Non-immunized natural human heavy chain CDR3 repertoires allow the isolation of high affinity peptides mimicking a human influenza hemagglutinin epitope.
In this study we constructed two phage libraries displaying non-immunized natural human IgM derived HCDR3 repertoires. One library was structurally constrained by a Gly to Cys substitution at position 104 enabling the formation of a disulfide bridge with the Cys at position 92. Panning of these libraries on an anti-human influenza hemagglutinin (HA) antibody resulted in the selection of 16 different HCDR3 loops displaying different degrees of sequence homology with the HA epitope. The specificity of the HCDR3 loops recovered from the structurally constrained library was confirmed by competition assays using the HA epitope. Only one of these HCDR3 peptides contained Cys104. Structural analysis of these sequences revealed that the loss of Cys104 was associated with an increased preference for the formation of the type I beta-turn required for high affinity binding to the antibody. Affinity studies confirmed that the HCDR3 peptides containing the sequence YDVPDY and Gly104 had affinities in the nanomolar range (K(d)=7.6 nM) comparable to the HA epitope. These findings provided evidence that the recovered HCDR3 sequences may bind to their target in a conformation that is unreachable by the parental antibody from which the HCDR3 was derived. Furthermore, the isolation of target-specific and high affinity binders demonstrates the value of HCDR3 libraries as a source of 'biologically randomized' sequences of human origin for the identification of peptidic lead molecules.
Deroo
Sabrina
S
Laboratoire de Rétrovirologie, Centre de Recherche Public-Santé, 84 Val Fleuri, L-1526 Luxembourg, Luxembourg. deroo.s@retrovirology.lu
Fischer
Aurélie
A
Beaupain
Nadia
N
Counson
Manuel
M
Boutonnet
Nathalie
N
Pletinckx
Jurgen
J
Loverix
Stefan
S
Beirnaert
Els
E
De Haard
Hans
H
Schmit
Jean-Claude
JC
Lasters
Ignace
I
D000595:Amino Acid Sequence; D000906:Antibodies; D000915:Antibody Affinity; D022801:Complementarity Determining Regions; D000939:Epitopes; D006388:Hemagglutinins / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D007251:Influenza, Human / Q000276:immunology*; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000276:immunology* / Q000302:isolation & purification; D017433:Protein Structure, Secondary
D000906:Antibodies; D022801:Complementarity Determining Regions; D000939:Epitopes; D006388:Hemagglutinins; D007143:Immunoglobulin Heavy Chains; D007075:Immunoglobulin M; D019151:Peptide Library; D010455:Peptides