Spatially addressed combinatorial protein libraries for recombinant antibody discovery and optimization.
Antibody discovery typically uses hybridoma- or display-based selection approaches, which lack the advantages of directly screening spatially addressed compound libraries as in small-molecule discovery. Here we apply the latter strategy to antibody discovery, using a library of ∼10,000 human germline antibody Fabs created by de novo DNA synthesis and automated protein expression and purification. In multiplexed screening assays, we obtained specific hits against seven of nine antigens. Using sequence-activity relationships and iterative mutagenesis, we optimized the binding affinities of two hits to the low nanomolar range. The matured Fabs showed full and partial antagonism activities in cell-based assays. Thus, protein drug leads can be discovered using surprisingly small libraries of proteins with known sequences, questioning the requirement for billions of members in an antibody discovery library. This methodology also provides sequence, expression and specificity information at the first step of the discovery process, and could enable novel antibody discovery in functional screens.
Fabrus LLC, La Jolla, California, USA.
Hongyuan
H
Mao
James J
JJ
Graziano
Tyson M A
TM
Chase
Cornelia A
CA
Bentley
Omar A
OA
Bazirgan
Neil P
NP
Reddy
Byeong Doo
BD
Song
Vaughn V
VV
Smider
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D016466:CHO Cells; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D008163:Luminescent Measurements; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013329:Structure-Activity Relationship
D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019151:Peptide Library; D011994:Recombinant Proteins; C064045:delta protein