A Generic Antibody-Blocking Protein That Enables pH-Switchable Activation of Antibody Activity

Abstract

Molecular strategies that allow for reversible control of antibody activity have drawn considerable interest for both therapeutic and diagnostic applications. Protein M is a generic antibody-binding protein that binds to the Fv domain of IgGs and, in doing so, blocks antigen binding. However, the dissociation of protein M is essentially irreversible, which has precluded its use as an antibody affinity reagent and molecular mask to control antibody activity. Here, we show that introduction of 8 histidine residues on the Fv binding interface of protein M results in a variant that shows pH-switchable IgG binding. This protein M-8his variant provides an attractive and universal affinity resin for the purification of IgGs, antibody fragments (Fab and single-chain variable fragments (scFv)), and antibody conjugates. Moreover, protein M-8his enables the pH-dependent blocking of therapeutic antibodies, allowing the selective targeting of cells at pH 6.0.

Figure 1

(a) Protein M (red) in complex with a Fab fragment (PDB: 4nzr[19]). The heavy chain is colored dark green, the light chain light green, and residues mutated to His yellow. (b) Elution of infliximab from protein-M-variant-functionalized superparamagnetic beads at various pHs. Protein-M-variant-functionalized superparamagnetic beads were incubated with 20 μL of 2 μM infliximab for 30 min. Subsequently, infliximab was eluted by incubating the beads in buffer with decreasing pH for 2 min. The concentration of infliximab in each elution fraction was determined by a Bradford protein assay.

Figure 2

Purification of IgG, F(ab′)₂, Fab and ScFv’s using M-8his affinity resin. (a) Nonreducing SDS PAGE gel of full-length cetuximab (150 kDa) spiked in bacterial lysate (Lys.) and M-8his affinity resin purified cetuximab (El.) (b) Reusability of the M-8his affinity resin. For 6 cycles, an excess cetuximab was incubated with M-8his affinity resin. The bound fraction was eluted with 40 mM sodium formate pH 4.0, followed by a regeneration step with 40 mM sodium formate pH 2.5. The cetuximab concentration in each fraction was determined by a Bradford protein assay. Error bars indicate standard deviation of three technical replicates. (c) Nonreducing SDS PAGE gel of F(ab′)₂ and Fab purified with M-8his affinity resin after digestion of Cetuximab with pepsin and papain respectively. (d) Nonreducing SDS PAGE gel of periplasmic extraction from Escherichia coli expressing anti-HER scFv (Per.) and M-8his affinity resin purification (El.).

Figure 3

RAPPID assay with unpurified and purified infliximab-Gx-LB and Gx-SB conjugates. (a) Nonreducing SDS PAGE of the photoconjugation of 1.5 μM Infliximab with 12 μM Gx-LB and Gx-SB and subsequent purification with M-8his affinity resin. (b) Luminescence signal of purified and unpurified infliximab-Gx-LB and Gx-SB conjugates after incubation with 0–50 nM TNFα. The data are represented as the absolute luminescence signal (left) and the relative signal (right) compared to the signal in the absence of TNFα.

Figure 4

SPR analysis of the kinetics of the interaction between cetuximab Fab fragments and protein M. (a) SPR traces of binding and dissociation of protein M WT and M-8his to immobilized cetuximab Fab at pH 7.5. (b) Dissociation of protein M WT and M-8his from immobilized cetuximab Fab at pH 5.0. 500 nM protein M WT or M-8his was flown over the cetuximab Fab chip for 9 min, and the SPR signal was normalized to 100 RU for comparison. Pulses of 10 or 60 s with buffer of the indicated pH were alternated with running buffer (pH 7.5) washes to remove nonspecifically bound protein M. Individual data points and fit to eq S1 are shown. (c) Dissociation of M-8his from immobilized cetuximab Fab at pH 7.5, 6.0, and 5.0.

Figure 5

pH-induced antibody activation. FACS analysis of A431 cells labeled with (a) 1 nM aAxl and 1 nM wt protein M-bound aAxl or (b) 1 nM M-8his-bound aAxl in the presence of 100 nM IgGs at pH 7.4 or pH 6.0. (c) FACS analysis of A431, PD-1-transfected CHO, or SK-BR-3 cells labeled with anti-EpCam (5 nM) and M-8his-complexed anti-EpCam, Nivolumab (1 nM) and M-8his-complexed Nivolumab, Trastuzumab (1 nM) and M-8his-complexed Trastuzumab, respectively, at pH 7.4 or pH 6.0. All antibodies were labeled with Alexa647-NHS.