Cell lines, ligands and antibody fragments for use in pharmaceutical compositions for preventing and treating haemostasis disorders

Abstract

A ligand derived from, e.g., a Fab fragment of a monoclonal antibody obtainable from the cell line deposited with the Belgian Coordinated Collections of Micro-Organisms under accession number LMBP 5108CB binds to the human platelet glycoprotein GPIb and prevents the binding of von Willebrand factor to said GPIb without inducing thrombocytopenia. The said ligand is useful, in admixture with a pharmaceutically acceptable carrier, in a pharmaceutical composition, optionally further comprising a thrombolytic agent, for preventing and / or treating haemostasis disorders.

Description

[0001]The present invention relates to novel cell lines and to ligands, namely human and / or humanized monoclonal antibodies, as well as fragments such as Fab or single variable domains and derivatives and combinations thereof, obtainable from the said cell line. It also relates to pharmaceutical compositions comprising said ligands or antibody fragments and to methods of preventing and treating haemostasis disorders, in particular antithrombotic treatments in humans, by administration of the said ligands or antibody fragments to patients in need thereof. It further relates to a polynucleotide encoding for the antigen-binding Fab fragment of a monoclonal antibody derivable from the said cell line.BACKGROUND OF THE INVENTION[0002]The coagulation of blood involves a cascading series of reactions leading to the formation of fibrin. The coagulation cascade consists of two overlapping pathways required for hemostasis. The intrinsic pathway comprises protein factors present in circulating ...

AI Technical Summary

Benefits of technology

[0016]The essence of this invention is that by using a ligand such as a monovalent Fab fragment of a certain inhibitory human GPIb antibody, a marked prevention of platelet dependent thrombus formation targeted to high shear flow vessels and without incurring thrombocytopenia can be obtained. Moreover, this is so far the only anti-human GPIb monoclonal antibody for which the antithrombotic efficacy has been proven in vivo in an animal thrombosis model.

[0019]Secondly, the present invention is based on in vitro and in vivo studies of the antithrombotic efficacy of the monoclonal antibody, 6B4 (IgG1), raised against human platelet glycoprotein Ib. In vitro, 6B4 potently inhibits the binding of vWF to human GPIb both under static and flow conditions, as further illustrated by the following examples, and it also binds to baboon platelets. When 6B4 was injected into baboons, both the intact monoclonal antibody and its F(ab′)2 fragments caused immediate and severe thrombocytopenia, whereas Fab fragments of 6B4 did not. Furthermore, Fab fragments studied in the two baboon models effectively prevented platelet-dependent arterial thrombosis.

Problems solved by technology

Further it has been speculated that a low dose (30 mg daily) might be more antithrombotic but attempts to identify the optimal dosage have yielded conflicting results.

However, aspirin in any daily dose of 30 mg or higher reduces the risk of major vascular events by 20% at most, which leaves much room for improvement.

However these agents still have significant shortcomings, including the need for large therapeutic doses, limited fibrin specificity, and significant associated bleeding tendency.

However it is known in the art that murine antibodies have characteristics which may severely limit their use in human therapy.

The need for re-administration in therapies of thromboembolic disorders increases the likelihood of such immune reactions.

While the use of human monoclonal antibodies would address this limitation, it has proven difficult to generate large amounts of such antibodies by conventional hybridoma technology.

Further, 4H12 does not inhibit ristocetin- or botrocetin-induced binding of von Willebrand factor to platelet cells, which indicates that this antibody does not prevent von Willebrand factor binding to GPIb.

However, in this particular study the antithrombotic effect of the F(ab′)2 fragments was not further investigated by e.g. testing the fragments in an animal thrombosis model.

Unfortunately, this antibody does not cross react with human platelets and therefore it has no further clinical relevance for human therapy.

Part of this rather surprising lack of in vivo studies is due to the low cross reactivity of the anti-human GPIb monoclonal antibodies with platelets from commonly used laboratory animals.

However, even then attempts to perform in vivo studies are hampered because injection of the anti-GPIb monoclonal antibodies, as well as the snake venom protein echicetin that reacts with GPIb, invariably causes severe thrombocytopenia.

One persistent concern with all available thrombolytic and anti-thrombotic agents, including aspirin, is to induce a risk of overdose and therefore of excessive and life-threatening bleeding.

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