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-␣ antibody, D2E7, which is a challenging test case, because D2E7 was highly optimized (Kd ‫؍‬ 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, and͞or three CDRs for each V H and VL 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-␣. These beneficial mutations in both VH and VL 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-␣ neutralization in an L929 bioassay. Thus, this LTM͞combinatorial 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.look-through mutagenesis ͉ maturation ͉ mutagenesis ͉ TNF-␣

ImmunoFET feasibility in physiological salt environments

“…In both species, MIG is a pro-inflammatory chemokine that is a chemoattractant for cytotoxic T-cells. MIG increases during inflammatory responses, rising from a normal concentration of 40-100 pM [31], to 1-2 orders of magnitude higher during acute inflammation [32]. In transplant biology, rising graft MIG concentrations precede allograft rejection and can be used as an early indicator of imminent rejection [33,34].…”

“…Neither does classical assessment consider differentiable ion permeability of various FET platforms. Ion permeation alters electrical properties of MOSFETs, impeding accurate sensing [31]. Less ionpermeable FETs (e.g.…”

Affinity maturation of antibodies assisted by in silico modeling

“…These strategies vary from substitution of specific selected residues within the complementarity determining region (CDR) loops to random mutagenesis of the entire variable fragment (Fv) sequence (12)(13)(14)(15). One of the main problems associated with affinity maturation by phage display is that of library completeness: It is practically unfeasible to generate all possible (combinations of) CDR residue mutants.…”

Probing Binding Mechanism of Interleukin-6 and olokizumab: In Silico Design of Potential lead Antibodies for Autoimmune and Inflammatory Diseases

“…Nevertheless, biologics have showed better efficacy in successful remission of autoimmune and inflammatory disease. Furthermore, site directed mutagenesis inducted on known antibodies have been shown to improve their binding affinity by ~10 -454 folds towards their desired target proteins in vitro [1][2][3][4]. These experimental insights, in fact encouraged us to implement molecular modelling approaches to explore if there exist any scope for improving existing therapeutic axis in silico.…”