Ribosome Display Selection of a Murine IgG1 Fab Binding Affibody Molecule Allowing Species Selective Recovery Of Monoclonal Antibodies

Abstract

Affinity reagents recognizing constant parts of antibody molecules are invaluable tools in immunotechnology applications, including purification, immobilization, and detection of immunoglobulins. In this article, murine IgG1, the primary isotype of monoclonal antibodies (mAbs) was used as target for selection of novel binders from a combinatorial ribosome display (RD) library of 1011 affibody molecules. Four rounds of selection using three different mouse IgG1 mAbs as alternating targets resulted in the identification of binders with broad mIgG1 recognition and dissociation constants (KD) in the low nanomolar to low micromolar range. For one of the binders, denoted Zmab25, competition in binding to full length mIgG1 by a streptococcal protein G (SPG) fragment and selective affinity capture of mouse IgG1 Fab fragments after papain cleavage of a full mAb suggest that an epitope functionally overlapping with the SPG-binding site in the CH1 domain of mouse IgG1 had been addressed. Interestingly, biosensor-based binding experiments showed that neither human IgG1 nor bovine Ig, the latter present in fetal bovine serum (FBS) was recognized by Zmab25. This selective binding profile towards murine IgG1 was successfully exploited in species selective recovery of two different mouse mAbs from complex samples containing FBS, resembling a hybridoma culture supernatant.

Purification Systems Based on Bacterial Surface Proteins

“…Another recent example of how novel specificity may be incorporated in small protein domains is illustrated by selection of Affibody molecules with increased affinity to mouse IgG1 (Grimm et al 2011). The original Z-domain has practically non-existing affinity against mouse IgG, which represents the most widely used within biotechnology.…”

“…The original Z-domain has practically non-existing affinity against mouse IgG, which represents the most widely used within biotechnology. The new specificity possessed by the mouse IgG1-specific binding molecule facilitates specific recovery of monoclonal mouse antibodies from hybridoma supernatants rich in bovine immunoglobulin that may cross-react with alternative capture agents (Grimm et al 2011). Furthermore, anti-ideotypic Affibody molecules have been generated using other affinity ligands or SPA itself as the target in the selections (Eklund et al 2002;Wallberg et al 2011).…”

Monitored whole gene in vitro evolution of an anti-hRaf-1 affibody molecule towards increased binding affinity

“…The GeneMorph 1 II Random Mutagenesis Kit (Stratagene) was used according to manufacturer's recommendations in combination with primers Zep-for (5 0 -AAGATGACGATGATAAAACGTCGAC-3 0 ; underlined: SalI recognition site), Zep-rev (5 0 -ATTGGCCTTGATATGAGCTC-GAG-3 0 ; underlined: SacI recognition site), 30 amplification cycles and 508C annealing temperature. 25 ng of resulting epPCR product was inserted between the SalI and SacI recognition sites of 50 ng pRD2-bla-H3 [11]. A small aliquot of the ligation reaction was transformed to E. coli to evaluate the mutation spectrum and frequency of the library and 25% were used as template for the preparation of library DNA cassettes for RD selection, using the primers RD-for and RD-rev9, as described previously [11].…”

“…25 ng of resulting epPCR product was inserted between the SalI and SacI recognition sites of 50 ng pRD2-bla-H3 [11]. A small aliquot of the ligation reaction was transformed to E. coli to evaluate the mutation spectrum and frequency of the library and 25% were used as template for the preparation of library DNA cassettes for RD selection, using the primers RD-for and RD-rev9, as described previously [11].…”

“…Library mRNA and ternary affibody-ribosome-mRNA (ARM) complexes were prepared as described previously [11]. ARM complexes were incubated for one hour with 80 nM (cycle 1) or 20 nM (cycle 2) biotinylated ABP-hRaf-1 5-143 at 48C (cycle 1 route I) or ambient room temperature (cycle 1 route II, cycle 2 route I/II) while shaking.…”