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

Academic Article
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Overview

authors

  • Rajpal, A.
  • Beyaz, N.
  • Haber, L.
  • Cappuccilli, G.
  • Yee, H.
  • Bhatt, R. R.
  • Takeuchi, T.
  • Lerner, Richard
  • Crea, R.

publication date

  • June 2005

journal

  • Proceedings of the National Academy of Sciences of the United States of America  Journal

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.

subject areas

  • Amino Acids
  • Antibodies
  • Antibody Affinity
  • Binding Sites, Antibody
  • Flow Cytometry
  • Gene Library
  • Mutagenesis, Site-Directed
  • Mutation
  • Neutralization Tests
  • Tumor Necrosis Factor-alpha
  • Yeasts
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Research

keywords

  • TNF-alpha
  • look-through mutagenesis
  • maturation
  • mutagenesis
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Identity

PubMed Central ID

  • PMC1143585

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.0503543102

PubMed ID

  • 15939870
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Additional Document Info

start page

  • 8466

end page

  • 8471

volume

  • 102

issue

  • 24

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