Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

doi:10.1016/S0304-4165(00)00165-3

Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 1524, Issues 2–3, 15 December 2000, Pages 238-246

https://doi.org/10.1016/S0304-4165(00)00165-3 Get rights and content

Abstract

The oncofoetal antigen 5T4 is a 72 kDa glycoprotein expressed at the cell surface. It is defined by a monoclonal antibody, mAb5T4, that recognises a conformational extracellular epitope in the molecule. Overexpression of 5T4 antigen by tumours of several types has been linked with disease progression and poor clinical outcome. Its restricted expression in non-malignant tissue makes 5T4 antigen a suitable target for the development of antibody directed therapies. The use of murine monoclonal antibodies for targeted therapy allows the tumour specific delivery of therapeutic agents. However, their use has several drawbacks, including a strong human anti-mouse immune (HAMA) response and limited tumour penetration due to the size of the molecules. The use of antibody fragments leads to improved targeting, pharmacokinetics and a reduced HAMA. A single chain antibody (scFv) comprising the variable regions of the mAb5T4 heavy and light chains has been expressed in Escherichia coli. The addition of a eukaryotic leader sequence allowed production in mammalian cells. The two 5T4 single chain antibodies, scFv5T4WT19 and LscFv5T4, described the same pattern of 5T4 antigen expression as mAb5T4 in normal human placenta and by FACS. Construction of a 5T4 extracellular domain–IgGFc fusion protein and its expression in COS-7 cells allowed the relative affinities of the antibodies to be compared by ELISA and measured in real time using a biosensor based assay. MAb5T4 has a high affinity, K_D=1.8×10⁻¹¹ M, as did both single chain antibodies, scFv5T4WT19 K_D=2.3×10⁻⁹ M and LscFv5T4 K_D=7.9×10⁻¹⁰ M. The small size of this 5T4 specific scFv should allow construction of fusion proteins with a range of biological response modifiers to be prepared whilst retaining the improved pharmacokinetic properties of scFvs.

Introduction

The use of tumour antigen specific antibodies for the delivery of therapeutic agents offers the possibility of specific therapy with reduced toxicity to normal tissues compared to conventional treatments [1]. However, the effectiveness of the use of whole monoclonal antibodies (mAbs) is restricted by several factors [2]. Repeated administration of murine antibodies generates a strong human anti-mouse immune (HAMA) response in up to 50% of patients after the first dose, and approximately 90% following a second treatment [2], [3]. This causes a rapid clearance of subsequent doses, and immune complex formation can lead to a severe anaphylactic response [4]. The severity of the HAMA response can be lessened by the use of antibody fragments lacking the Fc domain, in particular single chain Fv antibodies (scFvs) [5], [6]. ScFv antibodies may also show much greater target specificity for tumour localisation compared with whole IgG. The small size of scFv constructs allows increased diffusion into a solid tumour mass [7], and more rapid clearance from the blood [8]. Thus, although lower levels of antibody may localise at the site of the tumour, a higher labelling index (tumour:normal) can be achieved, as normal tissue labelling is minimised, in particular renal uptake [8], [9]. A further advantage of scFvs is that they greatly simplify the design of genetically modified constructs for therapy, compared with whole antibody molecules, and may be expressed in vivo by recombinant viral vectors.

The oncofoetal antigen 5T4 is defined by a mouse monoclonal antibody, mAb5T4, raised against wheat germ agglutinin purified placental glycoproteins that recognises a 72 kDa membrane molecule [10], [11]. 5T4 antigen is expressed in human trophoblast and many different carcinomas [10], but shows limited expression in normal adult tissue [12]. Studies in gastric [13], ovarian [14] and colorectal carcinoma [15] have demonstrated a prognostic significance for tumour associated 5T4 expression. The shared expression of 5T4 antigen between trophoblast and tumours is thought to reflect a common function possibly including tissue invasion. The cDNA sequences encoding both human and mouse 5T4 molecules have been determined [16], [17]; these predict conserved molecules comprising cytoplasmic and transmembrane domains and an extracellular domain containing two leucine rich repeat domains separated by a hydrophilic domain and seven potential N-linked glycosylation sites. Leucine rich repeats are involved in protein–protein interactions and are present in a wide range of proteins with diverse functions [18], [19], [20], [21], [22], [23], [24]. Overexpression of the gene in normal mouse mammary epithelial cells led to a reduction in cell–cell contacts, a rearrangement of the actin cytoskeleton, increased motility and a more dendritic morphology [25].

Here we report the isolation from the parent hybridoma of a single chain antibody with specificity for the oncofoetal antigen 5T4. This was adapted for expression in eukaryotic cells via a retroviral system. Affinities of the antibodies were measured in real time by surface plasmon resonance using a fusion protein constructed from the extracellular domain of 5T4 and the Fc of human IgG. The specificity and high affinity of this scFv make it a suitable platform for the development of tumour specific targeted therapies.

Section snippets

Cell culture

Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum and 2 mM L-glutamine at 37°C in a humidified atmosphere of 5% CO₂/air. Cultures were passaged as they reached visual confluence, using trypsin–EDTA. Murine L cells (A9 cells) transfected with human 5T4 (A95T4a) or control plasmid pCMVα (A9H12) [26] were maintained under antibiotic selection by the addition of 1 mg/ml G418.

Purification of monoclonal antibodies and preparation of 9E10 affinity matrix

MAb5T4 [10] was purified on protein A Sepharose in high salt loading buffer

Production of 5T4–IgGFc fusion protein

A fusion protein comprising the extracellular domain of human 5T4 and the Fc portion of human IgG₁ was expressed in COS-7 cells as a source of soluble 5T4 antigen. This was used to assess specificity and measure the affinities of single chain antibodies made from the mAb5T4 hybridoma. Production levels were typically 5–7 mg/l from 7 day cultures; purification on WGA–agarose and protein G Sepharose gave a yield of approximately 60%. The predicted size for the mature protein was 92 kDa. Silver

Discussion

The extracellular domain of human 5T4 expressed as a fusion with the Fc portion of human IgG was used to characterise single chain antibodies prepared from the mouse monoclonal antibody mAb5T4. The Fc was fused to the C-terminus of the 5T4 extracellular domain, preserving the conformation of the 5T4 molecule, and therefore its antigenicity (article in preparation).

Expressed in COS-7 cells, the fusion protein was affinity purified utilising properties of both component molecules, WGA lectin and

Acknowledgments

This work was funded by the Cancer Research Campaign and Oxford Biomedica.

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Isolation of a high affinity scFv from a monoclonal antibody recognising the oncofoetal antigen 5T4

Abstract

Introduction

Section snippets

Cell culture

Purification of monoclonal antibodies and preparation of 9E10 affinity matrix

Production of 5T4–IgGFc fusion protein

Discussion

Acknowledgments

Biochim. Biophys. Acta

Cell

Trends Biochem. Sci.

J. Immunol. Methods

Immunochemistry

J. Biol. Chem.

J. Immunol. Methods

Curr. Opin. Immunol.

Cancer Res.

J. Immunol.

Cancer Res.

Cancer Res.

Cancer Res.

Cancer Res.

J. Natl. Cancer Inst.

Cancer Res.

Br. J. Cancer

Int. J. Cancer

Br. J. Cancer

Br. J. Cancer

Int. J. Gynecol. Cancer

Br. J. Cancer

J. Biol. Chem.

Proc. Natl. Acad. Sci. USA