Expression of full-length immunoglobulins in Escherichia coli: rapid and efficient production of aglycosylated antibodies

Abstract

Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcγRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.

Introduction

Monoclonal antibodies have recently become promising therapeutic proteins (King and Adair, 1999), in addition to their conventional use as research tools. These proteins can be targeted to almost any extracellular or cell surface protein and, through the simple act of binding, promote the blocking or activation of specific biochemical steps Hori, 1991, Kim et al., 1993. Additionally, antibodies can couple their antigen to natural effector functions Dyer et al., 1989, Reff et al., 1994 or perhaps provide another activity through conjugation Hinman et al., 1993, Liu et al., 1996. One significant advantage of full-length IgGs is their long circulating half-life in mammals (Mariani and Strober, 1990), the result of both a large molecular size preventing clearance in the kidneys, and the ability of these proteins to avoid proteolysis in the endothelium by using a salvage pathway (Junghans, 1997). This pathway plays a major role in the slow clearance of IgGs and depends on binding of the immunoglobulin Fc-domain to the neonatal receptor (FcRn) (Simister and Mostov, 1989).

Full-length monoclonal antibodies have traditionally been produced in mammalian cell culture due to their original hybridoma source and due to the complexity of the molecule (King, 1998). More recently, transgenic plants offer an alternative route and potentially a more economical system of production Conrad and Fiedler, 1998, Giddings et al., 2000. Generally, Escherichia coli is the host system of choice for the expression of antibody fragments such as Fvs, scFvs, Fabs or F(ab')₂s Pluckthun et al., 1996, Kipriyanov and Little, 1999. These fragments can be made relatively quickly in large quantities with the retention of antigen binding activity. However, because antibody fragments lack the Fc domain, they do not bind the FcRn receptor and are cleared quickly; thus, they are only occasionally suitable as therapeutic proteins (Knight et al., 1995). Attempts to extend the heavy chain beyond the hinge have been successful with the expression of a C_H2-deleted antibody (Lo et al., 1992). The addition of the C_H3 domain promotes dimerization of the antigen binding arms, but the lack of the C_H2 voids any chance of the protein binding the FcRn receptor (Yasmeen et al., 1976). Full-length antibody chains can also be expressed in E. coli as insoluble aggregates and then refolded in vitro Boss et al., 1984, Cabilly et al., 1984, but the complexity of this method limits its usefulness.

Although the assembly of full-length antibodies in E. coli has not been reported, two reasons suggested the feasibility of this approach. First, fully functional antibody fragments through the hinge are routinely expressed as F(ab′)₂s Rodrigues et al., 1993, Koumenis et al., 2000, and second, the Fc fragment including the hinge has also been successfully produced as a dimer in E. coli (Kim et al., 1994). The aglycosylated Fc fragment bound to immobilized protein A, and displayed equivalent in vivo clearance to a full-length mammalian-derived glycosylated IgG. This data correlates well with previous studies showing that glycosylated and aglycosylated IgGs have equivalent in vitro FcRn binding and in vivo half-life in mammals Tao and Morrison, 1989, Hobbs et al., 1992. Since both halves of a full-length antibody can be expressed and assembled independently in E. coli, the folded full-length molecule itself could likely be produced, with advances in technology.

This study demonstrates that a full-length anti-tissue factor (αTF) IgG1 (Presta et al., 2001) can be expressed at high levels in E. coli, produced in large-scale fermentors, and captured on immobilized protein A. In vitro, the purified protein binds tightly to both the antigen and the FcRn receptor and, as predicted, fails to bind C1q or FcγRI indicating a lack of effector functions. Additionally, the circulating half-life of the E. coli-derived IgG1, determined in chimpanzees, appears similar to glycosylated αTF IgG2 and IgG4 produced in mammalian cells.