Immunohistological distribution of 5T4 antigen in normal and malignant tissues

Abstract

Summary A trophoblast cell surface antigen has been characterised by a monoclonal antibody (mAb) 5T4, raised following immunisation with solubilised wheat germ agglutinin binding glycoproteins from human syncytiotrophoblast plasma membrane (StMPM). The expression of the 72 kDa glycoprotein was assessed on cryostat sections of a range of neoplastic and non-neoplastic tissues, using an avidin-biotin immunoperoxidase technique. In products of conception, intense reactions were noted with villous syncytiotrophoblast membrane in normal early and term placenta, with weaker positivity of placental site trophoblast. Most normal or non-neoplastic tissues were negative, including liver, kidney, spleen, small intestine, ovary Specifically, sections were washed in two changes of tris buffered saline (TBS) pH 7.6 and then covered with 10% normal horse serum in TBS for 20 min. After draining, the slides were incubated with neat culture supernatant for 30 min in a moist chamber. Following three washes in TBS (5 min each), biotinylated anti-mouse Ig (Vector Laboratories) diluted 1/250 in TBS containing 10% normal human serum was applied. After 30 min incubation in the moist chamber, the slides were washed three times and incubated with the avidin-biotin peroxidase complexes reagent (Vector Laboratories) for 50 min. After three washes in TBS, peroxidase was visualised using a freshly prepared and filtered solution of diaminobenzidine tetrahydrochloride (DAB-Sigma) in TBS containing 0.03% hydrogen peroxide (6 min). Sections were washed in tap water and counterstained in Coles' haematoxylin, dehydrated, cleared and mounted (Ralmount-R.A. Lamb). The immunohistochemical results were interpreted with reference to a set of controls run in parallel with each test. These included sections treated with DAB only to show endogenous peroxidase, omission of the primary antibody and replacement of the primary antibody with one of the same class but of unrelated specificity. Reactivity of mAb 5T4 with fixed and paraffin wax embedded sections of term placental trophoblast was also assessed by immunoperoxidase. 5T4 mAb reactivity was assessed by P.J.S. and G.M.B. and the intensity of staining was scored on an arbitrary scale ( + to + + + ). Very weak or equivocal reactions were scored + /-.Flow cytometric analyses of human bone marrow cells labelled in PBS, 1% BSA 0. 1% azide with 5T4 mAb followed by 1/20 rabbit anti-mouse Ig-FITC (Serotec, Bicester, UK) was performed on a Becton-Dickenson FACS analyser. The purified mAb 5T4 at 10 ig ml-' was tested for interference with the growth of human pluripotential haematopoietic colonies as described by Welte et al. (1985). RadioimmunoassaySyncytiotrophoblast microvillous plasma membranes (StMPM) were prepared as previously described (Hole &

Expression of the 5T4 oncofoetal antigen in renal cell carcinoma: a potential target for T-cell-based immunotherapy

“…Importantly, the strong positive membrane expression on most of the tumour specimens makes RCC a good candidate for 5T4-targeted therapies. Previous reports of 5T4 have documented minimal expression on normal tissue (Southall et al, 1990) and we wished to confirm this was the case with respect to the kidney. We were able to analyse normal kidney taken greater than 2 cm from the tumour margin in five patients, however, the surrounding kidney in RCC frequently shows inflammatory change and our samples were no exception.…”

“…This is further supported by evidence that expression of 5T4 correlates with poorer survival in colorectal carcinoma (Starzynska et al, 1994). This protein has only limited expression in normal adult human tissues (Southall et al, 1990) making it a useful target for the immunotherapy of cancer.…”

“…5T4 is a 72 kDa glycoprotein identified by a murine monoclonal antibody produced by a hybridoma from splenocytes of mice immunised with syncytiotrophoblast microvillous membrane glycoproteins (Hole and Stern, 1988). It is highly expressed on placental trophoblast and a variety of human cancers (Southall et al, 1990). Transduction of the 5T4 cDNA into cell lines enhances cell motility and reduces cell -cell contacts suggesting that it may be mechanistically involved in the malignant phenotype (Carsberg et al, 1996).…”

Therapy of human non-small-cell lung carcinoma using antibody targeting of a modified superantigen

“…The murine antibody 5T4 recognizes a 72 kDa glycoprotein Stern, 1988, 1990) found in many carcinomas, especially non-small-cell lung and breast cancer, but at low levels in normal tissues (Southall et al, 1990). 5T4 tumourassociated labelling is also a marker of prognostic significance in colorectal (Starzynska et al, 1992, Mulder et al, 1997 and gastric carcinoma (Starzynska et al, 1998).…”

In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies

“…The scFv used for this study is specific to the tumour-associated antigen, 5T4, which is a human oncofetal protein expressed on a large number of malignancies but absent on most normal adult tissue. [21][22][23] ScFv fusion proteins specific to 5T4 have previously been described 24 including the 5T4 scFv fused to the murine homologue of the immunostimulatory molecule, B7.1 (scFvB7). In this study, we use this molecule to evaluate the ability of EIAV to genetically deliver a secreted protein, systemically, in vivo.…”