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CC BY 4.0 · Pharmaceutical Fronts 2020; 02(01): e64-e76
DOI: 10.1055/s-0040-1708527
Original Article
Georg Thieme Verlag KG Stuttgart · New York

Construction of Novel Bispecific Single-Domain Antibodies (BiSdAbs) with Potent Antiangiogenic Activities

Xianglei Liu
1   Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
,
Tianyuan Sun
1   Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
,
Qiuhan Ge
1   Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
,
Jianwei Zhu
1   Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China
2   Jecho Laboratories, Inc. Maryland, United States
3   Jecho Biopharmaceuticals Co., Ltd., Tianjin, China
› Author Affiliations Funding This work was supported by the China Postdoctoral Science Foundation (2016M600319) and Natural Science Foundation of China (81473127&81773621).
Further Information

Publication History

Publication Date:
31 March 2020 (online)

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Abstract

The development of bispecific antibodies (BsAbs) has had a profound impact on cancer immunotherapy. Single-domain antibodies (SdAbs) could offer advantages over other antibody formats for the generation of a BsAbs, such as small size (∼12–15 kDa), with high affinity and specificity, superior accessibility, and high yield expression in bacteria. In this study, VEGFR2 and CD16 were chosen as the targets to construct BsAbs. As the rationale, VEGFR2 is critical for tumor-associated angiogenesis, and CD16 expressed on natural killer cells is an important target on immune cells. Humanized anti-VEGFR2 SdAb 3VGR19 and anti-CD16 SdAb C21 were combined to construct several bispecific SdAbs (BiSdAbs). The biochemical properties of the BiSdAbs were characterized. They retained the high affinity for both targets, binding selectivity, and antiangiogenic activity such as inhibition of cell proliferation, migration, endothelial tube formation, angiogenesis, and cytotoxicity to cancer cells in vitro, indicating that BiSdAbs could be a potential alternative for cancer therapy.

Keywords

bispecific antibody - single-domain antibodies - nanobody - BiSdAb

Author Contributions

X.L. designed and conducted the experiments, analyzed the data, and drafted the manuscript; T.S. and Q. G. performed some of the experiments. J.Z. supervised project, analyzed data, critically discussed, and revised the manuscript. All authors read and approved the final manuscript.


Supplementary Material

 

