In vitro-engineered non-antibody protein therapeutics

Abstract

Antibodies have proved to be a valuable mode of therapy for numerous diseases, mainly owing to their high target binding affinity and specificity. Unfortunately, antibodies are also limited in several respects, chief amongst those being the extremely high cost of manufacture. Therefore, non-antibody binding proteins have long been sought after as alternative therapies. New binding protein scaffolds are constantly being designed or discovered with some already approved for human use by the FDA. This review focuses on protein scaffolds that are either already being used in humans or are currently being evaluated in clinical trials. Although not all are expected to be approved, the significant benefits ensure that these molecules will continue to be investigated and developed as therapeutic alternatives to antibodies. Based on the location of the amino acids that mediate ligand binding, we place all the protein scaffolds under clinical development into two general categories: scaffolds with ligand-binding residues located in exposed flexible loops, and those with the binding residues located in protein secondary structures, such as α-helices. Scaffolds that fall under the first category include adnectins, anticalins, avimers, Fynomers, Kunitz domains, and knottins, while those belonging to the second category include affibodies, β-hairpin mimetics, and designed ankyrin repeat proteins (DARPins). Most of these scaffolds are thermostable and can be easily produced in microorganisms or completely synthesized chemically. In addition, many of these scaffolds derive from human proteins and thus possess very low immunogenic potential. Additional advantages and limitations of these protein scaffolds as therapeutics compared to antibodies will be discussed.

Keywords

scaffold / multivalent / phage / yeast / ribosome / antibody surrogate

References

[1] A Study of Abicipar Pegol in Patients With Neovascular Age-related Macular Degeneration. https://www.clinicaltrialsgov/ct2/show/study/NCT02181517.

[2] Aboye TL, Stromstedt AA, Gunasekera S, Bruhn JG, El-Seedi H, Rosengren KJ, Goransson U (2015) A cactus-derived toxin-like cystine knot Peptide with selective antimicrobial activity. Chembiochem 16:1068–1077

[3] Ahlgren S, Tolmachev V (2010) Radionuclide molecular imaging using Affibody molecules. Curr Pharm Biotechnol 11:581–589

[4] Angelini A, Diderich P, Morales-Sanfrutos J, Thurnheer S, Hacker D, Menin L, Heinis C (2012) Chemical macrocyclization of peptides fused to antibody Fc fragments. Bioconjug Chem 23:1856–1863

[5] Avrutina O (2016) Synthetic cystine-knot miniproteins—valuable scaffolds for polypeptide engineering. Adv Exp Med Biol 917:121–144

[6] Bailon P, Won CY (2009) PEG-modified biopharmaceuticals. Expert Opin Drug Deliv 6:1–16

[7] Bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B (2007) Immunogenicity, efficacy and adverse events of adalimumab in RA patients. Rheumatol Int 27:269–274

[8] Binz HK, Stumpp MT, Forrer P, Amstutz P, Pluckthun A (2003) Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J Mol Biol 332:489–503

[9] Bode W, Huber R (1992) Natural protein proteinase inhibitors and their interaction with proteinases. Eur J Biochem 204:433–451

[10] Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15:553–557

[11] Bork P (1993) Hundreds of ankyrin-like repeats in functionally diverse proteins: mobile modules that cross phyla horizontally? Proteins 17:363–374

[12] Bryant AP, Busby RW, Bartolini WP, Cordero EA, Hannig G, Kessler MM, Pierce CM, Solinga RM, Tobin JV, Mahajan-Miklos S (2010) Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract. Life Sci 86:760–765

[13] Caliceti P, Veronese FM (2003) Pharmacokinetic and biodistribution properties of poly(ethylene glycol)–protein conjugates. Adv Drug Deliv Rev 55:1261–1277

[14] Campochiaro PA, Channa R, Berger BB, Heier JS, Brown DM, Fiedler U, Hepp J, Stumpp MT (2013) Treatment of diabetic macular edema with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study. Am J Ophthalmol 155(697–704):e692

[15] Chaudhury C, Brooks CL, Carter DC, Robinson JM, Anderson CL (2006) Albumin binding to FcRn: distinct from the FcRn–IgG interaction. Biochemistry 45:4983–4990

[16] Chauhan VP, Stylianopoulos T, Boucher Y, Jain RK (2011) Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies. Annu Rev Chem Biomol Eng 2:281–298

