Generation and Characterization of a Functional Nanobody Against Inflammatory Chemokine CXCL10, as a Novel Strategy for the Treatment of Multiple Sclerosis

Author(s): Tahereh Sadeghian-Rizi, Mahdi Behdani, Hossein Khanahmad, Hamid Mirmohammad Sadeghi, Ali Jahanian-Najafabadi*

Journal Name: CNS & Neurological Disorders - Drug Targets
Formerly Current Drug Targets - CNS & Neurological Disorders

Volume 18 , Issue 2 , 2019

Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Background & Objective: Chemokines and their receptors play a pivotal role in the pathogenesis of various autoimmune diseases such as multiple sclerosis, infectious diseases, and also in cancer metastasis via attraction of the pathogenic immune cells into the inflammation sites.

Methods: Inflammatory chemokine CXCL10 as a T helper (Th)1-chemokine directs chemotaxis of many cell subsets especially Th1 into the central nervous system (CNS) via its receptor CXCR3 and it has been put forward as a potential therapeutic target in the treatment of multiple sclerosis. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies occurring in camelids with unique biochemical and biophysical features which render them superior to conventional antibodies or antibody fragments. Here, we describe the generation, selection, and characterization of CXCL10-specific Nanobodies from camel immunized with CXCL10. The obtained Nanobodies displayed high affinity towards CXCL10 about 10-11-10-8 M.

Results: Then a Nanobody with the highest affinity named 3Nb12 was selected and investigated as a migration inhibitor of CXCR3+ cells. Chemotaxis assay results showed that 3Nb12 blocked CXCL10- CXCR3 binding and potently inhibited chemotaxis of CXCR3-transfected HEK293T cells.

Conclusion: The nanobody 3Nb12 might be a promising specific and powerful blocking agent of CXCL10 function, which can be used for diagnostic, therapeutic and research purposes in MS.

Keywords: CXCL10, CXCR3, Multiple sclerosis, Heavy chain antibody, Nanobody, Chemotaxis.

