噬菌体抗体展示技术及其在抗新冠病毒抗体发现中的应用

doi:10.13560/j.cnki.biotech.bull.1985.2021-0862

摘要/Abstract

摘要：

噬菌体抗体展示技术是第一个也是目前应用最广泛的体外抗体筛选技术,可用于发现治疗各种疾病的全人源抗体。虽然存在不同的抗体发现方法,但噬菌体抗体展示技术已成为发现和优化靶标特异性单克隆抗体不可或缺的工具。研究人员可通过在丝状噬菌体上创建组合抗体库,从而在几周内获得抗原特异性抗体。在当前冠状病毒病（COVID-19）大流行的情况下,对中和抗体的研究激励着研究人员寻找出抗SARS-CoV-2的候选治疗药物。通过对噬菌体抗体库的筛选,目前已有许多不同的SARS-CoV-2中和抗体被发现。因此,本文综述了噬菌体抗体展示技术的原理,抗体库的构建、分类及淘选流程,并对其优点及局限性进行了讨论。此外,还总结了利用该技术发现抗SARS-CoV-2中和抗体的最新进展。旨为今后该技术在抗体发现中的应用提供理论支持。

关键词: 单克隆抗体, 噬菌体抗体展示, 抗体库, 淘选, 新冠病毒

Abstract:

Phage antibody display technology is the first and most widely used of the in vitro antibody screening technologies,which enable the discovery of human antibodies for therapeutic applications associated with all kinds of diseases. Different antibody discovery approaches exist,but phage antibody display technology has become an indispensable tool for the discovery and optimization of target-specific monoclonal antibodies. The researchers can create combinatorial antibody libraries on filamentous phage to be utilized for generating antigen specific antibodies in a matter of weeks. In the current situation of pandemic of Coronavirus disease（COVID-19）,the research for neutralizing antibodies is motivating the researchers to find therapeutic candidates against SARS-CoV-2. By panning phage antibody libraries,many different SARS-CoV-2 neutralizing antibodies have been found at present. Therefore,we summarize the principles of phage antibody display technology,and the construction,classification and panning process of phage antibody library,also discuss the advantages and limitations. Furthermore,we summarize the recent advances in the use of this technology for discovering neutralizing antibody against SARS-CoV-2. It is aimed to provide the theoretical support for the application of this technology in antibody discovery in the coming years.

Key words: monoclonal antibody, phage antibody display, antibody library, panning, SARS-CoV-2

王加利, 和似琦, 康子茜, 王建勋. 噬菌体抗体展示技术及其在抗新冠病毒抗体发现中的应用[J]. 生物技术通报, 2022, 38(5): 248-256.

WANG Jia-li, HE Si-qi, KANG Zi-xi, WANG Jian-xun. Antibody Phage Display Technology and Its Application in the Discovery of Anti-SARS-CoV-2 Antibodies[J]. Biotechnology Bulletin, 2022, 38(5): 248-256.

