Identification and characterization of the epitopes of two monoclonal antibodies binding to the tissue-plasminogen activator rt-PA

The glycosylation of tissue plasminogen activator (t-PA) obtained from the Bowes melanoma cell line was re-examined using methods of serial lectin affinity chromatography coupled with Bio-Gel P-4 gel filtration chromatography and exoglycosidase sequencing. This study clarified an earlier discrepancy in the literature and confirmed that the major complex N-linked glycans on Bowes t-PA that carry sialic acid as their sole charged group are bi-antennary, core fucosylated, with terminal N-acetylgalactosamine residues. We also report the characterization of a series of related and previously unidentified sialylated glycans. Further we show that Bowes t-PA expresses glucuronic acid/sulphate containing N-linked glycans and is recognized by anti-carbohydrate L2/HNK-1 monoclonal antibodies. The presence on Bowes t-PA of glycans associated primarily with the nervous system is consistent with its expression in a cell line of neuroectodermal origin.

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Bispecific Monoclonal Antibodies Produced by Somatic Cell Fusion Increase the Potency of Tissue Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647297 ◽

1990 ◽

Vol 64 (02) ◽

pp. 260-266 ◽

Cited By ~ 23

Author(s):

Elizabeth E Branscomb ◽

Marschall S Runge ◽

Christopher E Savard ◽

Keith M Adams ◽

Gary R Matsueda ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Somatic Cell ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Cell Fusion ◽

Bispecific Antibody ◽

Solid Phase ◽

Bispecific Antibodies ◽

Somatic Cell Fusion ◽

Tissue Plasminogen

SummaryBispecific monoclonal antibodies that bind simultaneously to human fibrin and tissue plasminogen activator (tPA) enhance the fibrinolytic potency of tPA. Two bispecific antibodies (F36.23 and F32.1) were generated by somatic cell fusion. Antibody F36.23 derives its tPA binding from monoclonal anti-tPA antibody TCL8 and its fibrin binding from monoclonal antifibrin antibody 59D8. After purification from cell supernatants and ascites by two steps of affinity chromatography, hybrid-hybridoma bispecific antibody F36.23 simultaneously bound tPA and fibrin in solution and in solid-phase assays. In an assay for the lysis of human fibrin monomer, F36.23 increased the fibrinolytic potency of tPA by 5 to 10 fold, regardless of whether the bispecific antibody had been combined with the tPA before or during the assay. Bispecific F36.23 F(ab′)2 also bound tPA and fibrin simultaneously, and the enhancement in fibrinolysis in the presence of F36.23 F(ab′)2 was identical to that in the presence of intact F36.23. The second bispecific antibody, F32.1, was produced by an alternative strategy that has a wider potential for applicaton in other systems. Hybridoma bispecific antibody F32.1 was derived from the fusion of immune splenocytes (in mice immunized with a synthetic oligopeptide representing the amino terminus of the α-chain of human fibrin) with the anti-tPA cell line TCL8. The properties of hybridoma bispecific antibody F32.1 and its F(ab′)2 were indistinguishable from those of hybrid-hybridoma bispecific antibody F36.23 in solid-phase binding assays and in assays of fibrinolysis. Bispecific antibodies produced by somatic cell fusion, particularly in the form of F(ab′)2, may have potential for use in clinical thrombolysis.

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Monoclonal Antibodies Towards the Fast Inhibitor of Tissue Plasminogen Activator from Human Plasma and Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661569 ◽

1986 ◽

Vol 55 (03) ◽

pp. 383-387 ◽

Cited By ~ 5

Author(s):

G Urdén ◽

Ulla Johansson ◽

Joanna Chmielewska ◽

J Brandt ◽

B Wiman

Keyword(s):

Monoclonal Antibodies ◽

Human Plasma ◽

Plasminogen Activator ◽

Tissue Plasminogen Activator ◽

Hybridoma Cells ◽

Spleen Cells ◽

Inhibitor Complex ◽

Tissue Plasminogen ◽

Different Levels ◽

Mouse Myeloma

SummaryHybridoma cells were produced by fusing mouse myeloma cells (SP 2/0 - Ag 14) with spleen cells from a Balb/c mouse, previously immunized with the partially purified complex between tissue plasminogen activator (t-PA) and its fast inhibitor from human plasma (serum). Screening with a radioimmunoassay revealed a number of hybridomas secreting antibodies directed towards the complex. Of these, about 1/3 reacted both with the complex and t-PA, whereas about 2/3 reacted only with the complex. Three of the latter hybridomas, producing antibodies directed towards the inhibitor-moiety in the complex have been cloned and the antibodies were studied in detail. PA-inhibitor activity in plasma or serum and t-PA/PA-inhibitor complex could be specifically adsorbed on all three insolubilized monoclonal antibodies (MCI, MC2 and MC3). None of the antibodies seems to be directed against structures of vital importance for the functional activity of the PA-inhibitor. In accordance with this finding the antibody with the highest avidity (MCI) reacts equally well with the PA-inhibitor alone or in complex with t-PA. A radioimmunoassay was devloped with this antibody and significant displacement was obtained with samples with PA-inhibitor concentrations above 2 AU/mL. In 13 plasma samples with different levels of PA-inhibitory activity a significant correlation was obtained when comparing this activity with the PA-inhibitor antigen as measured with the radioimmunoassay (r = 0.88).

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Characterization of Monoclonal Antibodies to Human Tissue-Type Plasminogen Activator: Catalytic Inhibition and One-Two Chain Discriminatory Reactivities

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646894 ◽

1989 ◽

Vol 62 (02) ◽

pp. 742-747 ◽

Cited By ~ 5

Author(s):

Torgny Stigbrand ◽

Lars Frängsmyr ◽

Nils Bergsdorf ◽

Per Wallen

Keyword(s):

Monoclonal Antibodies ◽

Plasminogen Activator ◽

Conformational Epitope ◽

Tissue Type ◽

Type Plasminogen Activator ◽

Tissue Plasminogen ◽

A Chain ◽

The One ◽

Chain Form

SummaryA new set of monoclonal antibodies was generated against the tissue plasminogen activator (t-PA). One of the antibodies, 1:3 C5, was found to be able to distinguish between the one- and two-chain form of t-PA and also exerted significant amidolytic inhibitory activity. Several of the antibodies, as judged from their binding properties in immunosorbent tests, were found to be suitable for immunoaffinity purification purposes, i.e. 1:3C5, 1:3 G5, and 1:2 B9. Three of the mabs, 1:2 B9, 1:3 G5 and 2:2 BIO, were selectively reactive with the A-chain of t-PA, whereas indirect evidences indicated 1:3 C5 to be reactive with a conformational epitope on the B-chain. Three of the antibodies were reactive with porcine t-PA. This new set of antibodies should prove useful for structure-function investigations of t-PA.

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