Torkildsen O, Myhr KM, Bo L. Disease-modifying treatments for multiple sclerosis - a review of approved medications. Eur J Neurol 2016; 23: 18-27.
Castro-Borrero W, Graves D, Frohman TC, et al. Current and emerging therapies in multiple sclerosis: A systematic review. Ther Adv Neurol Disorder 2012; 5(4): 205-20.
Clement M, Pearson JA, Gras S, et al. Targeted suppression of autoreactive CD8 (+) T-cell activation using blocking anti-CD8 antibodies. Sci Rep 2016; 6: 35332.
Friese MA, Fugger L. Attractive CD8+ T cells in multiple sclerosis: a new target for therapy? Brain 2005; 128: 1747-63.
Sospedra M, Martin R. Immunology of multiple sclerosis. Annu Rev Immunol 2005; 23: 683-747.
Hickey WF. The pathology of multiple sclerosis: A historical perspective. J Neuroimmunol 1999; 98(1): 37-44.
Sadeghian-Rizi T, Khanahmad H, Jahanian-Najafabadi A. Therapeutic targeting of chemokines and chemokine receptors in multiple sclerosis: opportunities and challenges. CNS Neurol Disord Drug Targets 2018.
Chai Q, He WQ, Zhou M, Lu H, Fu ZF. Enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection. J Virol 2014; 88(9): 4698-710.
Sato W. Chemokine receptors on T cells in multiple sclerosis. Clin Exp Neuroimmunol 2014; 5(2): 162-74.
Holman DW, Klein RS, Ransohoff RM. The blood-brain barrier, chemokines and multiple sclerosis. Biochim Biophys Acta 2011; 1812(2): 220-30.
Engelhardt B. Immune cell entry into the central nervous system: involvement of adhesion molecules and chemokines. J Neurol Sci 2008; 274(1-2): 23-6.
Man S, Ubogu EE, Ransohoff RM. Inflammatory cell migration into the central nervous system: A few new twists on an old tale. Brain Pathol 2007; 17(2): 243-50.
Ubogu EE, Cossoy MB, Ransohoff RM. The expression and function of chemokines involved in CNS inflammation. Trends Pharmacol Sci 2006; 27(1): 48-55.
Mahad DJ, Lawry J, Howell SJ, Woodroofe MN. Longitudinal study of chemokine receptor expression on peripheral lymphocytes in multiple sclerosis: CXCR3 upregulation is associated with relapse. Mult Scler 2003; 9(2): 189-98.
Fife BT, Kennedy KJ, Paniagua MC, et al. CXCL10 (IFN-gamma-inducible protein-10) control of encephalitogenic CD4+ T cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis. J Immunol 2001; 166(12): 7617-24.
Simpson JE, Newcombe J, Cuzner ML, Woodroofe MN. Expression of the interferon-gamma-inducible chemokines IP-10 and Mig and their receptor, CXCR3, in multiple sclerosis lesions. Neuropathol Appl Neurobiol 2000; 26(2): 133-42.
Heliopoulos I, Patousi A. Therapeutic monoclonal antibodies and multiple sclerosis: the essentials. Med Chem 2018; 14(2): 144-54.
Khamehchian S, Zolfagharian H, Dounighi NM, Tebianian M, Madani R. Study on camel IgG purification: A new approach to prepare Naja Naja Oxiana antivenom as passive immunization for therapy. Hum Vaccin Immunother 2014; 10(6): 1633-8.
Lu CT, Zhao YZ, Wong HL, Cai J, Peng L, Tian X-Q. Current approaches to enhance CNS delivery of drugs across the brain barriers. Int J Nanomedicine 2014; 9: 2241-57.
Rissiek B, Koch-Nolte F, Magnus T. Nanobodies as modulators of inflammation: Potential applications for acute brain injury. Front Cell Neurosci 2014; 8: 344.
Sadeghian-Rizi T, Behdani M, Khanahmad H, et al. Production of novel camelid anti-CXCL10 specific polyclonal antibodies and evaluation of their bioreactivity. Int J Pept Res Ther 2018; 1-6.
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: 35-41.
Arezumand R, Mahdian R, Zeinali S, et al. Identification and characterization of a novel nanobody against human placental growth factor to modulate angiogenesis. Mol Immunol 2016; 78: 183-92.
Darvish M, Behdani M, Shokrgozar MA, Pooshang-Bagheri K, Shahbazzadeh D. Development of protective agent against Hottentotta saulcyi venom using camelid single-domain antibody. Mol Immunol 2015; 68: 412-20.
Lefranc MP, Pommie C, Kaas Q, et al. IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig super family C like domains. Dev Comp Immunol 2005; 29(3): 185-203.
Kazemi-Lomedasht F, Behdani M, Bagheri KP, et al. Inhibition of angiogenesis in human endothelial cell using VEGF specific nanobody. Mol Immunol 2015; 65: 58-67.
Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 1978; 100: 173-9.
Sadeqzadeh E, Rahbarizadeh F, Ahmadvand D, Rasaee MJ, Parhamifar L, Moghimi SM. Combined MUC1-specific nanobody-tagged PEG-polyethylenimine polyplex targeting and transcriptional targeting of tBid transgene for directed killing of MUC1 over-expressing tumor cells. J Control Release 2011; 156: 85-91.
Blanchetot C, Verzijl D, Mujić-Delić A, et al. Neutralizing nanobodies targeting diverse chemokines effectively inhibit chemokine function. J Biol Chem 2013; 288: 25173-82.
Deschacht N, De Groeve K, Vincke C, Raes G, De Baetselier P, Muyldermans S. A novel promiscuous class of camelid single-domain antibody contributes to the antigen-binding repertoire. J Immunol 2010; 184(10): 5696-704.
Nylander A, Hafler DA. Multiple sclerosis. J Clin Invest 2012; 122(4): 1180-8.
Choi J, Selmi C, Leung P, Kenny TP, Roskams T, Gershwin ME. Chemokine and chemokine receptors in autoimmunity: The case of primary biliary cholangitis. Expert Rev Clin Immunol 2016; 12: 661-72.
Griffith JW, Sokol CL, Luster AD. Chemokines and chemokine receptors: Positioning cells for host defense and immunity. Annu Rev Immunol 2014; 32: 659-702.
Manel J, Edurne P, Roger C. Chemokines and chemokine receptors. eLS 2013. doi: 10.1002/9780470015902.a0000933.pub3
Antonelli A, Ferrari SM, Giuggioli D, Ferrannini E, Ferri C, Fallahi P. Chemokine (C–X–C motif) ligand (CXCL)10 in autoimmune diseases. Autoimmun Rev 2014; 13: 272-80.
Lee EY, Lee ZH, Song YW. CXCL10 and autoimmune diseases. Autoimmun Rev 2009; 8: 379-83.
Liu M, Guo S, Stiles J. The emerging role of CXCL10 in cancer. Oncol Lett 2011; 2(4): 583-9.
Van Raemdonck K, Van den Steen PE, Liekens S, Van Damme J, Struyf S. CXCR3 ligands in disease and therapy. Cytokine Growth Factor Rev 2015; 26: 311-27.
Breser ML, Motrich RD, Sanchez LR, Mackern-Oberti JP, Rivero VE. Exprescersion of CXCR3 on specific T cells is essential for homing to the prostate gland in an experimental model of chronic prostatitis/chronic pelvic pain syndrome. J Immunol 2013; 190: 3121-33.
Mohan K, Issekutz TB. Blockade of chemokine receptor CXCR3 inhibits T cell recruitment to inflamed joints and decreases the severity of adjuvant arthritis. J Immunol 2007; 179: 8463-9.
Singh UP, Singh S, Taub DD, Lillard JW. Inhibition of IFN-γ-inducible protein-10 abrogates colitis in IL-10−/− mice. J Immunol 2003; 171: 1401-6.
Tsutahara K, Okumi M, Kakuta Y, Abe T, Yazawa K, Miyagawa S. The blocking of CXCR3 and CCR5 suppresses the infiltration of T lymphocytes in rat renal ischemia reperfusion. Nephrol Dial Transplant 2012; 27: 3799-806.
De Genst E, Silence K, Decanniere K, et al. Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci USA 2006; 103: 4586-91.
Harmsen MM, De Haar HJ. Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biotechnol 2007; 77: 13-22.
Kazemi-Lomedasht F. muyldermans S, Habibi-Anbouhi M, Behdani M. Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function. Iran J Basic Med Sci 2018; 21: 260-6.
Stijlemans B, Conrath K, Cortez-Retamozo V, et al. Efficient targeting of conserved cryptic epitopes of infectious agents by single domain antibodies: african trypanosomes as paradigm. J Biol Chem 2004; 279: 1256-61.
de Wit RH, Heukers R, Brink HJ, et al. CXCR4-specific nanobodies as potential therapeutics for WHIM syndrome. J Pharmacol Exp Ther 2017; 363: 35-44.
Jähnichen S, Blanchetot C, Maussang D, et al. CXCR4 nanobodies (VHH-based single variable domains) potently inhibit chemotaxis and HIV-1 replication and mobilize stem cells. Proc Natl Acad Sci 2010; 107: 20565-70.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [141 - 148]
Pages: 8
DOI: 10.2174/1871527317666181114134518
Price: $65

Article Metrics

PDF: 46
PRC: 1