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Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection

  • Authors:
    • Yuan Dong
    • Fanwei Meng
    • Zhiheng Wang
    • Tianyi Yu
    • An Chen
    • Song Xu
    • Jianming Wang
    • Moli Yin
    • Lu Tang
    • Chuanmin Hu
    • Huiyan Wang
    • Jianhui Cai
  • View Affiliations / Copyright

    Affiliations: Jilin Collaborative Innovation Center for Antibody Engineering, Jilin Medical University, Jilin, Jilin 132013, P.R. China, Clinical Laboratory, 944th Hospital of People's Liberation Army, Jiuquan, Gansu 735000, P.R. China, Department of Clinical Biochemistry, Faculty of Pharmacy and Laboratory Medicine, Army Medical University, Chongqing 400038, P.R. China, Research and Development Department, Chongqing Biomean Technology Co., Ltd., Chongqing 400715, P.R. China
    Copyright: © Dong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Pages: 708-718
    |
    Published online on: December 16, 2020
       https://doi.org/10.3892/ijmm.2020.4822
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Abstract

A large human natural single‑chain fragment variable (scFv) phage library was constructed based on Cre‑LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription‑quantitative PCR. Two‑step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre‑LoxP enzyme‑mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10‑10 M towards PCSK9. In vitro, the PCSK9/low‑density lipoprotein receptor (LDLR) pathway of Hep‑G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3‑fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre‑LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.
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1 

Zhang Y, Wang W, Lv M, Lin Z, Geng J, Li Y, Shen B, Ma Y, Li Y, Qiao C and Feng J: A single-chain antibody using LoxP511 as the linker enables large-content phage library construction via Cre/LoxP recombination. J Biomol Screen. 19:839–846. 2014. View Article : Google Scholar : PubMed/NCBI

2 

Zhang GM, Chen YP, Guan YZ, Wang Y and An YQ: Modification and identification of a vector for making a large phage antibody library. Chin Med J (Engl). 120:2011–2016. 2007. View Article : Google Scholar

3 

Baragetti A, Grejtakova D, Casula M, Olmastroni E, Jotti GS, Norata GD, Catapano AL and Bellosta S: Proprotein convertase subtilisin-Kexin type-9 (PCSK9) and triglyceride-rich lipo-protein metabolism: Facts and gaps. Pharmacol Res. 130:1–11. 2018. View Article : Google Scholar : PubMed/NCBI

4 

Abifadel M, Varret M, Rabès JP, Allard D, Ouguerram K, Devillers M, Cruaud C, Benjannet S, Wickham L, Erlich D, et al: Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet. 34:154–156. 2003. View Article : Google Scholar : PubMed/NCBI

5 

Cohen J, Pertsemlidis A, Kotowski IK, Graham R, Garcia CK and Hobbs HH: Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9. Nat Genet. 37:161–165. 2005. View Article : Google Scholar : PubMed/NCBI

6 

Huang CC, Fornage M, Lloyd-Jones DM, Wei GS, Boerwinkle E and Liu K: Longitudinal association of PCSK9 sequence variations with low-density lipoprotein cholesterol levels: The Coronary Artery Risk Development in Young Adults Study. Circ Cardiovasc Genet. 2:354–361. 2009. View Article : Google Scholar : PubMed/NCBI

7 

Frank-Kamenetsky M, Grefhorst A, Anderson NN, Racie TS, Bramlage B, Akinc A, Butler D, Charisse K, Dorkin R, Fan Y, et al: Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates. Proc Natl Acad Sci USA. 105:11915–11920. 2008. View Article : Google Scholar : PubMed/NCBI

8 

Ray KK, Stoekenbroek RM, Kallend D, Leiter LA, Landmesser U, Wright RS, Wijngaard P and Kastelein JJP: Effect of an siRNA therapeutic targeting PCSK9 on atherogenic lipoproteins: Prespecified secondary end points in ORION 1. Circulation. 138:1304–1316. 2018. View Article : Google Scholar : PubMed/NCBI

9 

Duff CJ, Scott MJ, Kirby IT, Hutchinson SE, Martin SL and Hooper NM: Antibody-mediated disruption of the interaction between PCSK9 and the low-density lipoprotein receptor. Biochem J. 419:577–584. 2009. View Article : Google Scholar : PubMed/NCBI

10 

Dubuc G, Chamberland A, Wassef H, Davignon J, Seidah NG, Bernier L and Prat A: Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia. Arterioscler Thromb Vasc Biol. 24:1454–1459. 2004. View Article : Google Scholar : PubMed/NCBI

11 

Careskey HE, Davis RA, Alborn WE, Troutt JS, Cao G and Konrad RJ: Atorvastatin increases human serum levels of proprotein convertase subtilisin/kexin type 9. J Lipid Res. 49:394–398. 2008. View Article : Google Scholar

12 

Tonikian R, Zhang Y, Boone C and Sidhu SS: Identifying specificity profiles for peptide recognition modules from phage-displayed peptide libraries. Nat Protoc. 2:1368–1386. 2007. View Article : Google Scholar : PubMed/NCBI

13 

Lakzaei M, Rasaee MJ, Fazaeli AA and Aminian M: A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin. J Cell Physiol. 234:9486–9494. 2019. View Article : Google Scholar

