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. 2015;7(2):294-302.
doi: 10.1080/19420862.2015.1006993.

De novo isolation of antibodies with pH-dependent binding properties

Pauline Bonvin  1 , Sophie Venet, Gaëlle Fontaine, Ulla Ravn, Franck Gueneau, Marie Kosco-Vilbois, Amanda Ei Proudfoot, Nicolas Fischer
Affiliations

De novo isolation of antibodies with pH-dependent binding properties

Pauline Bonvin et al. MAbs. 2015.

Abstract

pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.

Keywords: BLI, bio-layer interferometry; CDR, complementary determining region; CDRH, CDR of the heavy chain; CDRL, CDR of the light chain; ELISA, enzyme-linked immunosorbent assay; GPCR, G protein-coupled receptor; KB, kinetic buffer; PBS, phosphate buffered saline; SPR, surface plasmon resonance; antibody recycling; chemokine; histidine; mAb, monoclonal antibody; monoclonal antibody; pH-dependency; phage display; phage libraries; scFv, single-chain variable fragment.

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Figures

Figure 1.
Figure 1.
De novo isolation of pH-dependent antibodies. (A). A phage display library enriched in histidine in the CDRH3 was constructed by introducing degenerated oligonucleotides in an acceptor library. NHT and YAT codons, encoding for the indicated amino acids, were alternated at the indicated positions. Inserts encoding for 8 to 15 amino acids were used. (B). Scheme of selections against hCXCL10. Phage particles were added on beads coated with hCXCL10 at pH 7.4. Following seven washes, the pH was lowered to 5.4 and the supernatant of the mixture was recovered and amplified before being used for the subsequent round of selection.
Figure 2.
Figure 2.
Characterization of C7 IgG. (A). Cross-reactivity of C7. C7 binds to murine (⋄) and human (⧫) CXCL10 with similar affinities by ELISA (left panel) and inhibits both murine and human CXCL10-mediated chemotaxis of L1.2 cells transfected with hCXCR3 (right panel). The ELISA as well as the chemotaxis assay were performed at pH 7.4. Chemotaxis data are presented as a percentage of cell migration, with 100 % being the migration observed with either 5 nM hCXCL10 or 5 nM mCXCL10. In both graphs, data are presented as the mean of triplicates +/− SEM and are representative of at least 2 independent experiments. (B). Binding of C7 to hCXCL10 by SPR. The biotinylated chemokine was captured on a streptavidin chip and the antibody was used as analyte. Following association at pH 7.4, the complex between C7 and hCXCL10 dissociate faster at pH 6.0 (- -) than at pH 7.4 (—). The signal obtained following association and dissociation at pH 6.0 (.....) was lower than at pH 7.4, suggesting that the affinity of C7 for hCXCL10 is reduced at slightly acidic pH. In contrast, the binding of the reference antibody CF1 to hCXCL10 was not affected by variations in pH.
Figure 3.
Figure 3.
Optimization of C7. (A). pH-dependent screening of selection outputs by FMAT. Streptavidin beads coated with mCXCL10 were incubated either at pH 6.0 or 7.4 with scFv isolated from periplasmic extracts. Candidates were considered as positive if a binding was observed at pH 7.4 but not at pH 6.0 nor with an irrelevant protein (hIL6R). (B). CDRH sequences of 5 positive candidates, presented in comparison to the C7 clone. Randomized positions are underlined and histidines are shaded in gray. (−) : same amino acid as C7. (C). Sequences reformatted onto the hIgG1 backbone of hits described in B were tested for pH-dependency by ELISA. Biotinylated mCXCL10 was captured into streptavidin wells and following binding of the clones to the chemokine, an elution step was performed at the indicated pH and the binding remaining after one hour was assessed.
Figure 4.
Figure 4.
Characterization of candidates isolated from C7 optimization selections. (A). Inhibition of 5 nM mCXCL10-mediated chemotaxis of L1.2/CXCR3 cells with C7 (black), 1A4 (red), 1D10 (green) and 4A3 (blue). Data are presented as a percentage of cell migration, with 100 % being the migration observed with 5 nM mCXCL10 and as the mean of 3 measurements ± SEM. They are representative of 2 independent experiments. (B). Dose-response curves demonstrating the binding of C7 (open diamonds) and 1A4 (filled circles) to mCXCL10 in a pH-dependent ELISA. The chemokine was captured on the plate via a biotin tag and the binding of the antibodies to their target was followed by an elution step at pH 7.4 or 6.0. The EC50 of 1A4 shifts from 0.19 nM to 56.5 nM when the pH of the elution step is lowered to 6.0, while the EC50 of C7 increases from 1.4 nM to 34.4 nM. (C). Binding of mCXCL10 to C7 (left) or 1A4 (right) coated on Protein A sensors by BLI. A comparison was performed between dissociation at pH 7.2 (red) and at pH 6.0 (blue) following association at pH 7.2. The experiment was also performed with conditions at pH 6.0 (gray). (D). Binding of C7 (left) or 1A4 (right) to biotinylated mCXCL10 coated on streptavidin biosensors. The color code is similar to that used in C.
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