[17] Chey WD, Lembo AJ, Lavins BJ, Shiff SJ, Kurtz CB, Currie MG, MacDougall JE, Jia XD, Shao JZ, Fitch DA (2012) Linaclotide for irritable bowel syndrome with constipation: a 26-week, randomized, double-blind, placebo-controlled trial to evaluate efficacy and safety. Am J Gastroenterol 107:1702–1712

[18] Cicardi M, Levy RJ, McNeil DL, Li HH, Sheffer AL, Campion M, Horn PT, Pullman WE (2010) Ecallantide for the treatment of acute attacks in hereditary angioedema. N Engl J Med 363:523–531

[19] Colgrave ML, Craik DJ (2004) Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 43:5965–5975

[20] Cooke MP, Perlmutter RM (1989) Expression of a novel form of the fyn proto-oncogene in hematopoietic cells. New Biol 1:66–74

[21] Craik DJ, Mylne JS, Daly NL (2010) Cyclotides: macrocyclic peptides with applications in drug design and agriculture. Cell Mol Life Sci 67:9–16

[22] Dejnirattisai W, Supasa P, Wongwiwat W, Rouvinski A, Barba-Spaeth G, Duangchinda T, Sakuntabhai A, Cao-Lormeau VM, Malasit P, Rey FA (2016) Dengue virus sero-cross-reactivity drives antibody-dependent enhancement of infection with zika virus. Nat Immunol. 17:1102–1108

[23] Dennis MS, Zhang M, Meng YG, Kadkhodayan M, Kirchhofer D, Combs D, Damico LA (2002) Albumin binding as a general strategy for improving the pharmacokinetics of proteins. J Biol Chem 277:35035–35043

[24] Dineen SP, Sullivan LA, Beck AW, Miller AF, Carbon JG, Mamluk R, Wong H, Brekken RA (2008) The Adnectin CT-322 is a novel VEGF receptor 2 inhibitor that decreases tumor burden in an orthotopic mouse model of pancreatic cancer. BMC Cancer 8:352

[25] Dreier B, Pluckthun A (2011) Ribosome display: a technology for selecting and evolving proteins from large libraries. Methods Mol Biol 687:283–306

[26] Dunlevy FK, Martin SL, de Courcey F, Elborn JS, Ennis M (2012) Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor. J Cyst Fibros 11:300–304

[27] Ecker DM, Jones SD, Levine HL (2015) The therapeutic monoclonal antibody market. mAbs 7:9–14

[28] Elsadek B, Kratz F (2012) Impact of albumin on drug delivery–new applications on the horizon. J Control Release 157:4–28

[29] Eutamene H, Bradesi S, Larauche M, Theodorou V, Beaufrand C, Ohning G, Fioramonti J, Cohen M, Bryant AP, Kurtz C (2010) Guanylate cyclase C-mediated antinociceptive effects of linaclotide in rodent models of visceral pain. Neurogastroenterol Motil 22:e312–e384

[30] Fasan R, Dias RLA, Moehle K, Zerbe O, Vrijbloed JW, Obrecht D, Robinson JA (2004) Using a β-hairpin to mimic an α-helix: cyclic peptidomimetic inhibitors of the p53–HDM2 protein-protein interaction. Angew Chem Int Ed 43:2109–2112

[31] Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JMW, Yeung YA, Cochran JR, Heinzelman P, Colby D, Swers J (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotech 21:163–170

[32] Feldwisch J, Tolmachev V, Lendel C, Herne N, Sjoberg A, Larsson B, Rosik D, Lindqvist E, Fant G, Hoiden-Guthenberg I (2010) Design of an optimized scaffold for affibody molecules. J Mol Biol 398:232–247

[33] Ferrara N, Damico L, Shams N, Lowman H, Kim R (2006) Development of ranibizumab, an anti-vascular endothelial growth factor antigen binding fragment, as therapy for neovascular agerelated macular degeneration. Retina 26:859–870

[34] Gai SA, Wittrup KD (2007) Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17:467–473

[35] Gebauer M, Skerra A (2012) Anticalins small engineered binding proteins based on the lipocalin scaffold. Method Enzymol 503:157–188

[36] Getmanova EV, Chen Y, Bloom L, Gokemeijer J, Shamah S, Warikoo V, Wang J, Ling V, Sun L (2006) Antagonists to human and mouse vascular endothelial growth factor receptor 2 generated by directed protein evolution in vitro. Chem Biol 13:549–556

