An antibody loop replacement design feasibility study and a loop-swapped dimer structure.
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.
Clark
Louis A
LA
Biogen Idec Inc., Cambridge, MA 02142, USA. louie@alumni.northwestern.edu
Boriack-Sjodin
P Ann
PA
Day
Eric
E
Eldredge
John
J
Fitch
Christopher
C
Jarpe
Matt
M
Miller
Stephan
S
Li
You
Y
Simon
Ken
K
van Vlijmen
Herman W T
HW
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000915:Antibody Affinity; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005240:Feasibility Studies; D005453:Fluorescence; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D055503:Protein Multimerization; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D014364:Tryptophan / Q000378:metabolism
D000906:Antibodies; D022801:Complementarity Determining Regions; D007140:Immunoglobulin Fab Fragments; D011994:Recombinant Proteins; D014364:Tryptophan