Original articles
Wei Sun, Zhaona Yang, Heng Lin, Ming Liu, Chenxi Zhao, Xueying Hou, Zhuowei Hu, Bing Cui. Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting[J]. Acta Pharmaceutica Sinica B, 2019, 9(5): 960-972

Improvement in affinity and thermostability of a fully human antibody against interleukin-17A by yeast-display technology and CDR grafting
Wei Sun, Zhaona Yang, Heng Lin, Ming Liu, Chenxi Zhao, Xueying Hou, Zhuowei Hu, Bing Cui
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China
Monoclonal antibodies (mAbs) are widely used in many fields due to their high specificity and ability to recognize a broad range of antigens. IL-17A can induce a rapid inflammatory response both alone and synergistically with other proinflammatory cytokines. Accumulating evidence suggests that therapeutic intervention of IL-17A signaling offers an attractive treatment option for autoimmune diseases and cancer. Here, we present a combinatorial approach for optimizing the affinity and thermostability of a novel anti-hIL-17A antibody. From a large naïve phage-displayed library, we isolated the anti-IL-17A mAb 7H9 that can neutralize the effects of recombinant human IL-17A. However, the modest neutralization potency and poor thermostability limit its therapeutic applications. In vitro affinity optimization was then used to generate 8D3 by using yeast-displayed random mutagenesis libraries. This resulted in four key amino acid changes and provided an approximately 15-fold potency increase in a cell-based neutralization assay. Complementarity-determining regions (CDRs) of 8D3 were further grafted onto the stable framework of the huFv 4D5 to improve thermostability. The resulting hybrid antibody 9NT/S has superior stabilization and affinities beyond its original antibody. Human fibrosarcoma cellbased assays and in vivo analyses in mice indicated that the anti-IL-17A antibody 9NT/S efficiently inhibited the secretion of IL-17A-induced proinflammatory cytokines. Therefore, this lead anti-IL-17A mAb might be used as a potential best-in-class candidate for treating IL-17A related diseases.
Key words:    Monoclonal antibody    Antibody maturation    Phage display    Yeast surface display    CDR grafting    Antibody engineering   
Received: 2018-11-08     Revised: 2018-11-24
DOI: 10.1016/j.apsb.2019.02.007
Funds: This work was partially supported by National Key R&D Program of China under Grant 2017YFA0205400, National Natural Science Foundation of China under Grant 81874316, 81773781 and 81530093, National Drug Innovation Major Project of China under Grant 2018ZX09711001-003-001, Chinese Academy of Medical Sciences (CAMS, Beijing, China) Central Publicinterest Scientific Institution Basal Research Fund under 2017PT31046 and 2018RC350004, CAMS Innovation Found for Medical Sciences (2016-I2M-3-008 and 2016-I2M-1-007).
Corresponding author: Bing Cui     Email:cuibing@imm.ac.cn
Author description:
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Wei Sun
Zhaona Yang
Heng Lin
Ming Liu
Chenxi Zhao
Xueying Hou
Zhuowei Hu
Bing Cui

1. Veldhoen M. Interleukin 17 is a chief orchestrator of immunity. Nat Immunol 2017;18:612-21.
2. Mi S, Li Z, Yang HZ, Liu H, Wang JP, Ma YG, et al. Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-β1-dependent and -independent mechanisms. J Immunol 2011;187:3003-14.
3. Zhang XW, Mi S, Li Z, Zhou JC, Xie J, Hua F, et al. Antagonism of Interleukin-17A ameliorates experimental hepatic fibrosis by restoring the IL-10/STAT3-suppressed autophagy in hepatocytes. Oncotarget 2017;8:9922-34.
4. Cui B, Cao X, Zou W, Wan Y, Wang N, Wang Y, et al. Regulation of immune-related diseases by multiple factors of chromatin, exosomes, microparticles, vaccines, oxidative stress, dormancy, protein quality control, inflammation and microenvironment:a meeting report of 2017 International Workshop of the Chinese Academy of Medical Sciences (CAMS) Initiative for Innovative Medicine on Tumor Immunology. Acta Pharm Sin B 2017;7:532-40.
