An immunohistological study of testicular germ cell tumours using two different monoclonal antibodies against placental alkaline phosphatase

Epenetos, Agamemnon A.; Travers, Paul; Gatter, K C; Oliver, Rtd; Mason, David Y.; Bodmer, W F

doi:10.1038/bjc.1984.3

Cited by 46 publications

(14 citation statements)

References 15 publications

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“…However, our detailed immunohistochemical and previous biochemical analysis (Hole & Stern, 1988) shows that the antigen recognised is novel and distinct from the Psubunit of HCG or recently reported urinary gonadotrophin peptide (UGP) (Kardana et al, 1988), PLAP (Johnson, 1984), trophoblast-leukocyte common antigen (Stern et al, 1986) and those which react with mAb 18A/C4 and 18B/A5 (Loke et al, 1986). Some of (Epenetos et al, 1984), were almost all negative using mAb 5T4. The one case that showed some positivity was a seminoma containing syncytiotrophoblast giant cells, admixed with embryonal carcinoma.…”

Section: Discussionmentioning

confidence: 98%

Immunohistological distribution of 5T4 antigen in normal and malignant tissues

Southall

Boxer²,

Bagshawe³

et al. 1990

Br J Cancer

167

153

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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 &

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Section: Discussionmentioning

confidence: 98%

Immunohistological distribution of 5T4 antigen in normal and malignant tissues

Southall

Boxer²,

Bagshawe³

et al. 1990

Br J Cancer

167

153

View full text Add to dashboard Cite

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“…The scFv is directed against the tumour-associated antigen human placental-like alkaline phosphatase (PLAP), which is an isoform of the placental alkaline phosphatase. This antigen is expressed on many tumours, such as ovarian and testicular, as well as some bladder and head and neck cancers (Epenetos et al, 1984;Iles et al, 1994). The antibody used is the well-studied H17E2 Epenetos et al, 1986).…”

mentioning

confidence: 99%

Design, characterization and anti-tumour cytotoxicity of a panel of recombinant, mammalian ribonuclease-based immunotoxins

Deonarain

Epenetos²

1998

Br J Cancer

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Summary Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable antitumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 1 04-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia cofi (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.

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“…This suggests that although PLAP is expressed by all types of germ cell tumours of the testis (Epenetos et al, 1984) only seminomatous tumours actually release PLAP into the bloodstream. Elevated levels of PLAP (above 0.20.D.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously shown (Epenetos et al, 1984) that placental alkaline phosphatase (PLAP) as detected by monoclonal antibody H17E2 is expressed on the surface membrane of most testicular germ cell tumour cells. H17E2 immunoassay of sera of patients with testicular cancer as reported in the accompanying paper (Tucker et al, 1985) showed elevated PLAP levels in patients with seminoma.…”

mentioning

confidence: 99%