Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions

Chinatsu Shinozaki,Yoshito Abe,Makoto Nakakido,Yu Yoshikawa,Kazuhide Inoue,Mitsunori Shiroishi,Daisuke Takahashi,Kohei Tsumoto,Kenji Hamase,Makoto Tsuda,Keita Kohno,Tadashi Ueda

doi:10.1038/s41598-021-03784-w

Chinatsu Shinozaki, Yoshito Abe + Show 10 more

Open Access

https://doi.org/10.1038/s41598-021-03784-w

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Abstract

We have recently developed a mouse monoclonal antibody (12–10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12–10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the “hot spot” in the 12–10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12–10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the “hot spot” in the paratope.

Highlights

We have recently developed a mouse monoclonal antibody (12–10H) binding to the head domain region in rat P2X4 receptor expressed on the cell with the highest binding affinity (KD = 20 nM)
We conjugated the 12–10H antibody with Alexa Fluor 488 (the degree of labeling (DOL) was 2.1, indicating that one antibody molecule was labelled with two fluorescent dyes) and examined the ability of the fluorescent labeled 12–10H antibody to bind to rat P2X4R (rP2X4R)
These results indicate that the fluorescent labeled 12–10H antibody detects P2X4 receptor (P2X4R) expressed on living cells

Summary

Introduction

Detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the “hot spot” in the paratope. By expressing the head domain of rat P2X4R (rHD, Gln111–Val167) in E. coli, we showed an intact structure with three correctly formed S–S b onds[13] and developed five anti-rHD monoclonal antibodies that recognize the conformational epitope on the rHD Among these antibodies, the monoclonal antibody 12–10H showed the highest binding affinity and detected rat P2X4R (rP2X4R) expressed on the c ells[14]. We demonstrated that the engineered antibody bound rP2X4Rs expressed on the cell in primary culture microglia and in vivo samples in comparison with the negative control antibody, in which Tyr 33 and Arg[52] in the heavy chain and Tyr[31] in light chain in 12–10H were mutated to Ala, simultaneously, prepared based on our recent study[15]

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