GB2270695A - Anti-idiotypic antibodies that activate plasminogen - Google Patents

Abstract

Antibodies which activate plasminogen to plasmin and induce fibrinolysis am disclosed The antibodies are produced by immunising one species of animal with streptokinase and producing primary antibodies against the biologically active region of the protein. These primary antibodies are introduced into a second species to induce anti-idiotypic antibodies, some of which mimic the biological activity of streptokinase. Humanising the anti-idiotypic antibodies results in a product which may be introduced into the human body without inducing an immune reaction.

Description

Antibodies that Activate Plasminogen: The invention relates to antibodies which activate plasminogen and may be used in fibrinolytic and thrombolytic therapy. Fibrinolytic and thrombolytic therapies are of great importance in man, being used to treat occlusive thrombotic diseases, like venous thrombosis, arterial thrombosis, certain forms of cerebrovascular conditions, coronary artery occlusion, and in certain other conditions in which it is desirable to lower circulatory fibrinogen levels.

Under physiological conditions occlusive thrombi are dissolved by the fibrinolytic mechanism. The fibrinolytic mechanism is composed of the interactions of the enzymes tissue type plasminogen activator (t-PA), urokinase (u-PA), and the proenzyme plasminogen, which is converted to the thrombolytic enzyme plasma.

Plasmin digests fibrin and fibrinogen. t-PA and u-PA, and a bacterially derived protein, streptokinase (SK), are all used therapeutically to activate plasminogen and induce thrombolysis. (1, 2, 3) t-PA and u-PA are enzymes which convert plasminogen to plasmin by cleavage of a single peptide bond (Arg - Val), whereas streptokinase is a co-enzyme which has no enzymatic activity of its own but which binds tightly to plasminogen at a number of sites in the molecular structure.

Most of the sites in plasminogen to which SK binds are found in the amino acid sequence Val591 - Asn791, known as the B-chain region. Only a limited amount of SK structure is involved in the B-chain contacts.

When SK binds to human plasminogen the interaction produces a molecular distortion resulting in the exposure of the catalytic residues of plasmin but which adopt a new enzymatic specificity. This new specificity is such that the complex between SK and plasminogen becomes a plasminogen activator. SKplasmin(ogen) complex activator is a most clinically effective agent in the treatment of occlusive thrombosis.

Not all of the streptokinase 414 amino acids that form the protein structure are needed for this process and a portion of the protein structure may be removed without destroying plasminogen activating activity. A minimum essential protein structure must exist for this activity but it is not yet discovered. (4,5) Unfortunately because streptokinase is a bacterial protein it elicits an antigenic response in man. These induced antibodies quickly inactivate streptokinase and limits its clinical usefulness to approximately three days.

Therapeutic induction of antistreptokinase is a contra indication for the subsequent use of therapeutic SK for as long as one to four years. Pre-existing antistreptokinase antibodies, obtained from recent streptococcal infection can also reduce the thrombolytic efficacy of therapeutic SK. As more patients receive a first dose of therapeutic SK an increased proportion of the population are becoming compromised for subsequent SK based therapy. Ideally a nonantigenic SK molecule is desirable. (6) The process of antibody formation involves reaction of antigen with T-cell receptors, multiple gene splicing in the B cells, and synthesis and secretion of intact antibodies (IgG and IgM) with variable binding sites which bind to small regions, or epitopes of the original antigen. These variable antibody binding sites represent a complementary configuration to that found in the antigen.

Thus for SK some of the population of antibodies' combining sites induced by therapeutic administration are complementary in configuration to the structures in SK that are responsible for binding to plasminogen and inducing plasminogen activation activity. (7) Though antibodies from one species to another are grossly similar, small inter species differences of structure are sufficient to induce inter-species immunological responses when infused into the circulation. Part of the inter species amino acid differences (for example between sheep and mice) are generally similar for all classes of IgG, and are common for all IgG molecules in those species. A further origin of variability lies in the variable amino acid sequences of the combining sites directed against the original immunising antigen. (8, 9) In the case of anti-SK IgG raised in one species and injected into another e.g.

sheep to mouse, some of the mouse anti-sheep antibodies will be a complement to the sheep IgG SK binding epitopes. These mouse combining sites are direct images of the SK epitope. In this present case some of these newly generated antibodies express the biological activity of the original SK epitope. In this way antibodies which mimic the activity of SK can be generated. These SK mimic antibodies have a therapeutic potential for inducing plasminogen activation and thrombolysis. Antibodies against antibody combining sites are called antiidiotypic antibodies (7,9).

