Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

Ari Hemminki (Corresponding Author), Seija Niemi, Lasse Hautoniemi, Hans Söderlund, Kristiina Takkinen

Research output: Contribution to journalArticleScientificpeer-review

35 Citations (Scopus)

Abstract

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.
Objectives: 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. Study design: 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.
Original languageEnglish
Pages (from-to)59-69
JournalImmunotechnology
Volume4
Issue number1
DOIs
Publication statusPublished - 1998
MoE publication typeA1 Journal article-refereed

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Antibody Binding Sites
Testosterone
Anti-Idiotypic Antibodies
Immunoglobulin Fab Fragments
Mutagenesis
Dehydroepiandrosterone Sulfate
Bacteriophages
Clone Cells
Steroids
Monoclonal Antibodies
Complementarity Determining Regions
Light
Streptavidin

Cite this

Hemminki, Ari ; Niemi, Seija ; Hautoniemi, Lasse ; Söderlund, Hans ; Takkinen, Kristiina. / Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs. In: Immunotechnology. 1998 ; Vol. 4, No. 1. pp. 59-69.
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abstract = "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. Objectives: 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. Study design: 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.",
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Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs. / Hemminki, Ari (Corresponding Author); Niemi, Seija; Hautoniemi, Lasse; Söderlund, Hans; Takkinen, Kristiina.

In: Immunotechnology, Vol. 4, No. 1, 1998, p. 59-69.

Research output: Contribution to journalArticleScientificpeer-review

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T1 - Fine tuning of an anti-testosterone antibody binding site by stepwise optimisation of the CDRs

AU - Hemminki, Ari

AU - Niemi, Seija

AU - Hautoniemi, Lasse

AU - Söderlund, Hans

AU - Takkinen, Kristiina

PY - 1998

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N2 - 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. Objectives: 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. Study design: 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.

AB - 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. Objectives: 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. Study design: 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.

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