  • References

  • 1 Weiner GJ. Building better monoclonal antibody-based therapeutics. Nat Rev Cancer 2015; 15 (06) 361-370
    CrossrefPubMedGoogle Scholar
  • 2 Sharma P, Hu-Lieskovan S, Wargo JA, Ribas A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 2017; 168 (04) 707-723
    CrossrefPubMedGoogle Scholar
  • 3 Klinger M, Benjamin J, Kischel R, Stienen S, Zugmaier G. Harnessing T cells to fight cancer with BiTE® antibody constructs--past developments and future directions. Immunol Rev 2016; 270 (01) 193-208
    CrossrefPubMedGoogle Scholar
  • 4 Han L, Chen J, Ding K. , et al. Efficient generation of bispecific IgG antibodies by split intein mediated protein trans-splicing system. Sci Rep 2017; 7 (01) 8360
    CrossrefPubMedGoogle Scholar
  • 5 Wu Z, Cheung NV. T cell engaging bispecific antibody (T-BsAb): from technology to therapeutics. Pharmacol Ther 2018; 182: 161-175
    CrossrefPubMedGoogle Scholar
  • 6 Godar M, de Haard H, Blanchetot C, Rasser J. Therapeutic bispecific antibody formats: a patent applications review (1994-2017). Expert Opin Ther Pat 2018; 28 (03) 251-276
    CrossrefPubMedGoogle Scholar
  • 7 Frampton JE. Catumaxomab: in malignant ascites. Drugs 2012; 72 (10) 1399-1410
    CrossrefPubMedGoogle Scholar
  • 8 Przepiorka D, Ko CW, Deisseroth A. , et al. FDA approval: blinatumomab. Clin Cancer Res 2015; 21 (18) 4035-4039
    CrossrefPubMedGoogle Scholar
  • 9 Scott LJ, Kim ES. Emicizumab-kxwh: first global approval. Drugs 2018; 78 (02) 269-274
    CrossrefPubMedGoogle Scholar
  • 10 Thakur A, Huang M, Lum LG. Bispecific antibody based therapeutics: Strengths and challenges. Blood Rev 2018; 32 (04) 339-347
    CrossrefPubMedGoogle Scholar
  • 11 Sedykh SE, Prinz VV, Buneva VN, Nevinsky GA. Bispecific antibodies: design, therapy, perspectives. Drug Des Devel Ther 2018; 12: 195-208
    CrossrefPubMedGoogle Scholar
  • 12 Nuñez-Prado N, Compte M, Harwood S. , et al. The coming of age of engineered multivalent antibodies. Drug Discov Today 2015; 20 (05) 588-594
    CrossrefPubMedGoogle Scholar
  • 13 Klingemann H, Boissel L, Toneguzzo F. Natural killer cells for immunotherapy - advantages of the NK-92 cell line over blood NK cells. Front Immunol 2016; 7: 91
    PubMedGoogle Scholar
  • 14 Rezvani K, Rouce RH. The application of natural killer cell immunotherapy for the treatment of cancer. Front Immunol 2015; 6: 578
    PubMedGoogle Scholar
  • 15 Ferrari de Andrade L, Tay RE, Pan D. , et al. Antibody-mediated inhibition of MICA and MICB shedding promotes NK cell-driven tumor immunity. Science 2018; 359 (6383): 1537-1542
    CrossrefPubMedGoogle Scholar
  • 16 Smits NC, Coupet TA, Godbersen C, Sentman CL. Designing multivalent proteins based on natural killer cell receptors and their ligands as immunotherapy for cancer. Expert Opin Biol Ther 2016; 16 (09) 1105-1112
    CrossrefPubMedGoogle Scholar
  • 17 Chen S, Li J, Li Q, Wang Z. Bispecific antibodies in cancer immunotherapy. Hum Vaccin Immunother 2016; 12 (10) 2491-2500
    CrossrefPubMedGoogle Scholar
  • 18 Wu J, Fu J, Zhang M, Liu D. AFM13: a first-in-class tetravalent bispecific anti-CD30/CD16A antibody for NK cell-mediated immunotherapy. J Hematol Oncol 2015; 8: 96
    CrossrefPubMedGoogle Scholar
  • 19 Pahl J, Reusch U, Gantke T. , et al. AFM13 is the most advanced bispecific NK-cell engaging antibody in clinical development substantially enhancing NK-cell effector function and proliferation. Blood 2016; 128: 1764
    CrossrefGoogle Scholar
  • 20 Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438 (7070): 967-974
    Google Scholar
  • 21 Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407 (6801): 249-257
    CrossrefPubMedGoogle Scholar
  • 22 Abdullah SE, Perez-Soler R. Mechanisms of resistance to vascular endothelial growth factor blockade. Cancer 2012; 118 (14) 3455-3467
    CrossrefPubMedGoogle Scholar
  • 23 Ferrara N, Hillan KJ, Gerber HP, Novotny W. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 2004; 3 (05) 391-400
    CrossrefPubMedGoogle Scholar
  • 24 Verdaguer H, Tabernero J, Macarulla T. Ramucirumab in metastatic colorectal cancer: evidence to date and place in therapy. Ther Adv Med Oncol 2016; 8 (03) 230-242
    CrossrefPubMedGoogle Scholar
  • 25 Hamers-Casterman C, Atarhouch T, Muyldermans S. , et al. Naturally occurring antibodies devoid of light chains. Nature 1993; 363 (6428): 446-448
    CrossrefPubMedGoogle Scholar
  • 26 Allegra A, Innao V, Gerace D, Vaddinelli D, Allegra AG, Musolino C. Nanobodies and cancer: current status and new perspectives. Cancer Invest 2018; 36 (04) 221-237
    CrossrefPubMedGoogle Scholar
