A general method for greatly improving the affinity of antibodies by using combinatorial libraries

Abstract

Look-through mutagenesis (LTM) is a multidimensional mutagenesis method that simultaneously assesses and optimizes combinatorial mutations of selected amino acids. The process focuses on a precise distribution within one or more complementarity determining region (CDR) domains and explores the synergistic contribution of amino acid side-chain chemistry. LTM was applied to an anti-TNF-alpha antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (K(d) = 1 nM) by others. We selected and incorporated nine amino acids, representative of the major chemical functionalities, individually at every position in each CDR and across all six CDRs (57 aa). Synthetic oligonucleotides, each introducing one amino acid mutation throughout the six CDRs, were pooled to generate segregated libraries containing single mutations in one, two, and/or three CDRs for each V(H) and V(L) domain. Corresponding antibody libraries were displayed on the cell surface of yeast. After positive binding selection, 38 substitutions in 21 CDR positions were identified that resulted in higher affinity binding to TNF-alpha. These beneficial mutations in both V(H) and V(L) were represented in two combinatorial beneficial mutagenesis libraries and selected by FACS to produce a convergence of variants that exhibit between 500- and 870-fold higher affinities. Importantly, these enhanced affinities translate to a 15- to 30-fold improvement in in vitro TNF-alpha neutralization in an L929 bioassay. Thus, this LTM/combinatorial beneficial mutagenesis strategy generates a comprehensive energetic map of the antibody-binding site in a facile and rapid manner and should be broadly applicable to the affinity maturation of antibodies and other proteins.

Fig. 1.

Triple LTM example. Nine amino acids, representative of the 20, were selected for the LTM process based on their side-chain chemical functionalities (Inset Left). Discrete CDR oligonucleotides are synthesized to produce a mutagenized CDR with one target amino acid mutation at each CDR position (Inset Right). In the triple CDR library, all combinations of CDR1, CDR2, and CDR3 oligonucleotides are combined to produce libraries with three simultaneously mutagenized CDRs.

Fig. 2.

Dot plot of yeast displayed anti-TNF-α scFv. (Left) D2E7 staining was used to thresholds for improved binders. (Center) The LTM libraries. Shown is FACS profile for the unsorted library. (Right) The dot plot profile for the sorted library.

Fig. 3.

BIAcore dissociation profiles of D2E7, LTM (broken lines), and CBM clones.

Fig. 4.

Affinity-matured anti-TNF-α antibodies have improved neutralizing abilities. L929 cells were treated with actinomycin and TNF-α, as indicated for 24 h, in the absence and presence of purified anti-TNF-α scFv antibodies. WST-1 was added and incubated for an additional 4 h, and OD₄₅₀ was recorded. Fifty percent neutralizing dose values were 141 pM for D2E7 (open green circle), 3.5 pM for A1 (red square), 6.1 pM for cb1-3 (red triangle), and 6.5 pM for cb2-6 (red diamond). Data represent mean ± SEM with each measurement performed in quadruplicate.