图/表 1

参考文献 66

[1]	Winter G, Milstein C. Man-made antibodies[J]. Nature, 1991, 349(6307):293-299. doi: 10.1038/349293a0 URL
[2]	Alfaleh MA, Alsaab HO, Mahmoud AB, et al. Phage display derived monoclonal antibodies:from bench to bedside[J]. Front Immunol, 2020, 11:1986. doi: 10.3389/fimmu.2020.01986 URL
[3]	Throsby M, van den Brink E, Jongeneelen M, et al. Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells[J]. PLoS One, 2008, 3(12):e3942. doi: 10.1371/journal.pone.0003942 URL
[4]	de Carvalho Nicacio C, Williamson RA, Parren PW, et al. Neutralizing human Fab fragments against measles virus recovered by phage display[J]. J Virol, 2002, 76(1):251-258. doi: 10.1128/JVI.76.1.251-258.2002 URL
[5]	Kramer R, Marissen W, Goudsmit J, et al. The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries[J]. Eur J Immunol, 2005, 35(7):2131-2145. pmid: 15971273
[6]	Sokullu E, Soleymani Abyaneh H, Gauthier MA. Plant/bacterial virus-based drug discovery, drug delivery, and therapeutics[J]. Pharmaceutics, 2019, 11(5):211. doi: 10.3390/pharmaceutics11050211 URL
[7]	Specthrie L, Bullitt E, Horiuchi K, et al. Construction of a microphage variant of filamentous bacteriophage[J]. J Mol Biol, 1992, 228(3):720-724. pmid: 1469710
[8]	van Wezenbeek PM, Hulsebos TJ, Schoenmakers JG. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome:comparison with phage fd[J]. Gene, 1980, 11(1/2):129-148. doi: 10.1016/0378-1119(80)90093-1 URL
[9]	Kehoe JW, Kay BK. Filamentous phage display in the new millennium[J]. Chem Rev, 2005, 105(11):4056-4072. pmid: 16277371
[10]	Smith GP. Filamentous fusion phage:novel expression vectors that display cloned antigens on the virion surface[J]. Science, 1985, 228(4705):1315-1317. doi: 10.1126/science.4001944 pmid: 4001944
[11]	Hoogenboom HR, Griffiths AD, Johnson KS, et al. Multi-subunit proteins on the surface of filamentous phage:methodologies for displaying antibody(Fab)heavy and light chains[J]. Nucleic Acids Res, 1991, 19(15):4133-4137. pmid: 1908075
[12]	Rakonjac J, Jovanovic G, Model P. Filamentous phage infection-mediated gene expression:construction and propagation of the gIII deletion mutant helper phage R408d3[J]. Gene, 1997, 198(1/2):99-103. doi: 10.1016/S0378-1119(97)00298-9 URL
[13]	Ledsgaard L, Kilstrup M, Karatt-Vellatt A, et al. Basics of antibody phage display technology[J]. Toxins, 2018, 10(6):236. doi: 10.3390/toxins10060236 URL
[14]	Rondot S, Koch J, Breitling F, et al. A helper phage to improve single-chain antibody presentation in phage display[J]. Nat Biotechnol, 2001, 19(1):75-78. pmid: 11135557
[15]	Lowman HB, Bass SH, Simpson N, et al. Selecting high-affinity binding proteins by monovalent phage display[J]. Biochemistry, 1991, 30(45):10832-10838. pmid: 1932005
[16]	Tohidkia MR, Barar J, Asadi F, et al. Molecular considerations for development of phage antibody libraries[J]. J Drug Target, 2012, 20(3):195-208. doi: 10.3109/1061186X.2011.611517 URL
[17]	Dübel S, Stoevesandt O, Taussig MJ, et al. Generating recombinant antibodies to the complete human proteome[J]. Trends Biotechnol, 2010, 28(7):333-339. doi: 10.1016/j.tibtech.2010.05.001 URL
[18]	Schirrmann T, Meyer T, Schütte M, et al. Phage display for the generation of antibodies for proteome research, diagnostics and therapy[J]. Molecules, 2011, 16(1):412-426. doi: 10.3390/molecules16010412 pmid: 21221060
[19]	Kim S, Park I, Park SG, et al. Generation, diversity determination, and application to antibody selection of a human naïve fab library[J]. Mol Cells, 2017, 40(9):655-666.
[20]	Bradbury ARM, Marks JD. Antibodies from phage antibody libraries[J]. J Immunol Methods, 2004, 290(1/2):29-49. doi: 10.1016/j.jim.2004.04.007 URL
[21]	Tsuruta LR, Dos ML, Moro AM. Display technologies for the selection of monoclonal antibodies for clinical use[M]// Böldicke T. Antibody Engineering.London:intechopen, 2018:47-73.
[22]	Lai JY, Lim TS. Infectious disease antibodies for biomedical applications:a mini review of immune antibody phage library repertoire[J]. Int J Biol Macromol, 2020, 163:640-648. doi: 10.1016/j.ijbiomac.2020.06.268 URL
[23]	Kessler C, Pardo A, et al. Novel PSCA targeting scFv-fusion proteins for diagnosis and immunotherapy of prostate cancer[J]. J Cancer Res Clin Oncol, 2017, 143(10):2025-2038. doi: 10.1007/s00432-017-2472-9 URL
[24]	Barbas CF, Bain JD, Hoekstra DM, et al. Semisynthetic combinatorial antibody libraries:a chemical solution to the diversity problem[J]. PNAS, 1992, 89(10):4457-4461. pmid: 1584777
[25]	Griffiths AD, Williams SC, Hartley O, et al. Isolation of high affinity human antibodies directly from large synthetic repertoires[J]. EMBO J, 1994, 13(14):3245-3260. doi: 10.1002/j.1460-2075.1994.tb06626.x pmid: 8045255
[26]	Sheets MD, Amersdorfer P, Finnern R, et al. Efficient construction of a large nonimmune phage antibody library:the production of high-affinity human single-chain antibodies to protein antigens[J]. PNAS, 1998, 95(11):6157-6162. pmid: 9600934
[27]	Chan CE, Lim AP, et al. The role of phage display in therapeutic antibody discovery[J]. Int Immunol, 2014, 26(12):649-657. doi: 10.1093/intimm/dxu082 URL
[28]	Andris-Widhopf J, Steinberger P, Fuller R, et al. Generation of human scFv antibody libraries:PCR amplification and assembly of light- and heavy-chain coding sequences[J]. Cold Spring Harb Protoc, 2011, 2011(9):pdb.prot065573.
[29]	Chen W, Dimitrov DS. Human monoclonal antibodies and engineered antibody domains as HIV-1 entry inhibitors[J]. Curr Opin HIV AIDS, 2009, 4(2):112-117. doi: 10.1097/COH.0b013e328322f95e URL
[30]	Beck A, Goetsch L, Dumontet C, et al. Strategies and challenges for the next generation of antibody-drug conjugates[J]. Nat Rev Drug Discov, 2017, 16(5):315-337. doi: 10.1038/nrd.2016.268 URL
[31]	Mattes MJ. Radionuclide-antibody conjugates for single-cell cytotoxicity[J]. Cancer, 2002, 94(4 suppl):1215-1223. pmid: 11877748
[32]	Alewine C, Hassan R, Pastan I. Advances in anticancer immunotoxin therapy[J]. Oncologist, 2015, 20(2):176-185. doi: 10.1634/theoncologist.2014-0358 URL
[33]	Bradbury AR, Sidhu S, Dübel S, et al. Beyond natural antibodies:the power of in vitro display technologies[J]. Nat Biotechnol, 2011, 29(3):245-254. doi: 10.1038/nbt.1791 pmid: 21390033