  • 27 Steeland S, Vandenbroucke RE, Libert C. Nanobodies as therapeutics: big opportunities for small antibodies. Drug Discov Today 2016; 21 (07) 1076-1113
    CrossrefPubMedGoogle Scholar
  • 28 Kijanka M, Dorresteijn B, Oliveira S, van Bergen en Henegouwen PM. Nanobody-based cancer therapy of solid tumors. Nanomedicine (Lond) 2015; 10 (01) 161-174
    Google Scholar
  • 29 Unciti-Broceta JD, Del Castillo T, Soriano M, Magez S, Garcia-Salcedo JA. Novel therapy based on camelid nanobodies. Ther Deliv 2013; 4 (10) 1321-1336
    CrossrefPubMedGoogle Scholar
  • 30 Hassanzadeh-Ghassabeh G, Devoogdt N, De Pauw P, Vincke C, Muyldermans S. Nanobodies and their potential applications. Nanomedicine (Lond) 2013; 8 (06) 1013-1026
    Google Scholar
  • 31 Ayyar BV, Arora S, O'Kennedy R. Coming-of-age of antibodies in cancer therapeutics. Trends Pharmacol Sci 2016; 37 (12) 1009-1028
    CrossrefPubMedGoogle Scholar
  • 32 Duggan S. Caplacizumab: first global approval. Drugs 2018; 78 (15) 1639-1642
    CrossrefPubMedGoogle Scholar
  • 33 Ren X, Xie W, Wang Y. , et al. VEGFR2-targeted fusion antibody improved NK cell-mediated immunosurveillance against K562 cells. Immunol Res 2016; 64 (04) 1060-1070
    CrossrefPubMedGoogle Scholar
  • 34 Xie W, Liu F, Wang Y. , et al. VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer. Oncotarget 2016; 7 (13) 16445-16461
    CrossrefPubMedGoogle Scholar
  • 35 Behdani M, Zeinali S, Khanahmad H. , et al. Generation and characterization of a functional Nanobody against the vascular endothelial growth factor receptor-2; angiogenesis cell receptor. Mol Immunol 2012; 50 (1–2): 35-41
    CrossrefPubMedGoogle Scholar
  • 36 Ma L, Gu K, Zhang CH. , et al. Generation and characterization of a human nanobody against VEGFR-2. Acta Pharmacol Sin 2016; 37 (06) 857-864
    CrossrefPubMedGoogle Scholar
  • 37 Behar G, Sibéril S, Groulet A. , et al. Isolation and characterization of anti-FcgammaRIII (CD16) llama single-domain antibodies that activate natural killer cells. Protein Eng Des Sel 2008; 21 (01) 1-10
    PubMedGoogle Scholar
  • 38 Geng S, Wang Y, Wang L. , et al. A light-responsive self-assembly formed by a cationic azobenzene derivative and SDS as a drug delivery system. Sci Rep 2017; 7: 39202
    CrossrefPubMedGoogle Scholar
  • 39 Lefranc MP, Ehrenmann F, Ginestoux C, Giudicelli V, Duroux P. Use of IMGT(®) databases and tools for antibody engineering and humanization. Methods Mol Biol 2012; 907: 3-37
    PubMedGoogle Scholar
  • 40 Vincke C, Loris R, Saerens D, Martinez-Rodriguez S, Muyldermans S, Conrath K. General strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold. J Biol Chem 2009; 284 (05) 3273-3284
    CrossrefPubMedGoogle Scholar
  • 41 Lo BK. Antibody humanization by CDR grafting. Methods Mol Biol 2004; 248: 135-159
    PubMedGoogle Scholar
  • 42 Biasini M, Bienert S, Waterhouse A. , et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 2014; 42 (Web Server issue): W252-8
    PubMedGoogle Scholar
  • 43 Pardon E, Laeremans T, Triest S. , et al. A general protocol for the generation of nanobodies for structural biology. Nat Protoc 2014; 9 (03) 674-693
    CrossrefPubMedGoogle Scholar
  • 44 Saerens D, Pellis M, Loris R. , et al. Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies. J Mol Biol 2005; 352 (03) 597-607
    CrossrefPubMedGoogle Scholar
  • 45 Könning D, Zielonka S, Grzeschik J. , et al. Camelid and shark single domain antibodies: structural features and therapeutic potential. Curr Opin Struct Biol 2017; 45: 10-16
    CrossrefPubMedGoogle Scholar
  • 46 Kim JH, Hong HJ. Humanization by CDR grafting and specificity-determining residue grafting. Methods Mol Biol 2012; 907: 237-245
    PubMedGoogle Scholar
  • 47 Spiess C, Zhai Q, Carter PJ. Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol 2015; 67 (2, Pt A): 95-106
    CrossrefPubMedGoogle Scholar
  • 48 Cerwenka A, Lanier LL. Natural killers join the fight against cancer. Science 2018; 359 (6383): 1460-1461
    CrossrefPubMedGoogle Scholar
  • 49 Dong B, Zhou C, He P. , et al. A novel bispecific antibody, BiSS, with potent anti-cancer activities. Cancer Biol Ther 2016; 17 (04) 364-370
    CrossrefPubMedGoogle Scholar
  • 50 Folkins C, Man S, Xu P, Shaked Y, Hicklin DJ, Kerbel RS. Anticancer therapies combining antiangiogenic and tumor cell cytotoxic effects reduce the tumor stem-like cell fraction in glioma xenograft tumors. Cancer Res 2007; 67 (08) 3560-3564
    CrossrefPubMedGoogle Scholar
  • 51 Behdani M, Zeinali S, Karimipour M. , et al. Development of VEGFR2-specific nanobody Pseudomonas exotoxin A conjugated to provide efficient inhibition of tumor cell growth. N Biotechnol 2013; 30 (02) 205-209
    CrossrefPubMedGoogle Scholar