[37] Gille H, Hulsmeyer M, Trentmann S, Matschiner G, Christian HJ, Meyer T, Amirkhosravi A, Audoly LP, Hohlbaum AM, Skerra A (2016) Functional characterization of a VEGF-A-targeting Anticalin, prototype of a novel therapeutic human protein class. Angiogenesis 19:79–94

[38] Grabulovski D, Kaspar M, Neri D (2007) A novel, non-immunogenic Fyn SH3-derived binding protein with tumor vascular targeting properties. J Biol Chem 282:3196–3204

[39] Gracy J, Le-Nguyen D, Gelly JC, Kaas Q, Heitz A, Chiche L (2008) KNOTTIN: the knottin or inhibitor cystine knot scaffold in 2007. Nucleic Acids Res 36:D314–D319

[40] Hackel BJ, Kapila A, Wittrup KD (2008) Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling. J Mol Biol 381:1238–1252

[41] Holt LJ, Basran A, Jones K, Chorlton J, Jespers LS, Brewis ND, Tomlinson IM (2008) Anti-serum albumin domain antibodies for extending the half-lives of short lived drugs. Protein Eng Des Sel 21:283–288

[42] Hosse RJ, Rothe A, Power BE (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci 15:14–27

[43] Humphrey W, Dalke A, Schulten K (1996) VMD: visual molecular dynamics. J Mol Graph 14(33–38):27–38

[44] Jost C, Pluckthun A (2014) Engineered proteins with desired specificity: DARPins, other alternative scaffolds and bispecific IgGs. Curr Opin Struct Biol 27:102–112

[45] Karpova D, Dauber K, Spohn G, Chudziak D, Wiercinska E, Schulz M, Pettit AR, Levesque JP, Romagnoli B, Patel K (2013) The novel CXCR4 antagonist POL5551 mobilizes hematopoietic stem and progenitor cells with greater efficiency than Plerixafor. Leukemia 27:2322–2331

[46] Kelley B (2009) Industrialization of mAb production technology the bioprocessing industry at a crossroads. mAbs 1:443–452

[47] Kintzing JR, Cochran JR (2016a) Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles. Curr Opin Chem Biol 34:143–150

[48] Kintzing JR, Cochran JR (2016b) Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles. Curr Opin Chem Biol 34:143–150

[49] Kolmar H (2009) Biological diversity and therapeutic potential of natural and engineered cystine knot miniproteins. Curr Opin Pharmacol 9:608–614

[50] Kontermann RE (2011) Strategies for extended serum half-life of protein therapeutics. Curr Opin Biotechnol 22:868–876

[51] Kristipati R, Nádasdi L, Tarczy-Hornoch K, Lau K, Miljanich GP, Ramachandran J, Bell JR (1994) Characterization of the binding of omega-conopeptides to different classes of non-L-type neuronal calcium channels. Mol Cell Neurosci 5:219–228

[52] Layer P, Stanghellini V (2014) Review article: Linaclotide for the management of irritable bowel syndrome with constipation. Aliment Pharmacol Ther 39:371–384

[53] Levy RJ, Lumry WR, McNeil DL, Li HH, Campion M, Horn PT, Pullman WE (2010) EDEMA4: a phase 3, double-blind study of subcutaneous ecallantide treatment for acute attacks of hereditary angioedema. Ann Allergy Asthma Immunol 104:523–529

[54] Lipovsek D (2011) Adnectins: engineered target-binding protein therapeutics. Protein Eng Des Sel 24:3–9

[55] Lipovsek D, Pluckthun A (2004) In-vitro protein evolution by ribosome display and mRNA display. J Immunol Methods 290:51–67

[56] Madireddi M, Malone H, Kukral D, Chimalakonda A, Kozhich A, Xiling Y, Swain J, Yamniuk A, Ahlijanian M (2016) BMS-986089 is a high affinity anti-myostatin adnectin that increases muscle volume in three preclinical species. Neuromuscul Disord 26:S94–S95

[57] Maillere B, Mourier G, Herve M, Cotton J, Leroy S, Menez A (1995) Immunogenicity of a disulphide-containing neurotoxin: presentation to T-cells requires a reduction step. Toxicon 33:475–482

