title: In vivo binding and retention of CD4-specific DARPin 57.2 in macaques.
abstract: BACKGROUND
  The recently described Designed Ankyrin Repeat Protein (DARPin) technology can produce highly selective ligands to a variety of biological targets at a low production cost.
  
  
  RESULTS
  To investigate the in vivo use of DARPins for future application to novel anti-HIV strategies, we identified potent CD4-specific DARPins that recognize rhesus CD4 and followed the fate of intravenously injected CD4-specific DARPin 57.2 in rhesus macaques. The human CD4-specific DARPin 57.2 bound macaque CD4(+) cells and exhibited potent inhibitory activity against SIV infection in vitro. DARPin 57.2 or the control E3_5 DARPin was injected into rhesus macaques and the fate of cell-free and cell-bound CD4-specific DARPin was evaluated. DARPin-bound CD4(+) cells were detected in the peripheral blood as early as 30 minutes after the injection, decreasing within 6 hours and being almost undetectable within 24 hours. The amount of DARPin bound was dependent on the amount of DARPin injected. CD4-specific DARPin was also detected on CD4(+) cells in the lymph nodes within 30 minutes, which persisted with similar kinetics to blood. More extensive analysis using blood revealed that DARPin 57.2 bound to all CD4(+) cell types (T cells, monocytes, dendritic cells) in vivo and in vitro with the amount of binding directly proportional to the amount of CD4 on the cell surface. Cell-free DARPins were also detected in the plasma, but were rapidly cleared from circulation.
  
  
  CONCLUSIONS
  We demonstrated that the CD4-specific DARPin can rapidly and selectively bind its target cells in vivo, warranting further studies on possible clinical use of the DARPin technology.
authors.affiliation: Center for Biomedical Research, Population Council, New York, New York, United States of America.
authors.forename: Pavel
authors.initials: P
authors.lastname: Pugach
authors.forename: Anders
authors.initials: A
authors.lastname: Krarup
authors.forename: Agegnehu
authors.initials: A
authors.lastname: Gettie
authors.forename: Marcelo
authors.initials: M
authors.lastname: Kuroda
authors.forename: James
authors.initials: J
authors.lastname: Blanchard
authors.forename: Michael
authors.initials: M
authors.lastname: Piatak
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Lifson
authors.forename: Alexandra
authors.initials: A
authors.lastname: Trkola
authors.forename: Melissa
authors.initials: M
authors.lastname: Robbiani
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat; D015704:CD4 Antigens / Q000378:metabolism*; D005260:Female; D006678:HIV / Q000187:drug effects; D006801:Humans; D007275:Injections, Intravenous; D007700:Kinetics; D008253:Macaca mulatta; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics; D013379:Substrate Specificity
chemical_list: D015704:CD4 Antigens; D011506:Proteins
references.citation: Trends Biochem Sci. 1999 Aug;24(8):311-6
references.pmid: 10431175
references.citation: PLoS One. 2008 Sep 08;3(9):e3162
references.pmid: 18776937
references.citation: Antimicrob Agents Chemother. 2009 Feb;53(2):450-7
references.pmid: 19015347
references.citation: J Biol Chem. 2004 Jan 9;279(2):1541-5
references.pmid: 14573599
references.citation: J Virol. 2001 Jul;75(14):6710-3
references.pmid: 11413340
references.citation: Nature. 2005 Nov 3;438(7064):99-102
references.pmid: 16258536
references.citation: Nat Rev Immunol. 2010 May;10(5):317-27
references.pmid: 20414205
references.citation: PLoS One. 2009 Nov 30;4(11):e8060
references.pmid: 20011586
references.citation: Nat Rev Immunol. 2010 May;10(5):301-16
references.pmid: 20414204
references.citation: J Med Primatol. 2005 Oct;34(5-6):303-12
references.pmid: 16128925
references.citation: J Biol Chem. 2006 Nov 17;281(46):35167-75
references.pmid: 16963452
references.citation: J Immunol. 2006 Dec 15;177(12):8531-41
references.pmid: 17142751
references.citation: Virology. 2004 Mar 30;321(1):8-22
references.pmid: 15033560
references.citation: Curr Opin Drug Discov Devel. 2007 Mar;10(2):153-9
references.pmid: 17436550
references.citation: J Biol Chem. 2009 Oct 30;284(44):30718-26
references.pmid: 19740746
references.citation: J Virol. 2009 Oct;83(20):10358-65
references.pmid: 19656878
references.citation: PLoS Pathog. 2008 Jul 25;4(7):e1000109
references.pmid: 18654624
references.citation: Cancer Res. 2010 Feb 15;70(4):1595-605
references.pmid: 20124480
references.citation: AIDS Res Hum Retroviruses. 1997 Jul 20;13(11):933-43
references.pmid: 9223409
references.citation: Protein Eng Des Sel. 2009 Jun;22(6):357-66
references.pmid: 19389717
references.citation: Nat Biotechnol. 2005 Oct;23(10):1257-68
references.pmid: 16211069
references.citation: J Infect Dis. 2004 Jan 15;189(2):286-91
references.pmid: 14722894
references.citation: J Mol Biol. 2003 Sep 12;332(2):489-503
references.pmid: 12948497
references.citation: Mol Immunol. 1993 May;30(7):649-57
references.pmid: 8098132