[34]	Dumoulin M, Conrath K, Van Meirhaeghe A, et al. Single-domain antibody fragments with high conformational stability[J]. Protein Sci, 2002, 11(3):500-515. doi: 10.1110/ps.34602 URL
[35]	Zhao AZ, Tohidkia MR, et al. Phage antibody display libraries:a powerful antibody discovery platform for immunotherapy[J]. Crit Rev Biotechnol, 2016, 36(2):276-289. doi: 10.3109/07388551.2014.958978 URL
[36]	Hoogenboom HR. Selecting and screening recombinant antibody libraries[J]. Nat Biotechnol, 2005, 23(9):1105-1116. pmid: 16151404
[37]	Shen W, Li SQ, et al. Blocking agent optimization for nonspecific binding on phage based magnetoelastic biosensors[J]. J Electrochem Soc, 2012, 159(10):B818-B823.
[38]	Jones ML, Seldon T, Smede M, et al. A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies[J]. J Immunol Methods, 2010, 354(1/2):85-90. doi: 10.1016/j.jim.2010.02.001 URL
[39]	Bazan J, Całkosiński I, Gamian A. Phage display—A powerful technique for immunotherapy[J]. Hum Vaccines Immunother, 2012, 8(12):1817-1828.
[40]	Smith GP, Petrenko VA. Phage display[J]. Chem Rev, 1997, 97(2):391-410. doi: 10.1021/cr960065d URL
[41]	Zhou M, Meyer T, Koch S, et al. Identification of a new epitope for HIV-neutralizing antibodies in the gp41 membrane proximal external region by an Env-tailored phage display library[J]. Eur J Immunol, 2013, 43(2):499-509. doi: 10.1002/eji.201242974 URL
[42]	Nizak C, Monier S, del Nery E, et al. Recombinant antibodies to the small GTPase Rab6 as conformation sensors[J]. Science, 2003, 300(5621):984-987. doi: 10.1126/science.1083911 URL
[43]	Liang WC, Wu XM, et al. Cross-species vascular endothelial growth factor(VEGF)-blocking antibodies completely inhibit the growth of human tumor xenografts and measure the contribution of stromal VEGF[J]. J Biol Chem, 2006, 281(2):951-961. doi: 10.1074/jbc.M508199200 URL
[44]	Frenzel A, Kügler J, et al. Designing human antibodies by phage display[J]. Transfus Med Hemother, 2017, 44(5):312-318. doi: 10.1159/000479633 URL
[45]	Froude JW, Pelat T, et al. Generation and characterization of protective antibodies to Marburg virus[J]. MAbs, 2017, 9(4):696-703. doi: 10.1080/19420862.2017.1299848 URL
[46]	Schofield DJ, Pope AR, Clementel V, et al. Application of phage display to high throughput antibody generation and characterization[J]. Genome Biol, 2007, 8(11):R254. doi: 10.1186/gb-2007-8-11-r254 pmid: 18047641
[47]	Löfblom J, Wernérus H, et al. Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display[J]. FEMS Microbiol Lett, 2005, 248(2):189-198. pmid: 15964717
[48]	Hoffmann M, Kleine-Weber H, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS₂ and is blocked by a clinically proven protease inhibitor[J]. Cell, 2020, 181(2):271-280. e8. doi: S0092-8674(20)30229-4 pmid: 32142651
[49]	Fan X, Cao D, Kong L, et al. Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein[J]. Nat Commun, 2020, 11(1):3618. doi: 10.1038/s41467-020-17371-6 URL
[50]	Lv Z, Deng YQ, Ye Q, et al. Structural basis for neutralization of SARS-CoV-2 and SARS-CoV by a potent therapeutic antibody[J]. Science, 2020, 369(6510):1505-1509. doi: 10.1126/science.abc5881 URL
[51]	Li W, Drelich A, Martinez DR, et al. Potent neutralization of SARS-CoV-2 in vitro and in an animal model by a human monoclonal antibody[J]. bioRxiv, 2020, 093088.
[52]	Zhu L, Deng YQ, Zhang RR, et al. Double lock of a potent human therapeutic monoclonal antibody against SARS-CoV-2[J]. Natl Sci Rev, 2021, 8(3):nwaa297. doi: 10.1093/nsr/nwaa297 URL
[53]	Parray HA, Chiranjivi AK, Asthana S, et al. Identification of an anti-SARS-CoV-2 receptor-binding domain-directed human monoclonal antibody from a naïve semisynthetic library[J]. J Biol Chem, 2020, 295(36):12814-12821. doi: 10.1074/jbc.AC120.014918 pmid: 32727845
[54]	Detalle L, Stohr T, Palomo C, et al. Generation and characterization of ALX-0171, a potent novel therapeutic nanobody for the treatment of respiratory syncytial virus infection[J]. Antimicrob Agents Chemother, 2016, 60(1):6-13. doi: 10.1128/AAC.01802-15 URL
[55]	Stalin Raj V, Okba NMA, Gutierrez-Alvarez J, et al. Chimeric camel/human heavy-chain antibodies protect against MERS-CoV infection[J]. Sci Adv, 2018, 4(8):eaas9667. doi: 10.1126/sciadv.aas9667 URL
[56]	Hufton SE, Risley P, Ball CR, et al. The breadth of cross sub-type neutralisation activity of a single domain antibody to influenza hemagglutinin can be increased by antibody valency[J]. PLoS One, 2014, 9(8):e103294. doi: 10.1371/journal.pone.0103294 URL
[57]	Ibañez LI, De Filette M, Hultberg A, et al. Nanobodies with in vitro neutralizing activity protect mice against H5N1 influenza virus infection[J]. J Infect Dis, 2011, 203(8):1063-1072. doi: 10.1093/infdis/jiq168 URL
[58]	Laursen NS, Friesen RHE, Zhu X, et al. Universal protection against influenza infection by a multidomain antibody to influenza hemagglutinin[J]. Science, 2018, 362(6414):598-602. doi: 10.1126/science.aaq0620 pmid: 30385580
[59]	Wrapp D, De Vlieger D, Corbett KS, et al. Structural basis for potent neutralization of betacoronaviruses by single-domain camelid antibodies[J]. Cell, 2020, 181(5):1004-1015.e15. doi: 10.1016/j.cell.2020.04.031 URL
[60]	Huo J, Le Bas A, Ruza RR, et al. Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2[J]. Nat Struct Mol Biol, 2020, 27(9):846-854. doi: 10.1038/s41594-020-0469-6 URL
[61]	Hanke L, Vidakovics Perez L, Sheward DJ, et al. An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction[J]. Nat Commun, 2020, 11(1):4420. doi: 10.1038/s41467-020-18174-5 pmid: 32887876
[62]	Gai J, Ma L, Li G, et al. A potent neutralizing nanobody against SARS-CoV-2 with inhaled delivery potential[J]. MedComm:Beijing, 2021, 2(1):101-113.
[63]	Dong J, Huang B, Jia Z, et al. Development of multi-specific humanized llama antibodies blocking SARS-CoV-2/ACE2 interaction with high affinity and avidity[J]. Emerg Microbes Infect, 2020, 9(1):1034-1036. doi: 10.1080/22221751.2020.1768806 URL
[64]	Koenig PA, Das H, Liu H, et al. Structure-Guided Multivalent Nanobodies Block Sars-Cov-2 Infection and Suppress Mutational Escape[J]. Science, 2021, 371(6530):eabe6230. doi: 10.1126/science.abe6230 URL
[65]	Wilson PC, Andrews SF. Tools to therapeutically harness the human antibody response[J]. Nat Rev Immunol, 2012, 12(10):709-719.
[66]	Sun Y, Ho M. Emerging antibody-based therapeutics against SARS-CoV-2 during the global pandemic[J]. Antib Ther, 2020, 3(4):246-256.

