Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding.
BACKGROUND
Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors.


OBJECTIVE
To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands.


METHODS
Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding.


RESULTS
In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH).


CONCLUSIONS
Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.
Davies
J
J
MRC Laboratory of Molecular Biology, Cambridge, UK.
Riechmann
L
L
D000595:Amino Acid Sequence; D000941:Antigens / Q000378:metabolism*; D001666:Binding Sites, Antibody; D004247:DNA / Q000235:genetics / Q000378:metabolism; D006241:Haptens / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012680:Sensitivity and Specificity
D000941:Antigens; D006241:Haptens; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D004247:DNA