Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: Inhibitory effects on receptormediated uPA activity in vitro and in vivo

Jesper Pass; Annika Jögi; Ida K. Lund; Birgitte Rønø; Morten G. Rasch; Henrik Gårdsvoll; Leif R. Lund; Michael Ploug; John Rømer; Keld Danø; Gunilla Høyer-Hansen

doi:10.1160/TH06-11-0644

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2007; 97(06): 1013-1022
DOI: 10.1160/TH06-11-0644

Cellular Proteolysis and Oncology

Schattauer GmbH

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: Inhibitory effects on receptormediated uPA activity in vitro and in vivo

Jesper Pass

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

²Present address: Novo Nordisk A/S, Novo Nordisk Park A9.1. 28, DK-2760 Måløv, Denmark

,

Annika Jögi

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Ida K. Lund

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Birgitte Rønø

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Morten G. Rasch

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Henrik Gårdsvoll

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Leif R. Lund

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Michael Ploug

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

John Rømer

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Keld Danø

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

,

Gunilla Høyer-Hansen

¹Finsen Laboratory, Copenhagen Biocenter, Copenhagen N, Denmark

› Author Affiliations

Abstract

Summary

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC50 value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, a ~50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.

Keywords

Plasminogen activation - uPAR knock-out mice - mouse antimouse mAbs - inhibitors of uPA-uPAR interaction - anthrax toxin assay

Full Text

References

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