抗体名称 Antibody description	噬菌体抗体库 Phage antibody library	靶抗原 Target antigen	抗体类型 Antibody format	中和活性 Neutralization activity	参考文献 Reference
H014	免疫Fab文库	RBD	人源化Fab	假病毒 3 nmol/L 活病毒 38 nmol/L	[50]
IgG1 ab1	天然Fab、scFv、VH文库	RBD	IgG1	活病毒200 ng/mL	[51]
HB27	免疫Fab文库	RBD	IgG1	活病毒0.22 nmol/L	[52]
II62	天然半合成scFv文库	RBD	scFv、scFv- Fc、IgG1	—	[53]
VHH-72	免疫VHH文库	S	VHH-Fc	假病毒 0.2 μg/mL	[59]
H11-D4、H11-H4	天然VHH文库	RBD	VHH-Fc	活病毒18 nmol/L（H11-D4）、4-6 nmol/L（H11-H4）	[60]
Ty1	免疫VHH文库	RBD	VHH、VHH-Fc	假病毒0.77 µg/mL（Ty1） 12 ng/mL（Ty1-Fc）	[61]
Nb11-59	免疫VHH文库	RBD	VHH	活病毒0.55 µg/mL	[62]
双特异性VHH	天然和合成VHH文库	S1	VHH-Fc	—	[63]
VHH EEE	免疫VHH文库	RBD	三价VHH	假病毒 0.52 nmol/L	[64]