[58] Mitchell T, Chao G, Sitkoff D, Lo F, Monshizadegan H, Meyers D, Low S, Russo K, DiBella R, Denhez F (2014) Pharmacologic profile of the Adnectin BMS-962476, a small protein biologic alternative to PCSK9 antibodies for low-density lipoprotein lowering. J Pharmacol Exp Ther 350:412–424

[59] Moebius U, Feuerer W, Fenzl E, van Swelm R, Swinkels DW, Hohlbaum A (2015) A Phase I study investigating the safety, tolerability, pharmacokinetics and pharmacodynamic activity of the hepcidin antagonist PRS-080#022. Results from a randomized, placebo controlled, double-blind study following single administration to healthy subjects. Blood 126:536

[60] Molecular Partners (2015). http://www.molecularpartners.com/preliminary-phase-i-results-demonstrate-potential-utility-of-darpinsin-anticancer-treatment/.

[61] Moore SJ, Cochran JR (2012) Engineering knottins as novel binding agents. Methods Enzymol 503:223–251

[62] Mross K, Richly H, Fischer R, Scharr D, Buchert M, Stern A, Gille H, Audoly LP, Scheulen ME (2013a) First-in-human phase I study of PRS-050 (Angiocal), an Anticalin targeting and antagonizing VEGF-A, in patients with advanced solid tumors. PLoS ONE 8: e83232

[63] Mross K, Richly H, Fischer R, Scharr D, Büchert M, Stern A, Gille H, Audoly LP, Scheulen ME (2013b) First-in-human phase I study of PRS-050 (Angiocal), an Anticalin targeting and antagonizing VEGF-A, in patients with advanced solid tumors. PLoS ONE 8: e83232

[64] Nguyen A, Reyes AE II, Zhang M, McDonald P, Wong WL, Damico LA, Dennis MS (2006) The pharmacokinetics of an albuminbinding Fab (AB.Fab) can be modulated as a function of affinity for albumin. Protein Eng Des Sel 19:291–297

[65] Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren P-A (1996) Binding proteins selected from combinatorial libraries of an [alpha]-helical bacterial receptor domain. Nat Biotech 15:772–777

[66] Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA (1997) Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol 15:772–777

[67] Nussberger J, Cugno M, Amstutz C, Cicardi M, Pellacani A, Agostoni A (1998) Plasma bradykinin in angio-oedema. The Lancet 351:1693–1697

[68] Nygren PA (2008) Alternative binding proteins: affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 275:2668–2676

[69] Paul L, Carlin E, Jenkins M (2016) Dengue virus antibodies enhance Zika virus infection. bioRxiv (http://www.biorxivorg/content/early/2016/04/25/050112).

[70] Pieris Pharmaceuticals I (2015). http://www.pieris.com/newsand-events/press-releases/detail/524/pieris-pharmaceuticalspresents-clinical-data-for-its.

[71] Pluckthun A (2015) Designed ankyrin repeat proteins (DARPins): binding proteins for research, diagnostics, and therapy. Annu Rev Pharmacol Toxicol 55:489–511

[72] Ramamurthy V, Krystek SR Jr, Bush A, Wei A, Emanuel SL, Das Gupta R, Janjua A, Cheng L, Murdock M, Abramczyk B (2012) Structures of adnectin/protein complexes reveal an expanded binding footprint. Structure 20:259–269

[73] Rauck RL, Wallace MS, Leong MS, MineHart M, Webster LR, Charapata SG, Abraham JE, Buffington DE, Ellis D, Kartzinel R (2006) A randomized, double-blind, placebo-controlled study of intrathecal ziconotide in adults with severe chronic pain. J Pain Symptom Manag 31:393–406

[74] Reichert JM (2017) Antibodies to watch in 2017. mAbs 9:167–181

[75] Reichert JM, Beck A, Lugovskoy AA, Wurch T, Coats S, Brezski RJ (2014) 9th Annual European antibody congress, November 11–13, 2013, Geneva, Switzerland. mAbs 6:309–326

[76] Roberts BL, Markland W, Ley AC, Kent RB, White DW, Guterman SK, Ladner RC (1992) Directed evolution of a protein: selection of potent neutrophil elastase inhibitors displayed on M13 fusion phage. Proc Natl Acad Sci USA 89:2429–2433

