Elsevier

Molecular Immunology

Volume 42, Issue 12, August 2005, Pages 1445-1451
Molecular Immunology

Humanization of an anti-human TNF-α antibody by variable region resurfacing with the aid of molecular modeling

https://doi.org/10.1016/j.molimm.2005.01.015Get rights and content

Abstract

The murine monoclonal antibody Z12 is of therapeutic interest for its neutralizing biological activity against human tumor necrosis factor-α (hTNF-α). We attempted to humanize Z12 with variable domain resurfacing guided by computer modeling. First, the genes of heavy and light chain variable region (VH, VL) of Z12 were cloned and the whole three-dimensional structure of Fv fragment was constructed by using homology-based modeling and molecular docking methods. Then the complex model of Fv interacting with hTNF-α whose crystal structure derived from PDB database was gained with computer-guided docking program. Based on this model, a humanized version was designed. The humanized Fab antibody was constructed, expressed and purified in the pComb3H vector system and it showed unaltered binding affinity to the antigen as determined by ELISA and atomic force microscopy (AFM). The method described here can be used to humanize other anti-hTNF-α antibodies.

Introduction

Tumor necrosis factor-α (TNF-α), a pleiotropic cytokine primarily produced by activated macrophage, has been proved to be implicated in the pathogenesis of many diseases as an endogenous mediator (Kollias et al., 1999). Increased level of human TNF-α (hTNF-α), therefore, became a useful target of therapy for several autoimmune diseases. Anti-hTNF-α monoclonal antibody (mAb), as an agent blocking hTNF-α activity, has been reported for therapy of rheumatoid arthritis (Maini et al., 1995), septic shock (Vincent et al., 1992), inflammatory bowel disease (Sandborn and Hanauer, 1999) and graft-versus-host disease (Herve et al., 1992). The murine mAb gained by us, designated as Z12, which was prepared by conventional hybridoma technology, characterized as neutralizing hTNF-α biological activity is, therefore, of therapeutic interest. In the previous study, we identified the epitope recognized by Z12 was the position 141–146 of hTNF-α (Zhang et al., 2004), suggesting that Z12 neutralizes the activity of hTNF-α by blocking this active region (Yamagishi et al., 1990).

To date, the commonest antibody manipulation is the process of humanization because of the murine-derived mAb elicits an inherent immune response in human. One approach for humanization is to produce chimeric molecules which consist of variable regions of mouse antibodies joined to the constant regions of human antibodies (Morrison et al., 1984). However, the chimeric antibodies will produce undesirable anti-variable region responses (Bruggemann et al., 1989), and it is also necessary to humanize the murine variable regions (VRs). The first procedure for humanizing murine VRs was based on transferring complementarity-determining regions (CDRs) from a mouse antibody to a human antibody framework, also called CDRs-grafting (Verhoeyen et al., 1988). However, to preserve the binding affinity, the majority of CDRs-grafted antibodies require additional work for backmutation of several murine framework amino acids which are deemed to be critical for CDRs loop conformation (Queen et al., 1989). As an alternative to CDRs-grafting, humanization by variable region resurfacing has been proposed, through the replacement of surface exposed residues in the murine framework regions (FRs) with those residues usually found in human antibodies (Padlan, 1991, O’Connor et al., 1998). A premise of the resurfacing approach is that the immunogenicity of murine VRs originates with the surface residues (Padlan, 1991).

Although resurfacing of antibodies is relatively straightforward using the techniques of molecular biology, the design is often a difficult and iterative procedure, and there is no information available about the effects of humanization of anti-hTNF-α antibodies by resurfacing on the binding affinities. In this study, a humanized version of Z12 was constructed through resurfacing via computer-aided molecular design in combination with molecular biology experiments and it showed the same binding reactivity against hTNF-α as the original antibody.

Section snippets

Materials

hTNF-α was purified by ion exchange chromatography DEAE52 (Shen et al., 1993). Z12 hybridoma cells were originally prepared by conventional hybridoma technology (Kohler and Milstein, 1975) using spleen cells from a BALB/c mouse immunized with purified hTNF-α (Li et al., 1991). Molecular modeling was performed using INSIGHTII2000 (Molecular Simulations, Inc., San Diego, CA), a program package involving HOMOLOGY, DOCKING and DISCOVER et al., with SGI Octane2 R12000 graphics workstation

cDNA cloning of Fd and κ chain gene of Z12

cDNAs encoding the Fd and κ chain were amplified by RT-PCR from mouse anti-hTNF-α hybridoma Z12 cells. The definition and numbering of VH, Vκ and CDRs were based on the work of Kabat et al. (1991). The translated amino acids of VH and Vκ are indicated in Fig. 1. Sequencing analysis with Kabat database showed that the heavy chain of Z12 belongs to mouse heavy chain subgroup IIID, whereas the light chain belongs to κ chain subgroup I.

Humanization of Z12

An optimized model of the mouse Z12 antibody Fv fragment was

Discussion

Engineered antibodies or antibody fragments with humanization and/or improved affinity have attracted much attention because of their therapeutic interest with desirable properties. The amount knowledge available with regards to antibody structure and interaction surface between antibody and antigen, even if approximate, would be instrumental in antibody engineering. Homology-based modeling has been used for antibody–antigen interaction studies and a complex model of an anti-hTNF-α mAb with its

Acknowledgement

The research work is supported by National Sciences Fund (No. 30490240), “973” fund (Nos. 001CB510005, 2003CB515508) and “863” fund (No.: 2002AA232021) of China.

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