Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs.
BACKGROUND
We have previously reported specificity improvement of an anti-testosterone monoclonal antibody (3-C4F5) by random mutagenesis of the third complementarity determining regions (CDR3s) and by phage display selection.


OBJECTIVE
Here we extend the mutagenesis strategy to the other CDRs and select the mutant libraries using two different approaches in order to further fine-tune the binding properties of this recombinant Fab fragment.


METHODS
To improve the affinity the new mutant libraries were selected by using limiting, decreasing concentrations of biotinylated testosterone (TES) in solution and capturing the binders on streptavidin-coated microtiter plate. The specificity was improved by preincubating the mutant libraries in solution with a high concentration of the most problematic cross-reacting steroid, dehydroepiandrosterone sulfate (DHEAS).


RESULTS
In two different light chain CDR1 mutant clones isolated from the affinity pannings, the relative TES affinity was increased over 10-fold while the cross-reactivities to related steroids were preserved at the same level as in the parental combined CDR3 mutant clone. New heavy chain CDR1 and light chain CDR2 mutants showing slightly decreased cross-reactivities were isolated from specificity selections. By combining compatible mutant CDRs together we were able to create a Fab fragment with over 12-fold higher relative TES affinity and significantly lower cross-reactivity to DHEAS when compared to the original monoclonal antibody 3-C4F5.


CONCLUSIONS
Our results demonstrate that a high-affinity and selective recombinant Fab fragment working over a wide TES concentration range with clinical samples could be generated by CDR mutagenesis and phage display selection.
Hemminki
A
A
VTT Biotechnology and Food Research, Espoo, Finland. ari.hemminki@vtt.fi
Niemi
S
S
Hautoniemi
L
L
Söderlund
H
H
Takkinen
K
K
D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D003429:Cross Reactions; D003687:Dehydroepiandrosterone / Q000276:immunology; D005069:Evaluation Studies as Topic; D015723:Gene Library; D006801:Humans; D007118:Immunoassay / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D007700:Kinetics; D016296:Mutagenesis; D009154:Mutation; D013739:Testosterone / Q000276:immunology*
D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D013739:Testosterone; D003687:Dehydroepiandrosterone