[77] Rodon J, Omlin A, Herbschleb KH, Garcia-Corbacho J, Steiner J, Dolado I, Zitt C, Feurstein D, Turner D, Dawson KM (2015) Abstract B25: First-in-human Phase I study to evaluate MP0250, a DARPin blocking HGF and VEGF, in patients with advanced solid tumors. Mol Cancer Ther 14:B25

[78] Sandstrom M, Lindskog K, Velikyan I, Wennborg A, Feldwisch J, Sandberg D, Tolmachev V, Orlova A, Sorensen J, Carlsson J (2016) Biodistribution and radiation dosimetry of the Anti-HER2 affibody molecule 68Ga-ABY-025 in breast cancer patients. J Nucl Med 57:867–871

[79] Schiff D, Kesari S, de Groot J, Mikkelsen T, Drappatz J, Coyle T, Fichtel L, Silver B, Walters I, Reardon D (2015) Phase 2 study of CT-322, a targeted biologic inhibitor of VEGFR-2 based on a domain of human fibronectin, in recurrent glioblastoma. Investig New Drugs 33:247–253

[80] Schilling J, Schoppe J, Pluckthun A (2014) From DARPins to LoopDARPins: novel LoopDARPin design allows the selection of low picomolar binders in a single round of ribosome display. J Mol Biol 426:691–721

[81] Schlatter D, Brack S, Banner DW, Batey S, Benz J, Bertschinger J, Huber W, Joseph C, Rufer AC, van der Klooster A (2012) Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain. mAbs 4:497–508

[82] Schmoldt HU, Wentzel A, Becker S, Kolmar H (2005) A fusion protein system for the recombinant production of short disulfide bond rich cystine knot peptides using barnase as a purification handle. Protein Expr Purif 39:82–89

[83] Schneider L, Lumry W, Vegh A, Williams AH, Schmalbach T (2007) Critical role of kallikrein in hereditary angioedema pathogenesis: a clinical trial of ecallantide, a novel kallikrein inhibitor. J Allergy Clin Immunol 120:416–422

[84] Screaton G, Mongkolsapaya J, Yacoub S, Roberts C (2015) New insights into the immunopathology and control of dengue virus infection. Nat Rev Immunol 15:745–759

[85] Shah DK, Betts AM (2013) Antibody biodistribution coefficients. mAbs 5:297–305

[86] Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328:333–363

[87] Silacci M, Baenziger-Tobler N, Lembke W, Zha W, Batey S, Bertschinger J, Grabulovski D (2014) Linker length matters, Fynomer-Fc fusion with an optimized linker displaying picomolar IL-17A inhibition potency. J Biol Chem 289:14392–14398

[88] Silacci M, Lembke W, Woods R, Attinger-Toller I, Baenziger-Tobler N, Batey S, Santimaria R, von der Bey U, Koenig-Friedrich S, Zha W (2016) Discovery and characterization of COVA322, a clinical-stage bispecific TNF/IL-17A inhibitor for the treatment of inflammatory diseases. mAbs 8:141–149

[89] Silverman J, Liu Q, Bakker A, To W, Duguay A, Alba BM, Smith R, Rivas A, Li P, Le H (2005) Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 23:1556–1561

[90] Skerra A (2000) Engineered protein scaffolds for molecular recognition. J Mol Recognit 13:167–187

[91] Skerra A (2008) Alternative binding proteins: anticalins – harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 275:2677–2683

[92] Smith HS, Deer TR (2009) Safety and efficacy of intrathecal ziconotide in the management of severe chronic pain. Ther Clin Risk Manag 5:521–534

[93] Smith BJ, Popplewell A, Athwal D, Chapman AP, Heywood S, West SM, Carrington B, Nesbitt A, Lawson AD, Antoniw P (2001) Prolonged in vivo residence times of antibody fragments associated with albumin. Bioconjug Chem 12:750–756

[94] Sorensen J, Velikyan I, Sandberg D, Wennborg A, Feldwisch J, Tolmachev V, Orlova A, Sandstrom M, Lubberink M, Olofsson H (2016) Measuring HER2-receptor expression in metastatic breast cancer using [68Ga]ABY-025 Affibody PET/CT. Theranostics 6:262–271

[95] Souied EH, Devin F, Mauget-Faÿsse M, Kolář P, Wolf-Schnurrbusch U, Framme C, Gaucher D, Querques G, Stumpp MT, Wolf S (2014a) Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study. Am J Ophthalmol 158(724–732):e722