Unfortunately these fibrinolytic antibodies will suffer some of the limitations described for SK, particularly the induction of human anti-mouse antibodies, for example, and these antibodies may also limit therapeutic efficacy. The choice of mouse is particularly good since mouse plasminogen does not react with streptokinase and large quantitites of monoclonal antibodies can be developed.

To avoid this consequence, the minimum necessary structure for monoclonal mouse antibody combining sites may be determined by cDNA isolation and sequencing and spliced to "humanised" antibodies by the techniques described by Sambrook et al (10,11).

Technical Details Purified streptokinase, isolated from the culture media of Streptococcus equisimilis, or from commercially available Kabikinase (KabiAB) or Streptase (Hoechst) was used to prepare high titer polyclonal antisera in mammalian species and avian species, preferably sheep, mouse, rat and chicken. Or in man following therapeutic intravenous use of SK. Purified streptokinase is emulsified with an adjuvent suitable for the induction of antibodies, like Freunds incomplete adjuvent, or other similar adjuvents for the production of high titre antibodies in the absence of other bacterial proteins. Between 10 - 1000yg quantities are injected at regular intervals, subcutaneously, intradermally or intramuscularly over a period of 30 - 130 days.Or in the case of man, a single therapeutic dose of 15mg streptokinase given intravenously and serum collected between 15 - 130 days later (3, 6, 7).

A small blood sample should be drawn and assessed for the presence of antistreptokinase antibodies using Methods 1. and 2. When the antibody titre is sufficiently high, as indicated by clearly visible immunoprecipitin bands and a neutralising activity some 100 times greater than pre-immune levels then blood is drawn in a practical volume and allowed to clot. The resulting serum removed by centrifugation and the antibody fraction precipitated by the addition of (NH4)2SO4 to final concentration of 65 %. Following redissolution at 2-Smg/ml, aprotinin is added to a final concentration of 20KIU/ml and the specific antistreptokinase antibodies are purified using an affinity column containing immobilised streptokinase prepared at a ratio of 0.1 - 1.Omg/g immobilised phase.

The antibody fraction is passed through the column which is then washed with sufficient phosphate-0.05mo1/l, NaCl-0.2 mol/l aprotinin 20KIU/ml buffer pH7.4 until the absorbance of the effluent is below 0.05 OD units. Bound antistreptokinase antibodies may be displaced by washing the column with acid buffer, e.g. glycine pH2.5, or chaotropic buffer containing 1.5mol/l KSCN at neutral pH, or urea-Smol/l or high salt concentration 14mol/l MgCl2.

The displaced antibodies are immediately pooled, the pH adjusted to pH7 and the antibody fraction desalted into phosphate-saline buffer pH7.4. The preparation is then concentrated to 2 - 4mg/ml using a pressure dialysis concentrator.

Alternatively the antibody fraction may be desalted into a glycine-0.lmol/l buffer, pH3.0 and the antibody digested with a 100* molar ratio of pepsin for 10 hours. The pepsin digested antibody is then gel filtered on a column of Sephadex G100 to separate the FAB fractions. The FAB fraction carries the antibody combining site. The FAB fractions are then checked for their streptokinase reactivity by measuring SK resistance (method 1), and direct binding using method 3 (7).

Monospecific antistreptokinase antibodies or FAB fractions are used to immunise other animal species, preferably those with low biological and fibrinolytic reactivity to streptokinase e.g. mouse, rat, sheep, according to a similar schedule as described for preparing anti-SK antibodies.

Serum is prepared from the hyper-immune donor, and antibody fraction prepared. Purified antibody is then screened by method 4 for the presence of human plasminogen activating activity.

Polyclonal antibody from a larger species, e.g. sheep, may be isolated on immobilised human plasminogen (Glu or Lys). In the event of plasminogen activating activity from mouse or rat due to the small volumes of collectable serum their spleen cells may be fused and cloned by standard techniques for the preparation of monoclonal antibodies. Monoclonal antibodies that mimic streptokinase must be screened by methods 2 and 4 and 5 due to the restrictive nature of the epitope.

Humanised antibodies: SK active antibodies will then need to be sequenced at and around the combining sites, comparison made with streptokinase sequences, and humanised antibodies prepared using synthetic cDNA by the techniques and in the manner described (10, 11).

Example 1: Preparation of sheep anti-streptokinase Antibody: Streptokinase was purified from commercially available Streptase-R as described by (12) and adjusted to a protein concentration of 1.0mg/ml in phosphate 0.05moll, NaCl-0.lSmol/l buffer, pH7.0 using El%lcm value of 9.5 (13). An equal volume of streptokinase was emulsified with Freund's incomplete adjuvent (Sigma Chemical Co., UK) and multiple subcutaneous injections of a total of 100gSK injected on each occasion into Westmoreland sheep on days 0, 10, 21 and at bimonthly intervals thereafter.

Whole blood was drawn on day 28, allowed to clot and the serum separated.

Anti-streptokinase antibody was detected by Methods 1. and 2.

Table 1.

Ratio SK units neutralised/ml of sheep serum Pre-immune neutralising titre 1.0 32 Post-immunisation neutralising titre 1000 3200 Total IgG was precipitated by the addition of dry ammonium sulphate to a final saturation- of 65 % to sheep serum with continuous mixing. The precipitated antibodies were recovered by centrifugation, and redissolved by dialysis against 5L phosphate-0.O5mol/l, NaCl-0. 15mol/1 pH7.0. The dialysed antibodies were adjusted to 1.0mg/ml using El%lcm value of 10.0 and shown to be gamma globulin fraction by agar gel electrophoresis at pH8.6 (14). Aprotinin was added to 20KIU/ml.

Isolation of anti-steptolzinase antibody: Streptokinase was immobilised to cyanogen bromide activated sepharose (15) at a ratio of 0.1 mg/ml settled volume gel.

20ml sheep anti-streptokinase gamma globulin fraction at 2.5mg/ml was applied to a column (15 x 1.5cm) of immobilised streptokinase at a flow rate of 0.3ml/minute. The column was washed with phosphate-0.O5mol/l, NaCl 0.15mol/l buffer pH7.0 until the effluent had an absorbance at 280nm of 0.05.

Bound anti-streptokinase antibodies were eluted by the application of Mg Cl2- 4.0mol/l and collected and immediately diluted ten fold with water. (Fig. 1).

The antibody solution was dialysed against three changes of 5.0L phosphate 0.05mol/l, NaCl-0. 15mol/l buffer pH7.0 for 24 hours and concentrated to 1.0mg/ml using an Amicon pressure concentration fitted with a PM10 membrane.

Monospecific sheep anti-streptokinase antibody was checked by methods 1. and 2. and shown to retain activity for streptokinase.

Preparation of Plasminogen Activating Antibody Rabbits/Sheep: Monospecific anti-streptokinase antibodies were injected into rabbits, rats and mice. For rabbits, the immunisation protocol was the same as that described above for sheep, with injection of 1.0mug IgG per occasion.

Rabbit serum was prepared between day 30 - 45 following immunisation and IgG fraction isolated by chromatography on immobilised protein-A (15). The IgG fraction was dialysed against phosphate-0.05mol/l, NaCl-0.15 molll buffer, pH7.0 and screened for plasminogen activating activity using Methods 4 and5.

Table 2a) OD/h Fibrin-Agar t-PA IU/ml Pre-immune rabbit IgG 0.05 Trace lysis Immune rabbit IgG 0.70 150 No IgG 0.01 Table 2b) DFP-treated IgG OD/h Fibrin-Agar t-PA 1U/ml Pre-immune rabbit IgG 0.01 0 Immune rabbit IgG 0.76 140 Positive plasminogen activating activity was found (Table 2a). To ensure that this was not due to contaminating rabbit plasminogen activator, the IgG fraction was treated with diisoflurophosphate which had little effect on activity (Table 2b).

That the rabbit antibody contained a fraction that reacted with human plasminogen was shown by mixing l25I-labelled human plasminogen with the rabbit IgG fraction in the presence of 6-amino hexanoic acid 0.05moll and applying the mixture to immobilised protein A (Figure 2). The plasminogen was retained by Protein A and displaced by acid pH.

Example 2: Preparation of Plasminogen Activating Antibody: S}=pMouse Monospecific sheep anti-streptokinase antibodies were injected into BALB/C mice. The immunisation protocol was similar to that described in example 1 except that a total of 1.0mug IgG fraction was injected over a period of 28 days.

Serum was collected between days 30 - 45 following immunisation and screened using method 2.

On detection of plasminogen binding antibodies the spleen cells were fused with myeloma P3X63 cells and the standard procedure for the production of monoclonal antibodies followed. Positive antibody producing cell cultures were cloned and clones selected for plasminogen binding antibodies.

Monoclonal antibodies, present in ascites fluid were screened directly for ability to activate human plasminogen by methods 4 and 5.

Table3 OD/h FIbrin Agar t-PA IU/ml Control ascites fluid 0.15 5.0 Positive Antibody clone 0.80 100 DFP-treated ascites 0.50 0 Example 3 Preparation of Plasminogen Activating Antibody Human / Mouse Serum was collected from 10 subjects who had received intravenous streptokinase (15mg) for the treatment of acute myocardial infarction. Serum was collected between 10 and 100 days after infusion and the IgG fraction prepared by chromatography on Protein A (16). All sera showed greatly increased levels of SK neutralizing activity. The IgG fraction was passed through a column of immobilised lysine to remove traces of plasminogen then applied to a column of immobilised SK as described in Example 1. The IgG fraction was applied at a concentration of 2mg/ml and flow rate of 0.3ml/minute.The column was washed with phosphate - 0.05mol/l, NaCl-0.15moltl buffer, pH7.4 and bound antibody displaced with MgCl2-4.0mol/l.

The monospecific human IgG fraction showed antistreptokinase activity using Method 1.

Preparation of Plasminogen Activating Antibody Human / Mouse.

Monoclonal antihuman antibodies that reacted with human plasminogen were prepared as described in Example 2. A clone with plasminogen activating activity was developed as described for Example 2.

Table4.

OD/h Fibrin Agar t-PA IU/ml Control ascites fluid 0.15 5.0 Positive antibody clone 0.40 80 DFP treated ascites 0.4 0 Appendix: Method 1: Strcptokinase resi:ce test: To measure streptokinase neutralization titers, human fibrinogen (Kabi L Flow Laboratories, UK) was dissolved in HEPES-buffered saline, pH 7.4 (HBS), to a concentration of lmg/ml, and a trace label of 125I-labelled human fibrinogen (Amersham International Limited, UK) added to give a final radioactive count of 20,000cpm. ml.

Test sera (O.lml) were incubated in triplicate with streptokinase (O.lml) solutions of varying concentrations from 1-50,OOOiu/ml at 370C for 10 minutes, then mixed with l25I-labelled fibrinogen (0.Sml) and clotted by the immediate addition of 0.3ml bovine thrombin (16.7iu/ml) in CaCl2-16.7mmol. Clotting was observed in every case, and fibrinolysis stopped exactly 10 minutes after the addition of fibrinogen by addition of urea-Smol/l adjusted to pH4.0.

The proportion of solubulized clot radioactivity was determined and expressed as a percentage of the total clot radioactivity present. In every case, at least one of the streptokinase concentrates induced complete lysis within 10 minutes, and from a plot of percent lysis versus streptokinase concentration (iu), the concentration that induced 50% lysis was determined and used for the calculation of the neutralization titer.

Method 2: Immnnoprccjpitation: Briefly 1% agar gel (Indubiose, Sigma Chemicals) is prepared in phosphate 0.O5mol/l, NaCl-O.lSmol/l, buffer pH7.0 containing 20KIU/ml aprotinin to prevent streptokinase degradation. A pattern of six circular wells around one central well is cut in the agar. Streptokinase of varying concentrations from 2.0 0. lmg/ml are placed in the central wells and varying dilutions of serum or IgG fractions placed in the outer wells. The plates are incubated in a humid chamber for 18h and inspected for the presence of immunoprecipitation bands. The limit of detectability of a visible reaction being called the antibody titre. Gels may be soaked to remove non-fixed proteins, dried and stained to form a permanent record.

Method 3: Direct binding of antibody or antibody fragments to streptokinase.

Streptokinase (200y1) at 10yg/ml in NaHCO3 buffer pH9.6 is pipetted into wells of microtitre plates and incubated overnight at 40C. The wells are then thoroughly washed with NaCl-O.lSmol/l containing 0.01% Tween 80. The wells are blotted dry and test samples (200y1) containing IgG or IgG fragments under investigation are added to the wells and incubated for 60 minutes at ambient temperature. The wells are thoroughly re-washed with NaCl-O. 15mop/1, Tween-0.01 % and 100yl of phosphatase conjugated anti-IgG (Sigma Chemicals) added for 60 minutes. The enzyme conjugate must be directed against the IgG species under investigation.The wells are thoroughly re-washed and 200,u1 p Nitrophenyl-phosphate (5mg/ml) added. Anti-streptokinase antibody is detected by development of a yellow colour after 60 minutes incubation at 370C. The level of yellow colour must be substantially greater than control wells containing no added test antibody or no SK coating. The resulting colour is treated as an arbitrary unit of antibody.

Method 4: Activation of human Plasminogen An equal volume (100cm1) of human lys-plasminogen (1004g/ml) is mixed with antibody preparation (varying concentrations) under investigation in the well of a micro-titre plate and incubated at 37"C for 60 minutes. Plasmin is detected by the addition of 200y1 chromogenic substrate (S2251, 2.0mmol/l) and the rate of substrate hydrolysis determined over a 30 minute incubation period at 370C.

Substrate hydrolysis is monitored with a plate reader at 405nm. Catalytic activity is standardised against varying concentrations of plasmin.

Method 5: Fibrin Agar Plate: Are prepared as described (17) except that human fibrinogen containing human plasminogen is used, and calibrated with varying concentrations of standardised t PA from 500 - 1 IU/ml. Varying concentrations of antibody under investigation are added and the plates incubated at 37"C for 18h.

References 1. Marsh, N.A. 1981. Fibrinolysis. John Wiley & Sons (Chichester - New York).

2. Reddy, K. N. 1988. Streptokinase--biochemistry and clinical application. Enzyme 40:79-89.

3. ISIS Steering Committee (Cederholm-Williams).

1987.Intravenous streptokinase given within 0-4 hours of onset of myocardial infarction reduced mortality in ISIS-2.

Lancetl:502.

4. Jackson, K.W., H. Malke, D. Gerlach, J.J. Ferretti, and J.Tang. 1986. Active streptokinase from the cloned gene in streptococcus sanguinis is without the carboxy terminal 32 residues. Biochemistry 25:108-114.

5. Misselwitz, R., R. Kraft, S. Kostka, H. Fabian, K. Welfle, W.Pfeil, H. Welfle, and D. Gerlach. 1992. Limited proteolysis of streptokinase and properties of some fragments.

Int.J.Biol.Macromol 14:107-116.

6. McGrath K, and Patterson R. 1985. Immunology of streptokinase in human subjects. Clin Exp Immunol62:421-426.

7. Kabat, E.A. 1968. Structural Concepts in Immunology and Immuno Chemistry. (Holt, Rinehart and Winston, N.Y.).

8. Greene, M.I., and A. Nisonoff. 1984. The biology of idiotypes. Plenum Publishing Corporation, New York.

9. Bona, C. 1981 Idiotypes and lymphocytes. AcademicPress, New York.

10. Levy, S., E. Mendel, and S. Kov. 1987. A rapid method for cloning and sequencing variable region genes of expressed immunoglobulins. Gene 54:167-173.

11. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989.

Molecular cloning - a laboratory manual. Cold Spring HarbourLaboratory Press.

12. Siefring, G.E., and F.J. Castellino. 1976. Interaction of streptokinase with plasminogen: isolation and characterisation of a streptokinase degradation product. J Biol Chem251:39133920.

13. Taylor, F.B., and J. Botts. 1968. Purification and charactersation of streptokinase with studies of streptokinase activation of plasminogen. Biochemistry 7:232-242.

14. Grabar, P., and P. Burtin. 1964. Immunoelectrophoretic analysis. Elsevier Publishing Company, Amsterdam.

15. Dean, P.D.G., W.S. Johnson, and F.A. Middle. 1991. Affinity chromatography - a practical approach. IRL Press,Oxford.

16. Ansari, A.A., and T.S. Chang. 1983. Immunochemical studies to purify rabbit and chicken immunoglobulin G antibody by protein-A Sepharose chromatography. Am J Vet Res44:901-906.

17. B., Holmstrom. 1965. Streptokinase-assay on large agar diffusion plates. Acta Chem Scand 19:1549-1554.

Claims (9)

1. An antibody or FAB fraction that activates (human) plasminogen.

2. An antibody or FAB fraction that induces fibrinolysis in (human) plasma.

3. An antibody as claimed in claim 1 or claim 2 that is suitable for (human) thrombolytic therapy.

4. An antibody as claimed in any one of claims 1 to 3, whose combining site is related to the structure of streptokinase or tissue-type plasminogen activator or urokinase.

5. An antibody as claimed in any one of claims 1 to 4, which is an anti-idiotype antibody to a streptokinase antibody.

6. An antibody as claimed in any one of claims 1 to 5, that produces no immune sensitisation reaction in humans.

7. A method or making an antibody according to any one of claims 1 to 6, which method comprises the steps of:a) using streptokinase to generate streptokinase antibodies in a first mammalian or avian species and recovering the streptokinase antibodies, b) using the streptokinase antibodies to generate anti-idiotype antibodies in a second mammalian or avian species and recovering the anti-idiotype antibodies, c) screening the anti-idiotype antibodies for activation of (human) plasminogen or induction of fibrinolysis in (human) plasma or other described property.

8. A method as claimed in claim 7, wherein the anti-idiotype antibody is produced by monoclonal antibody technology.

9. A method as claimed in claim 7 or claim 8, wherein the anti-idiotype antibody is produced by recombinant DNA technology.