Expression of single-chain Fv-Fc fusions in Pichia pastoris

doi:10.1016/S0022-1759(00)00290-8

Journal of Immunological Methods

Volume 251, Issues 1–2, 1 May 2001, Pages 123-135

https://doi.org/10.1016/S0022-1759(00)00290-8 Get rights and content

Abstract

Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (∼106 kDa vs. ∼25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t_1/2 of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.

Introduction

The modular nature of antibody molecules allows for an almost unlimited number of domain rearrangements and fusions. Antibody engineering allows the researcher to design and use a variety of binding domains, effector domains, as well as non-antibody fusion partners (Chamow and Ashkenazi, 1999, Hoogenboom, 1997). One of the most useful antibody fragments is the single-chain Fv (scFv) in which the isolated antibody V_H and V_L domains are joined by a short peptide linker (Bird et al., 1988, Huston et al., 1988). This monovalent, minimal binding fragment is favored for antibody phage display techniques and phage antibody library construction (Marks et al., 1991, McCafferty et al., 1990).

Once a scFv has been isolated from a phage display library, the binding domain can be characterized with respect to affinity, epitope, and biological activity, as well as subjected to further affinity maturation. The scFv can be used as is, or engineered into other formats (Fv, Fab, (Fab′)₂, IgG, or fused to other proteins).

For some applications the scFv itself is the desired format. The small size of the scFv, its rapid clearance from the blood, and tumor penetration properties make it the format of choice for tumor targeting and radioimmunoimaging applications (Adams et al., 1995, Adams et al., 1998, Yokata et al., 1992). The single-chain nature of the scFv is also best-suited for intracellular immunization (intrabody) applications (Marasco, 1997). However for other uses, it would be desirable to transfer the antigen-binding properties of the scFv into a full length IgG, to take advantage of avidity effects, effector functions, and the prolonged serum half-life of an immunoglobulin. Increasing the serum half-life of scFv is particularly important for efficient in vivo antigen neutralization since the t_1/2β of a typical scFv is only 3.5 h in mice (Huston et al., 1996).

One approach is to directly engineer a phage displayed scFv into a full length IgG and express it in mammalian cells (Persic et al., 1997). However this approach requires separate cloning steps for the V_H and V_L domains, and establishing a stable antibody secreting cell line can be time consuming. An alternate approach would be to engineer the scFv into a more ‘IgG-like’ structure that can be accomplished in a single cloning step, and that can be expressed at high levels in a eucaryotic microorganism such as yeast. We have constructed a vector to rapidly express an scFv-Fc fusion (wherein the scFv is fused to the hinge, C_H2, and C_H3 domains of human IgG1) in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion can easily be constructed in a single cloning step from a scFv and is secreted at high levels as a glycosylated dimer from Pichia cultures. The scFv-Fc fusion retains the affinity and specificity of the parent scFv, combined with the bivalency, prolonged serum half-life, and the Fc-mediated ADCC (antibody-dependent cellular cytotoxicity) of an IgG. These yeast-produced scFv-Fc fusions will be useful to rapidly characterize candidate scFv isolated from phage antibody libraries before converting to a full-length IgG, or as an alternative format in its own right.

Section snippets

Cells and media

Pichia pastoris strain GS115 was obtained from Invitrogen, San Diego, CA. SKBR-3 and SK-OV-3 cells were obtained from the ATCC (American Type Culture Collection, Rockville, MD). Media used: YPD, yeast extract peptone dextrose medium (1% yeast extract, 2% peptone, 2% dextrose); YPDS, YPD medium plus 1 M sorbitol; BMGY, buffered glycerol complex medium (1% yeast extract, 2% peptone, 100 mM phosphate buffer pH 6.0, 1.34% YNB, 4×10⁻⁵% biotin, 1% glycerol); BMMY, buffered methanol complex medium (1%

Plasmid constructions

We have constructed a plasmid for the expression of single-chain antibody Fv domains (scFv) fused to the human IgG1 Fc domain to make a bivalent, ∼106,000 molecular weight scFv-Fc fusion (Fig. 1). Plasmid pPIgG1 for the expression of the scFv-Fc fusions (comprising the scFv followed by the hinge, C_H2 and C_H3 domains of human IgG1) is based on the Pichia pastoris expression plasmid pPICZαA (Invitrogen). This plasmid uses the alcohol oxidase (AOX1) promoter for high level expression of

Discussion

The single-polypeptide, minimally sized scFv antibody fragment is useful for the design and construction of phage display antibody libraries, as fusion proteins, and as intrabodies; in addition the small size is best suited to tumor targeting due to superior tissue penetration (Yokata et al., 1992). However useful, the scFv format suffers from a number of key limitations. The lack of avidity due to monovalent binding can limit the effectiveness and/or sensitivity of scFv fragments in many

Acknowledgements

We would like to thank Mr. Tom Purcell of Invitrogen Co. for helpful discussions, Mr. Steve Chen for technical assistance, and Dr. Chris Turk of UCSF for N-terminal sequence analysis. This work was partially funded by DAMD17-94-C-4034, DAMD17-94-J-4433, and NIH CA65559.

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Recombinant TechnologyExpression of single-chain Fv-Fc fusions in Pichia pastoris

Abstract

Introduction

Section snippets

Cells and media

Plasmid constructions

Discussion

Acknowledgements

Immunotechnology

Anal. Biochem.

Immunotechnology

J. Pharm. Sci.

Mol. Immunol.

J. Mol. Biol.

Anal. Biochem.

Gene

Immunotechnology

Immunotechnology

J. Mol. Biol.

Trends Biotechnol.

Highly specific in vivo tumor targeting by monovalent and divalent forms of 741F8 anti-c-erbB-2 single chain Fv

Cancer Res.

Enhanced tumor specificity with stably iodinated 741F8-2 (sFv′)2, an anti-c-erbB-2 single chain Fv dimer

J. Nucl. Med.

Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies

Cancer Res.

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries

Infect. Immun.

Single-chain antigen-binding proteins

Science

Recombinant Technology
Expression of single-chain Fv-Fc fusions in Pichia pastoris