[96] Souied EH, Devin F, Mauget-Faysse M, Kolar P, Wolf-Schnurrbusch U, Framme C, Gaucher D, Querques G, Stumpp MT, Wolf S (2014b) Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study. Am J Ophthalmol 158(724–732):e722

[97] Sparrow E, Friede M, Sheikh M, Torvaldsen S (2017) Therapeutic antibodies for infectious diseases. Bull World Health Org 95:1–81

[98] Stein EA, Kasichayanula S, Turner T, Kranz T, Arumugam U, Biernat L, Lee J (2014) LDL cholesterol reduction with BMS-962476, an adnectin inhibitor of PCSK9: results of a single ascending dose study. J Am Coll Cardiol 63:2889–2934

[99] Strebhardt K, Ullrich A (2008) Paul Ehrlich’s magic bullet concept: 100 years of progress. Nat Rev Cancer 8:473–480

[100] Tam JP, Wang S, Wong KH, Tan WL (2015) Antimicrobial peptides from plants. Pharmaceuticals (Basel) 8:711–757

[101] Thell K, Hellinger R, Sahin E, Michenthaler P, Gold-Binder M, Haider T, Kuttke M, Liutkeviciute Z, Goransson U, Grundemann C (2016) Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis. Proc Natl Acad Sci USA 113:3960–3965

[102] Tolcher AW, Sweeney CJ, Papadopoulos K, Patnaik A, Chiorean EG, Mita AC, Sankhala K, Furfine E, Gokemeijer J, Iacono L (2011) Phase I and pharmacokinetic study of CT-322 (BMS-844203), a targeted adnectin inhibitor of VEGFR-2 based on a domain of human fibronectin. Clin Cancer Res 17:363–371

[103] Veronese FM, Pasut G (2005) PEGylation, successful approach to drug delivery. Drug Discov Today 10:1451–1458

[104] Walker RG, Willingham AT, Zuker CS (2000) A Drosophila mechanosensory transduction channel. Science 287:2229–2234

[105] Wallace MS, Charapata SG, Fisher R, Byas-Smith M, Staats PS, Mayo M, McGuire D, Ellis D, The Ziconotide Nonmalignant Pain Study, G. (2006) Intrathecal ziconotide in the treatment of chronic nonmalignant pain: a randomized, double-blind, placebo-controlled clinical trial. Neuromodulation 9:75–86

[106] Wang YX, Pettus M, Gao D, Phillips C, Scott Bowersox S (2000) Effects of intrathecal administration of ziconotide, a selective neuronal N-type calcium channel blocker, on mechanical allodynia and heat hyperalgesia in a rat model of postoperative pain. Pain 84:151–158

[107] Weidle UH, Auer J, Brinkmann U, Georges G, Tiefenthaler G (2013) The emerging role of new protein scaffold-based agents for treatment of cancer. Cancer Genom Proteom 10:155–168

[108] Werle M, Schmitz T, Huang H-L, Wentzel A, Kolmar H, Bernkop-Schnürch A (2006) The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in oral peptide drug delivery. J Drug Target 14:137–146

[109] Williams A, Baird LG (2003) DX-88 and HAE: a developmental perspective. Transfus Apher Sci 29:255–258

[110] Wong CT, Rowlands DK, Wong CH, Lo TW, Nguyen GK, Li HY, Tam JP (2012) Orally active peptidic bradykinin B1 receptor antagonists engineered from a cyclotide scaffold for inflammatory pain treatment. Angew Chem Int Ed Engl 51:5620–5624

[111] Xiang J, Hurchla MA, Fontana F, Su X, Amend SR, Esser AK, Douglas GJ, Mudalagiriyappa C, Luker KE, Pluard T (2015) CXCR4 protein epitope mimetic antagonist POL5551 disrupts metastasis and enhances chemotherapy effect in triple-negative breast cancer. Mol Cancer Ther 14:2473–2485

[112] Zhu S, Darbon H, Dyason K, Verdonck F, Tytgat J (2003) Evolutionary origin of inhibitor cystine knot peptides. FASEB J 17:1765–1767

RIGHTS & PERMISSIONS

2017 The Author(s) 2017. This article is published with open access at Springerlink.com and journal.hep.com.cn