5. Mease PJ, McInnes IB, Kirkham B, Kavanaugh A, Rahman P, van der Heijde D, et al. Secukinumab inhibition of interleukin-17A in patients with psoriatic arthritis. N Engl J Med 2015;373:1329-39.
6. Frieder J, Kivelevitch D, Menter A. Secukinumab:a review of the antiIL-17A biologic for the treatment of psoriasis. Ther Adv Chronic Dis 2018;9:5-21.
7. Syed YY. Ixekizumab:a review in moderate to severe plaque psoriasis. Am J Clin Dermatol 2017;18:147-58.
8. Miossec P, Kolls JK. Targeting IL-17 and TH17 cells in chronic inflammation. Nat Rev Drug Discov 2012;11:763-76.
9. Wang EA, Suzuki E, Maverakis E, Adamopoulos IE. Targeting IL-17 in psoriatic arthritis. Eur J Rheumatol 2017;4:272-7.
10. Cui B, Eyers PA, Dobens LL, Tan NS, Mace PD, Link WA, et al. Highlights of the 2nd International Symposium on Tribbles and Diseases:tribbles tremble in therapeutics for immunity, metabolism, fundamental cell biology and cancer. Acta Pharm Sin B 2019;9:443-54.
11. Lerner RA. Combinatorial antibody libraries:new advances, new immunological insights. Nat Rev Immunol 2016;16:498-508.
12. Zhao C, Hu Z, Cui B. Recent advances in monoclonal antibody-based therapeutics. Acta Pharm Sin 2017;52:837-47.
13. Guan X. Cancer metastases:challenges and opportunities. Acta Pharm Sin B 2015;5:402-18.
14. Jiang W, Cai G, Hu PC, Wang Y. Personalized medicine in non-small cell lung cancer:a review from a pharmacogenomics perspective. Acta Pharm Sin B 2018;8:530-8.
15. Igawa T, Tsunoda H, Kuramochi T, Sampei Z, Ishii S, Hattori K. Engineering the variable region of therapeutic IgG antibodies. MAbs 2011;3:243-52.
16. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD. Isolating and engineering human antibodies using yeast surface display. Nat Protoc 2006;1:755-68.
17. Baker MP, Reynolds HM, Lumicisi B, Bryson CJ. Immunogenicity of protein therapeutics:the key causes, consequences and challenges. Self Nonself 2010;1:314-22.
18. Frenzel A, Schirrmann T, Hust M. Phage display-derived human antibodies in clinical development and therapy. MAbs 2016;8:1177-94.
19. Lonberg N. Fully human antibodies from transgenic mouse and phage display platforms. Curr Opin Immunol 2008;20:450-9.
20. Hou X, Hu Z, Cui B. Recent advances of phage display techniques for drug discovery. Acta Pharm Sin 2018;53:1279-88.
21. Bradbury AR, Marks JD. Antibodies from phage antibody libraries. J Immunol Methods 2004;290:29-49.
22. Ewert S, Huber T, Honegger A, Pluckthun A. Biophysical properties of human antibody variable domains. J Mol Biol 2003;325:531-53.
23. Ewert S, Honegger A, Pluckthun A. Stability improvement of antibodies for extracellular and intracellular applications:CDR grafting to stable frameworks and structure-based framework engineering. Methods 2004;34:184-99.
24. Sun W, Lin H, Hua F, Hu ZW. Optimizing the host bacteria to make a large naive phage antibody library in the recombination system. Acta Pharm Sin 2013;48:66-70.
25. Chen CG, Fabri LJ, Wilson MJ, Panousis C. One-step zero-background IgG reformatting of phage-displayed antibody fragments enabling rapid and high-throughput lead identification. Nucleic Acids Res 2014;42:e26.
26. Ferreira MU, Katzin AM. The assessment of antibody affinity distribution by thiocyanate elution:a simple dose-response approach. J Immunol Methods 1995;187:297-305.
27. Garber E, Demarest SJ. A broad range of Fab stabilities within a host of therapeutic IgGs. Biochem Biophys Res Commun 2007;355:751-7.