抗体名称 Antibody description	噬菌体抗体库 Phage antibody library	靶抗原 Target antigen	抗体类型 Antibody format	中和活性 Neutralization activity	参考文献 Reference
H014	免疫Fab文库	RBD	人源化Fab	假病毒 3 nmol/L 活病毒 38 nmol/L	[50]
IgG1 ab1	天然Fab、scFv、VH文库	RBD	IgG1	活病毒200 ng/mL	[51]
HB27	免疫Fab文库	RBD	IgG1	活病毒0.22 nmol/L	[52]
II62	天然半合成scFv文库	RBD	scFv、scFv- Fc、IgG1	—	[53]
VHH-72	免疫VHH文库	S	VHH-Fc	假病毒 0.2 μg/mL	[59]
H11-D4、H11-H4	天然VHH文库	RBD	VHH-Fc	活病毒18 nmol/L（H11-D4）、4-6 nmol/L（H11-H4）	[60]
Ty1	免疫VHH文库	RBD	VHH、VHH-Fc	假病毒0.77 µg/mL（Ty1） 12 ng/mL（Ty1-Fc）	[61]
Nb11-59	免疫VHH文库	RBD	VHH	活病毒0.55 µg/mL	[62]
双特异性VHH	天然和合成VHH文库	S1	VHH-Fc	—	[63]
VHH EEE	免疫VHH文库	RBD	三价VHH	假病毒 0.52 nmol/L	[64]