J L, Liu D X, Zhen S J, et al. A novel anti-p21Ras scFv antibody reacting specifically with human tumour cell lines and primary tumour tissues. BMC Cancer, 2016, 16(1): 1-9. doi: 10.1186/s12885-015-2026-y

[50]	Wang P, Pan X Y, Feng Q, et al. The immunoreactivity of the anti-p21Ras single-chain fragment variant KGH-R1 and its predicted binding sites to p21Ras. Immunotherapy, 2020, 12(12): 879-890. doi: 10.2217/imt-2019-0222 pmid: 32664770

[51]	Huang C C, Liu F R, Feng Q, et al. 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): 1-14. doi: 10.1186/s12885-020-07763-8

[52]	Tong Q, Liu K, Lu X M, et al. Construction and characterization of a novel fusion protein MG7-scFv/SEB against gastric cancer. Journal of Biomedicine & Biotechnology, 2010, 2010: 121094.

[53]	Gattenlöhner S, Jöriβen H, Huhn M, et al. A human recombinant autoantibody-based immunotoxin specific for the fetal acetylcholine receptor inhibits rhabdomyosarcoma growth in vitro and in a murine transplantation model. Journal of Biomedicine and Biotechnology, 2010, 2010: 1-11.

[54]	Mikiewicz D, Łukasiewicz N, Zieliński M, et al. Bacterial expression and characterization of an anti-CD 22 single-chain antibody fragment. Protein Expression and Purification, 2020, 170: 105594. doi: 10.1016/j.pep.2020.105594

[55]	Patil S S, Railkar R, Swain M, et al. Novel anti IGFBP 2 single chain variable fragment inhibits glioma cell migration and invasion. Journal of Neuro-Oncology, 2015, 123(2): 225-235. doi: 10.1007/s11060-015-1800-7

[56]	Mohammadi M, Nejatollahi F, Ghasemi Y, et al. Anti-metastatic and anti-invasion effects of a specific anti-MUC 18 scFv antibody on breast cancer cells. Applied Biochemistry and Biotechnology, 2017, 181(1): 379-390. doi: 10.1007/s12010-016-2218-1 pmid: 27565656

[57]	Gao R J, Li L, Shang B Y, et al. A gelatinases-targeting scFv-based fusion protein shows enhanced antitumour activity with endostar against hepatoma. Basic & Clinical Pharmacology & Toxicology, 2015, 117(2): 105-116.

[58]	Duan Y T, Chen R Q, Huang Y J, et al. Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy. Cellular and Molecular Life Sciences, 2022, 79(1): 14. doi: 10.1007/s00018-021-04089-x

[59]	Kim D W, Cho J Y. Recent advances in allogeneic CAR-T cells. Biomolecules, 2020, 10(2): 263. doi: 10.3390/biom10020263

[60]	Vishwasrao P, Li G B, Boucher J C, et al. Emerging CAR T cell strategies for the treatment of AML. Cancers, 2022, 14(5): 1241. doi: 10.3390/cancers14051241

[61]	Zhou S J, Liu M G, Ren F, et al. The landscape of bispecific T cell engager in cancer treatment. Biomarker Research, 2021, 9(1): 1-23. doi: 10.1186/s40364-020-00251-y

[62]	Wathikthinnakon M, Luangwattananun P, Sawasdee N, et al. Combination gemcitabine and PD-L1xCD 3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells. Scientific Reports, 2022, 12: 6154. doi: 10.1038/s41598-022-09964-6 pmid: 35418130

[63]	Demarest Stephen J, Glaser Scott M. Antibody therapeutics, antibody engineering, and the merits of protein stability. Current Opinion in Drug Discovery & Development, 2008, 11(5): 675-87.

[64]	Bates A, Power C A. David vs. Goliath: the structure, function, and clinical prospects of antibody fragments. Antibodies, 2019, 8(2): 28. doi: 10.3390/antib8020028

[65]	Turki I, Hammami A, Kharmachi H, et al. Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency. Molecular Immunology, 2014, 57(2): 66-73. doi: 10.1016/j.molimm.2013.08.009 pmid: 24091293

[66]	Zuber C, Mitteregger G, Schuhmann N, et al. Delivery of single-chain antibodies (scFvs) directed against the 37/ 67 kDa laminin receptor into mice via recombinant adeno-associated viral vectors for prion disease gene therapy. Journal of General Virology, 2008, 89(8): 2055-2061. doi: 10.1099/vir.0.83670-0

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