PEDS Advance Access published online on December 10, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzn072
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An antibody loop replacement design feasibility study and a loop-swapped dimer structure
Biogen Idec Inc., Cambridge, MA 02142, USA
2 To whom correspondence should be addressed. E-mail: louie{at}alumni.northwestern.edu
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 Å 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°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.
Keywords: antibody loop design/antibody structure/binding affinity/protein design/protein stability
Received June 1, 2008; revised October 4, 2008; accepted November 4, 2008.
1 Present address: Codexis Inc., 200 Penobscot drive, Redwood City, CA 94063, USA.