Directed evolution of high-affinity antibody mimics using mRNA display.
We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.
Xu
Lihui
L
Phylos, Inc., Lexington, MA 02421, USA.
Aha
Patti
P
Gu
Ke
K
Kuimelis
Robert G
RG
Kurz
Markus
M
Lam
Terence
T
Lim
Ai Ching
AC
Liu
Hongxiang
H
Lohse
Peter A
PA
Sun
Lin
L
Weng
Shawn
S
Wagner
Richard W
RW
Lipovsek
Dasa
D
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D019020:Directed Molecular Evolution / Q000379:methods*; D005353:Fibronectins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D040081:Protein Array Analysis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000906:Antibodies; D005353:Fibronectins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha