Calcium-dependent antigen binding as a novel modality for antibody recycling by endosomal antigen dissociation

Abstract

The pH-dependent antigen binding antibody, termed a recycling antibody, has recently been reported as an attractive type of second-generation engineered therapeutic antibody. A recycling antibody can dissociate antigen in the acidic endosome, and thus bind to its antigen multiple times. As a consequence, a recycling antibody can neutralize large amounts of antigen in plasma. Because this approach relies on histidine residues to achieve pH-dependent antigen binding, which could limit the epitopes that can be targeted and affect the rate of antigen dissociation in the endosome, we explored an alternative approach for generating recycling antibodies. Since calcium ion concentration is known to be lower in endosome than in plasma, we hypothesized that an antibody with antigen-binding properties that are calcium-dependent could be used as recycling antibody. Here, we report a novel anti-interleukin-6 receptor (IL-6R) antibody, identified from a phage library that binds to IL-6R only in the presence of a calcium ion. Thermal dynamics and a crystal structure study revealed that the calcium ion binds to the heavy chain CDR3 region (HCDR3), which changes and possibly stabilizes the structure of HCDR3 to make it bind to antigen calcium dependently (PDB 5AZE). In vitro and in vivo studies confirmed that this calcium-dependent antigen-binding antibody can dissociate its antigen in the endosome and accelerate antigen clearance from plasma, making it a novel approach for generating recycling antibody.

Keywords: Antibody engineering; antigen clearance; antigen dissociation; calcium ion; recycling antibody.

Figure 1.

Scheme of identification of calcium-dependent antigen-binding antibody. A calcium-dependent antigen-binding antibody was obtained from a human naïve antibody library as follows: Fab-expressing phages were bound to the antigen in the presence of a calcium ion and dissociated in the absence of a calcium ion. After several rounds of panning, several phages were cloned. Phage clones with a calcium-dependent antigen-binding property were converted to human IgG1 format.

Figure 2.

Calcium-dependent antigen-binding profile of 6RL#9 and 6RK#12. Calcium-dependent antigen binding of 6RL#9 (A) and 6RK#12 (B) was evaluated by SPR analysis.

Figure 3.

Effect of a calcium ion on the thermal stability of 6RL#9 and its antigen hsIL-6R. The thermal stability of hsIL-6R (A) and anti-hsIL-6R antibodies (B: NPH-hIgG1, C: 6RL#9-hIgG1) was determined by differential scanning calorimetry at 2 mM or 3 µM of a calcium ion. Dotted line represents 2 mM CaCl₂ condition and solid line shows 3 µM CaCl₂ condition.

Figure 4.

Calcium ion binding into 6RL#9-Fab from X-ray crystal structure. (A) Overall structure of Fv region of 6RL#9-Fab domain in the presence of calcium ion. (B) 2Fo-Fc electron density map of 6RL#9-Fab crystal in calcium ion including condition around HCDR3 is contoured at 1 times rmsd. The structure of 6RL#9-Fab is superimposed. (C) The detailed view of binding between calcium ion and HCDR3 of 6RL#9-Fab. Heavy chain is shown in green. Light chain is shown in magenta. Heavy chain CDR3 residues are indicated with sticks. Calcium ion is shown with a sphere and in yellow. Hydrogen bonds and a salt bridge with calcium ion are shown as dotted lines.

Figure 5.

Evaluation of endosomal antigen dissociation by confocal microscopy analysis. The calcium-dependent antigen-binding properties of an antibody affects the intracellular location of the antigen. FcRn expressing MDCK cells were pulsed with (A) labeled hsIL-6R alone or labeled hsIL-6R at a same amount of (B) a conventional antibody without calcium dependency (NPH) or (C) an antibody with calcium-dependent antigen binding (6RL#9). In (A), (B) and (C), upper and lower panels show 200 fold and 400 fold magnification, respectively. Cell images are shown with FcRn, hsIL-6R and nuclear in green, red and blue, respectively.

Figure 6.

Plasma concentration–time profile of hsIL-6R injected alone or in complex with anti-hsIL-6R antibodies. C57BL/6 mice were injected with either hsIL-6R alone or in complex with a conventional antibody without calcium dependency (NPH-hIgG1) or with a calcium-dependent antigen-binding antibody (6RL#9-hIgG1). Plasma concentration of hsIL-6R at the indicated time points was determined by ELISA, and mean values for each mouse group are presented. Error bar represents standard deviation.