Efficient construction of a highly useful phage-displayed human antibody repertoire Gertrudis Rojas * , Humberto Lamdan, Sheila Padron, Yasmiana Munoz, Marta Ayala, Jorge V. Gavilondo Recombinant Antibodies Laboratory, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Ave 31 e/ 158 y 190, Cubanaca ´ n, Playa, La Habana 10600, Cuba Received 31 August 2005 Available online 12 September 2005 Abstract We have constructed a highly useful phage-displayed human antibody repertoire with limited cloning efforts. Our strategy was to maximize diversity during the first steps of library construction through the use of various lymphoid sources from several donors, inclu- sion of different immunoglobulin isotypes, and performance of multiple separate amplification reactions with all possible combinations within a complex primer set. The resulting variable region collections were cloned to form a moderate size library, composed by 4.25 · 10 8 single chain antibody fragments. This repertoire was successfully used to retrieve binders to seven model antigens: six proteins and one 12 aa peptide. Binding affinities reached nanomolar and even subnanomolar range. Sequence diversity and V-gene usage var- iability among binders were proven. Our approach was not focused on absolute library size, but on a high quality sampling of variable regions from the human antibody repertoire. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Phage display; Antibody library; Repertoire size Libraries of phage-displayed antibody fragments give a unique opportunity to exploit the recognition potential of the human humoral immune system [1]. A critical issue is the size of antibody repertoires to be displayed. Both theo- retical considerations and practical experiences indicate that as more antibody fragments are included in a library, there are more chances to find particular binders among them. Small libraries (with as few as 10 6 members) contain- ing antibody variable regions from immune individuals have been useful to obtain binders to the immunizing anti- gen [2,3]. Library sizes are more important when trying to use a single universal library to pick antibody fragments to any antigen, by-passing the need for immunization. Small or moderate size libraries from non-immunized donors ren- der fewer binders and lower binding affinities in general [4]. Such findings have promoted a race to obtain larger libraries, and excellent results have been obtained with some of them [5–7]. Construction of large libraries is hampered by technical limitations, mainly the efficiency of cloning and bacterial transformation. Until now, there seems to be an upper lim- it of around 5 · 10 10 individuals to be cloned in a library, and the construction of such large libraries is a very labo- rious and time-consuming process. Final library size can be increased using in vivo recombination [8,9]. On the other hand, unlimited growth of libraries is not the ideal solution by itself, since the limits could then be imposed by our ability to completely sample library diversity during selection procedures [10]. Some groups have focused on the improvement of li- brary quality beyond the absolute size, based on the grow- ing knowledge about human immunoglobulinÕs structure and function. Synthetic repertoires can be optimized in terms of bacterial expression/phage display of variable 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.005 * Corresponding author. E-mail address: gertrudis.rojas@cigb.edu.cu (G. Rojas). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 336 (2005) 1207–1213 BBRC