An antibody loop replacement design feasibility study and a loop-swapped dimer structure

Protein Eng Des Sel. 2009 Feb;22(2):93-101. doi: 10.1093/protein/gzn072. Epub 2008 Dec 10.

Abstract

A design approach was taken to investigate the feasibility of replacing single complementarity determining region (CDR) antibody loops. This approach may complement simpler mutation-based strategies for rational antibody design by expanding conformation space. Enormous crystal structure diversity is available, making CDR loops logical targets for structure-based design. A detailed analysis for the L1 loop shows that each loop length takes a distinct conformation, thereby allowing control on a length scale beyond that accessible to simple mutations. The L1 loop in the anti-VLA1 antibody was replaced with the L2 loop residues longer in an attempt to add an additional hydrogen bond and fill space on the antibody-antigen interface. The designs expressed well, but failed to improve affinity. In an effort to learn more, one design was crystallized and data were collected at 1.9 A resolution. The designed L1 loop takes the qualitatively desired conformation; confirming that loop replacement by design is feasible. The crystal structure also shows that the outermost loop (residues Leu51-Ser68) is domain swapped with another monomer. Tryptophan fluorescence measurements were used to monitor unfolding as a function of temperature and indicate that the loop involved in domain swapping does not unfold below 60 degrees C. The domain-swapping is not directly responsible for the affinity loss, but is likely a side-effect of the structural instability which may contribute to affinity loss. A second round of design was successful in eliminating the dimerization through mutation of a residue (Leu51Ser) at the joint of the domain-swapped loop.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / chemistry
  • Antibodies / genetics*
  • Antibodies / metabolism
  • Antibody Affinity
  • Cloning, Molecular
  • Complementarity Determining Regions / chemistry
  • Complementarity Determining Regions / genetics*
  • Complementarity Determining Regions / metabolism
  • Computer Simulation
  • Crystallography, X-Ray
  • Escherichia coli / genetics
  • Feasibility Studies
  • Fluorescence
  • Humans
  • Immunoglobulin Fab Fragments / chemistry
  • Immunoglobulin Fab Fragments / genetics*
  • Immunoglobulin Fab Fragments / metabolism
  • Models, Molecular
  • Protein Conformation
  • Protein Engineering
  • Protein Folding
  • Protein Multimerization
  • Rats
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Sequence Alignment
  • Temperature
  • Tryptophan / metabolism

Substances

  • Antibodies
  • Complementarity Determining Regions
  • Immunoglobulin Fab Fragments
  • Recombinant Proteins
  • Tryptophan