De novo selection of high-affinity antibodies from synthetic fab libraries displayed on phage as pIX fusion proteins.
Filamentous phage was the first display platform employed to isolate antibodies in vitro and is still the most broadly used. The success of phage display is due to its robustness, ease of use, and comprehensive technology development, as well as a broad range of selection methods developed during the last two decades. We report here the first combinatorial synthetic Fab libraries displayed on pIX, a fusion partner different from the widely used pIII. The libraries were constructed on four V(L) and three V(H) domains encoded by IGV and IGJ germ-line genes frequently used in human antibodies, which were diversified to mirror the variability observed in the germ-line genes and antibodies isolated from natural sources. Two sets of libraries were built, one with diversity focused on V(H) by keeping V(L) in the germ-line gene configuration and the other with diversity in both V domains. After selection on a diverse panel of proteins, numerous specific Fabs with affinities ranging from 0.2 nM to 20 nM were isolated. V(H) diversity was sufficient for isolating Fabs to most antigens, whereas variability in V(L) was required for isolation of antibodies to some targets. After the application of an integrated maturation process consisting of reshuffling V(L) diversity, the affinity of selected antibodies was improved up to 100-fold to the low picomolar range, suitable for in vivo studies. The results demonstrate the feasibility of displaying complex Fab libraries as pIX fusion proteins for antibody discovery and optimization and lay the foundation for studies on the structure-function relationships of antibodies.
Shi
Lei
L
Centocor R&D, Inc., 145 King of Prussia Road, Radnor, PA 19087, USA.
Wheeler
John C
JC
Sweet
Raymond W
RW
Lu
Jin
J
Luo
Jinquan
J
Tornetta
Mark
M
Whitaker
Brian
B
Reddy
Ramachandra
R
Brittingham
Raymond
R
Borozdina
Lina
L
Chen
Qiang
Q
Amegadzie
Bernard
B
Knight
David M
DM
Almagro
Juan C
JC
Tsui
Ping
P
D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000915:Antibody Affinity; D001435:Bacteriophages / Q000235:genetics; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D008958:Models, Molecular; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D036022:Capsid Proteins; D007140:Immunoglobulin Fab Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins