Thrombolytic therapeutics have formerly been used for treatment of thrombotic diseases such as cardiac infarction, arterial embolism and cerebral infarction. At first, streptokinase (hereinafter occasionally referred to as SK) and urokinase (hereinafter occasionally referred to as UK) were clinically employed as useful thrombolytic agents. In particular, UK was relatively frequently employed because of its high fibrinolysis activity. However, UK has the disadvantage that its selectivity for fibrin is low, and that it also acts on fibrinogen resulting in the tendency to cause bleeding in patients who it is given to.
Then, considering such a disadvantage, tissue plasminogen activator (hereinafter occasionally referred to as TPA) and prourokinase (hereinafter occasionally referred to as ProUK) appeared as the second-generation thrombolytic agents. These agents had higher fibrin selectivity than that of UK, and were therefore expected to reduce the bleeding side effect observed in UK. Accordingly, many studies focused on these agents. In particular, mass production of theses agents by recombinant technology has recently begun. In addition, their clinical applications have been extensive (European Cooperative Study Group for Recombinant Tissue-Type Plasminogen Activator: The Lancet, Vol.1, 842 (1985)).
However, these clinical applications have revealed that TPA and related agents have also problems. Namely, (1) the half-life of TPA is very short (2 to 3 minutes), so that it is necessary for thrombolysis that TPA is given in large amounts for a long period of time, and (2) a reduction in bleeding tendency can not always be expected in such therapeutics in which TPA is given in large amounts.
As a result of these problems, more effective thrombolytic agents were studied and developed. These included the preparation of modified TPA and the hybrid protein of TPA and UK or ProUK. As to modified TPA, a TPA mutein partially lacking the sugar chain structure which is considered to be the cause of a reduction in half-life is prepared by genetic engineering techniques to avoid the capture with sugar chain receptors such as hepatocytes, thereby intending to improve the kinetics in blood. For the hybrid protein of UK and TPA, the strong thrombolytic activity of UK is used in combination with the fibrin affinity of TPA, thereby aiming at reducing the dosage. These thrombolytic agents can be expected to decrease the bleeding tendency slightly compared to those known in the art. However, significant improvement depends on future studies and developments.
Then, as the third-generation thrombolytic agents, protein complexes utilizing antibody targeting appeared. Namely, thrombolytic agents that decompose fibrin alone, without decomposing fibrinogen were developed by chemically binding antibodies substantially unreactive to fibrinogen and having high affinity for fibrinogen alone to UK (C. Bode et al., Science 229, 765 (1985)) or TPA (M. S. Runge et al., Proc. Natl. Acad. Sci. USA 84, 7659 (1987)). It has been reported that such antibody targeted thrombolytic agents exhibited the effect 3 to 100 times higher than that of single active ingredient preparations of UK or TPA in each of in vitro and in vivo experiments. In each of these protein complexes, however, an antibody is chemically bound to a thrombolytic enzyme. Therefore, these complexes have the disadvantages that (1) the chemical binding operation is accompanied by a reduction in antibody activity and in enzyme activity, (2) it is difficult to obtain the protein complexes having an antibody-enzyme ratio of 1:1 in good yields, and (3) as a result of protein denaturation, metabolism in the patients who are given the complexes is accelerated or immune response is induced.
Then, a thrombolytic agent specific for fibrin and having no side effects was developed by preparing a bispecific MoAb which could be bound at one binding site thereof to fibrin and at the other binding site thereof to a thrombolytic active substance, and immunologically binding the thrombolytic active substance to this antibody to prepare an immune complex having a bispecific MoAb-thrombolytic active substance ratio of 1:1, which was not accompanied by a reduction in antibody activity and in thrombolytic activity (see Japanese Patent Unexamined Publication No. 2-500321/1990, European Patent Unexamined Publication No. 363712/1990).
In the above-mentioned bispecific MoAb-thrombolytic active substance immune complex, the reactivity to fibrinogen is almost negligible. Hence, the immune complex specifically, efficiently acts on lysis of thrombi formed in vivo, which contain fibrin as a main constituent. However, almost all of the thrombi formed in vivo are accompanied by platelets, and some of them are formed containing a large amount of platelets (in a platelet-rich state).
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