Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
2000-02-02
2002-07-23
Clark, Deborah J. R. (Department: 1633)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C435S320100, C530S350000, C536S023100, C536S023400
Reexamination Certificate
active
06423316
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the inhibition of blood coagulation, especially during organ rejection.
BACKGROUND TO THE INVENTION
The surgical technique of organ transplantation has now been successfully practised for several decades and, because of its success, the procedure has become widespread and, arguably, routine. However, the supply of suitable transplant organs is not able to match ever-rising demands.
Because of the shortage of suitable human (ie. allogeneic) organs, the possibility of using animal (ie. xenogeneic) organs in human transplant operations (“xenografting” or “xenotransplantation”) has been receiving increased attention in recent years (eg.
Nature
1997; 385:285). Porcine donor organs are thought to be suitable candidates because pigs are anatomically and physiologically similar to humans and are in abundant supply.
Xenografting is currently hindered, however, by the severe and well-documented problems of rejection. This process can be divided into distinct stages, the first of which occurs within minutes of transplantation. This is known as the hyperacute response and is caused by existing antibodies in the recipient which recognise and react with foreign antigens on the endothelial cells (ECs) of the xenograft. This recognition triggers the complement cascade which in turn leads to lysis and death of ECs of the transplant.
This initial hyperacute rejection is then reinforced by the delayed vascular response (also known as acute vascular rejection or delayed xenograft rejection). The lysis and death of ECs during the hyperacute response is accompanied by oedema and the exposure of adventitial cells, which constitutively express tissue factor (TF) on their surface. Tissue factor is thought to be pivotal in the initiation of the in vivo coagulation cascade, and its exposure to plasma triggers the clotting reactions. Thrombin and TNF-&agr; become localised around the damaged tissue and this induces further synthesis and expression of TF by ECs.
The environment around resting ECs does not favour coagulation. Several natural coagulation inhibitors are associated with the extracellular proteoglycans of ECs, such as tissue factor pathway inhibitor, antithrombin III, and thrombomodulin. The recognition of the foreign tissue by xenoreactive natural antibodies (XNAs), however, causes the loss of these molecules.
Together with the exposure and induction of tissue factor, the anticoagulant environment around ECs thus becomes pro-coagulant.
The vascularised regions of the xenograft thus become sites of blood clots, a characteristic of damaged tissue. Blood flow is impaired and the transplanted organ becomes ischaemic. A fuller account of delayed vascular rejection can be found in Bach et al. (1996).
The use of xenogeneic organs in transplants is therefore hindered by an initial hyperacute rejection followed by a prolonged vascular rejection, possibly followed by T-cell mediated rejection. Inhibition of the mechanisms responsible for these rejections could facilitate the use of xenografts.
The simple administration of suitable inhibitors, however, is not a particularly suitable approach. Completely inhibiting complement in a recipient animal is tantamount to immunosuppression, leaving the subject prone to opportunistic infections. Similarly, inhibiting the coagulation cascade in a recipient will leave the animal susceptible to uncontrolled post-operative bleeding. Therefore the inhibitors should desirably be localised in the recipient to the site of the xenograft.
The prevention of hyperacute rejection is the subject of European patent 0495852 (Imutran). To make tissues more suitable for xenografting this patent teaches that they should be associated with homologous complement restriction factors, which prevent the complete activation of complement in the xenogeneic organ recipient.
This approach has been developed and applied in order to produce transgenic animals with organs designed to survive hyperacute rejection (Squinto, 1996). Transgenic mice expressing human CD59, a complement regulator, on cardiac ECs have been produced (Diamond, 1995). The human CD59 retained biological activity and complement was inhibited when transgenic hearts were perfused with human plasma.
Transgenic pigs expressing human DAF and/or CD59 have been reported (McCurry, 1996). Cardiac rejection took twice as long to occur with the transgenic xenografts than with controls.
Inhibiting delayed vascular rejection has not received the same attention, although inhibitors of the coagulation cascade are well known in the art and many have been well characterised.
For instance, tissue factor pathway inhibitor (TFPI) is known to inhibit the function of the active complex which is normally formed between tissue factor, factor VIIa, and factor Xa. TFPI is a 276 residue soluble polypeptide whose positively charged C-terminus binds to heparin sulphate in the proteoglycan layer of ECs. It has been notionally divided into three “Kunitz” domains: Kunitz domain I is responsible for binding tissue factor and factor VIIa; domain II binds factor Xa; but the functions of domain III are less clear (Hamamoto, 1993).
Tick anticoagulant peptide (TAP) is a specific and potent inhibitor of factor Xa. This 60 amino acid polypeptide has been purified from the soft tick
Ornithodoros moubata.
Many snake venoms also contain anticoagulant polypeptides. For instance, a 231 amino acid protein C activator has been purified from the venom of the snake
Agkistrodon contortrix contortrix
(McMullen, 1989; Kisiel, 1987).
Hirudin is the anticoagulant protein utilised by the leech
Hirudo medicinalis
when extracting blood from its victim. It is highly potent and binds to thrombin at a 1:1 ratio with a dissociation constant in the femtomolar range. The active site of thrombin is masked in the stable complex and so the hirudin prevents fibrinogen breakdown, thus inhibiting clot formation.
One possible approach for localising anticoagulants to the site of rejection is to link hirudin to antibodies against E-selectin, which is expressed on the surface of ECs during cell activation. This approach has been shown to be effective in inhibiting clot formation in vitro (Kiely, 1995). Other possible strategies were recently reviewed by Bach et al. (1996).
P-selectin (also known as CD62) is also expressed on the surface of ECs during cell activation. During synthesis it is targeted to secretory storage granules in platelets and endothelial cells by sequences residing in its cytoplasmic domain (Disdier, 1992). In response to cell agonists, such as thrombin, the granules are rapidly redistributed and P-selectin is expressed on the cell surface (Green, 1994).
It is an object of the present invention to provide membrane-bound anticoagulant proteins. These proteins are suitable for inhibiting the clotting cascade at the surface of ECs, thus inhibiting in vivo mechanisms responsible for organ rejection.
It is a further object to provide regulated expression of such molecules on the surface of ECs such that coagulation inhibition occurs locally during conditions of organ rejection. The rejection might be xenogeneic or allogeneic.
It is yet a further object of the invention to provide biological tissue suitable for transplantation, particularly for xenotransplantation.
DESCRIPTION OF THE INVENTION
According to a first aspect of the present invention there is provided a protein comprising a region with anticoagulant activity and a region which can anchor said protein to a cell membrane. Preferably this is a chimeric protein, that is to say the anchor region and anticoagulant region are derived from different proteins.
The anticoagulant region can comprise the sequence of any anticoagulant polypeptide. Examples of such anticoagulant polypeptides include heparin, TAPs, antithrombin, hirudins, and TFPIs, along with their functional derivatives, such as fragments and derivatives which retain anticoagulant activity. Anticoagulant derivatives of thrombin, normally a procoagulant, have also been reported (Dang, 1997).
Preferably the an
Dorling Anthony
George Andrew John Timothy
Lechler Robert Ian
Riesbeck Kristian
Chen Shin-Lin
Clark Deborah J. R.
Darby & Darby
Imperial College Innovative Limited
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