28. Sblattero D, Bradbury A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat Biotechnol 2000;18:75-80.
29. Silacci M, Lembke W, Woods R, Attinger-Toller I, Baenziger-Tobler N, Batey S, et al. Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases. MAbs 2016;8:141-9.
30. Pullen GR, Fitzgerald MG, Hosking CS. Antibody avidity determination by ELISA using thiocyanate elution. J Immunol Methods 1986;86:83-7.
31. Bottermann M, Lode HE, Watkinson RE, Foss S, Sandlie I, Andersen JT, et al. Antibody-antigen kinetics constrain intracellular humoral immunity. Sci Rep 2016;6:37457.
32. Wang Y, Keck ZY, Saha A, Xia J, Conrad F, Lou J, et al. Affinity maturation to improve human monoclonal antibody neutralization potency and breadth against hepatitis C virus. J Biol Chem 2011;286:44218-33.
33. Sela-Culang I, Kunik V, Ofran Y. The structural basis of antibodyantigen recognition. Front Immunol 2013;4:302.
34. Kawa S, Onda M, Ho M, Kreitman RJ, Bera TK, Pastan I. The improvement of an anti-CD22 immunotoxin:conversion to singlechain and disulfide stabilized form and affinity maturation by alanine scan. MAbs 2011;3:479-86.
35. Jung S, Pluckthun A. Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Eng 1997;10:959-66.
36. Willuda J, Honegger A, Waibel R, Schubiger PA, Stahel R, Zangemeister-Wittke U, et al. High thermal stability is essential for tumor targeting of antibody fragments:engineering of a humanized anti-epithelial glycoprotein-2(epithelial cell adhesion molecule) single-chain Fv fragment. Cancer Res 1999;59:5758-67.
37. Lee CV, Liang WC, Dennis MS, Eigenbrot C, Sidhu SS, Fuh G. Highaffinity human antibodies from phage-displayed synthetic Fab libraries with a single framework scaffold. J Mol Biol 2004;340:1073-93.
38. Holm L, Sander C. The FSSP database:fold classification based on structure-structure alignment of proteins. Nucleic Acids Res 1996;24:206-9.
39. Weitzner BD, Jeliazkov JR, Lyskov S, Marze N, Kuroda D, Frick R, et al. Modeling and docking of antibody structures with Rosetta. Nat Protoc 2017;12:401-16.
40. Zaretsky M, Etzyoni R, Kaye J, Sklair-Tavron L, Aharoni A. Directed evolution of a soluble human IL-17A receptor for the inhibition of psoriasis plaque formation in a mouse model. Chem Biol 2013;20:202-11.
41. Eisen HN. Affinity enhancement of antibodies:how low-affinity antibodies produced early in immune responses are followed by high-affinity antibodies later and in memory B-cell responses. Cancer Immunol Res 2014;2:381-92.
42. Schier R, McCall A, Adams GP, Marshall KW, Merritt H, Yim M, et al. Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. J Mol Biol 1996;263:551-67.
43. North B, Lehmann A, Dunbrack Jr. RL. A new clustering of antibody CDR loop conformations. J Mol Biol 2011;406:228-56.
44. McConnell AD, Zhang X, Macomber JL, Chau B, Sheffer JC, Rahmanian S, et al. A general approach to antibody thermostabilization. MAbs 2014;6:1274-82.
45. Jung S, Honegger A, Pluckthun A. Selection for improved protein stability by phage display. J Mol Biol 1999;294:163-80.
46. Steipe B. Consensus-based engineering of protein stability:from intrabodies to thermostable enzymes. Methods Enzymol 2004;388:176-86.
47. Gong R, Vu BK, Feng Y, Prieto DA, Dyba MA, Walsh JD, et al. Engineered human antibody constant domains with increased stability. J Biol Chem 2009;284:14203-10.
48. Chennamsetty N, Voynov V, Kayser V, Helk B, Trout BL. Design of therapeutic proteins with enhanced stability. Proc Natl Acad Sci U S A 2009;106:11937-42.
49. Schaefer JV, Pluckthun A. Transfer of engineered biophysical properties between different antibody formats and expression systems. Protein Eng Des Sel 2012;25:485-506.