Recombinant Technology Progressive epitope-blocked panning of a phage library for isolation of human RSV antibodies Ping Tsui a, * ,1 , Mark A. Tornetta a , Robert S. Ames a , Carol Silverman b , Terence Porter b , Cynthia Weston c , Sandra Griego c , Raymond W. Sweet c,1 a Department of Molecular Biology, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA b Department of Protein Biochemistry, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA c Department of Molecular Virology and Host Defense, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, P.O. Box 1539, King of Prussia, PA 19406, USA Received 27 April 2001; received in revised form 11 February 2002; accepted 11 February 2002 Abstract Epitope-blocked panning is an approach to mining antigen-specific diversity from phage display antibody libraries. Previously, we developed and used this method to recover a neutralizing antibody to respiratory syncytial virus (RSV) by blocking a dominant response to a nonneutralizing epitope on a recombinant derivative of the viral F antigen. We have extended this approach to the blocking of multiple epitopes simultaneously, which led to the recovery of new antibodies of different specificity, including one new neutralizing activity. A phage display Fab library was selected on recombinant F antigen in the presence of three representative antibodies recovered in the unblocked and subsequent single-blocked panning procedures. Restriction endonuclease fingerprinting of 13 F+ clones revealed seven unique Fabs. DNA sequence analysis of five of these clones revealed five new light chains in combination with different heavy chains, three of which were very similar or identical to Fabs previously isolated from this library. The blocking antibodies did not compete with the new Fabs, demonstrating effective masking of their binding sites in the panning procedure. Conversely, these Fabs did show variable inhibition of two of the blocking antibodies suggesting a close proximity or interdependence of their epitopes. One of the antibodies did inhibit virus infection, albeit with modest potency. These results demonstrate that epitope-blocked panning is a self-progressing approach to retrieving diverse antibodies from phage libraries. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Antibody engineering; Epitope blocked panning; RSV neutralization 1. Introduction Respiratory syncytial virus (RSV) is a highly con- tagious RNA virus of the paramyxovirus family. Infec- tion of the virus in infants and young children often causes lower respiratory diseases and can be fatal in those who have underlying cardiac or respiratory dis- ease condition (Chanok et al., 1992; Hall, 1987; LaVia 0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0022-1759(02)00032-7 * Corresponding author. E-mail address: tsuip@centocor.com (P. Tsui). 1 Current address: Department of Molecular and Cellular Biology, Centocor, Inc., 200 Great Valley Parkway, Malvern, PA 19355, USA. www.elsevier.com/locate/jim Journal of Immunological Methods 263 (2002) 123 – 132