14 

Tohidkia MR, Sepehri M, Khajeh S, Barar J and Omidi Y: Improved soluble ScFv ELISA screening approach for antibody discovery using phage display technology. SLAS Discov. 22:1026–1034. 2017.PubMed/NCBI

15 

Grozdanov PN, Petkov PM, Karagyozov LK and Dabeva MD: Expression and localization of PCSK9 in rat hepatic cells. Biochem Cell Biol. 84:80–92. 2006. View Article : Google Scholar : PubMed/NCBI

16 

Lipari MT, Li W, Moran P, Kong-Beltran M, Sai T, Lai J, Lin SJ, Kolumam G, Zavala-Solorio J, Izrael-Tomasevic A, et al: Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels. J Biol Chem. 287:43482–43491. 2012. View Article : Google Scholar : PubMed/NCBI

17 

Rodriguez F and Harrington RA: Cholesterol, cardiovascular risk, statins, PCSK9 inhibitors, and the future of LDL-C lowering. JAMA. 316:1967–1968. 2016. View Article : Google Scholar : PubMed/NCBI

18 

Harada LM, Carrilho AJ, Oliveira HC, Nakandakare ER and Quintão EC: Regulation of hepatic cholesterol metabolism in CETP/LDLr mice by cholesterol feeding and by drugs (chole-styramine and lovastatin) that lower plasma cholesterol. Clin Exp Pharmacol Physiol. 33:1209–1215. 2006. View Article : Google Scholar : PubMed/NCBI

19 

Cao Y, Yang H, Zhou X, Mao H, Gao T, Hu Z, He L, Pan F and Guo Z: Selection and characterization of human PCSK9 antibody from phage displayed antibody library. Biochem Bioph Res Commun. 463:712–718. 2015. View Article : Google Scholar

20 

Lie J, de Crom R, van Gent T, van Haperen R, Scheek L, Sadeghi-Niaraki F and van Tol A: Elevation of plasma phospholipid transfer protein increases the risk of atherosclerosis despite lower apolipoprotein B-containing lipoproteins. J Lipid Res. 45:805–811. 2004. View Article : Google Scholar : PubMed/NCBI

21 

Rashid S, Curtis DE, Garuti R, Anderson NN, Bashmakov Y, Ho YK, Hammer RE, Moon YA and Horton JD: Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci USA. 102:5374–5379. 2005. View Article : Google Scholar : PubMed/NCBI

22 

Schroeder KM, Beyer TP, Hansen RJ, Han B, Pickard RT, Wroblewski VJ, Kowala MC and Eacho PI: Proteolytic cleavage of antigen extends the durability of an anti-PCSK9 monoclonal antibody. J Lipid Res. 56:2124–2132. 2015. View Article : Google Scholar : PubMed/NCBI

23 

Barale C, Bonomo K, Frascaroli C, Morotti A, Guerrasio A, Cavalot F and Russo I: Platelet function and activation markers in primary hypercholesterolemia treated with anti-PCSK9 mono-clonal antibody: A 12-month follow-up. Nutr Metab Cardiovasc Dis. 30:282–291. 2020. View Article : Google Scholar

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Copy and paste a formatted citation
Spandidos Publications style
Dong Y, Meng F, Wang Z, Yu T, Chen A, Xu S, Wang J, Yin M, Tang L, Hu C, Hu C, et al: Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection. Int J Mol Med 47: 708-718, 2021.
APA
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S. ... Cai, J. (2021). Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection. International Journal of Molecular Medicine, 47, 708-718. https://doi.org/10.3892/ijmm.2020.4822
MLA
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S., Wang, J., Yin, M., Tang, L., Hu, C., Wang, H., Cai, J."Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection". International Journal of Molecular Medicine 47.2 (2021): 708-718.
Chicago
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S., Wang, J., Yin, M., Tang, L., Hu, C., Wang, H., Cai, J."Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection". International Journal of Molecular Medicine 47, no. 2 (2021): 708-718. https://doi.org/10.3892/ijmm.2020.4822
Copy and paste a formatted citation
x
Spandidos Publications style
Dong Y, Meng F, Wang Z, Yu T, Chen A, Xu S, Wang J, Yin M, Tang L, Hu C, Hu C, et al: Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection. Int J Mol Med 47: 708-718, 2021.
APA
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S. ... Cai, J. (2021). Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection. International Journal of Molecular Medicine, 47, 708-718. https://doi.org/10.3892/ijmm.2020.4822
MLA
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S., Wang, J., Yin, M., Tang, L., Hu, C., Wang, H., Cai, J."Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection". International Journal of Molecular Medicine 47.2 (2021): 708-718.
Chicago
Dong, Y., Meng, F., Wang, Z., Yu, T., Chen, A., Xu, S., Wang, J., Yin, M., Tang, L., Hu, C., Wang, H., Cai, J."Construction and application of a human scFv phage display library based on Cre‑LoxP recombination for anti‑PCSK9 antibody selection". International Journal of Molecular Medicine 47, no. 2 (2021): 708-718. https://doi.org/10.3892/ijmm.2020.4822
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