A PREPARATION FOR THE TREATMENT OF HEMOPHILIA A INHIBITOR PATIENTS AND A PROCESS FOR PRODUCING SUCH A PREPARATION

Abstract: A preparation for the treatment of hemophilia A inhibitor patients contains a protein or peptide having a specific Factor VIII:CAg activity of at least 0.5, preferably at least 1 VIII:CAg unit per mg protein, the ratio between the VIII:CAg activity and the VIII:C procoagulant activity being greater than 5:1, preferably greater than 10:1. A fragment of Factor VIII:C, which displays a doublet of a molecular weight of 80/77 kD in electrophoresis, is reactive hemophilia A inhibitor antibodies and has VIII:CAg activity. This fragment and more low-molecular fragments of Factor VIII:C are capable of neutralizing the coagulation inhibiting effect of all tested antibodies. Such fragments can therefore be used as active component in preparations for providing immunotolerance towards Factor VIII:C in high-dose treatment of inhibitor patients. The peptides are moreover useful as an immunosorbent in specific extracorporeal adsorption treatment of inhibitor patients. The inhibitor reactive peptides can e.g. be recovered from plasma fractions by affinity chromatography, hydrophobic interaction chromatography or cation exchange or they may by produced biosynthetically and recovered in a similar manner.

Description: A preparation for the treatment of hemophilia A inhibitor patients and a process for producing such a preparation The present invention relates to a preparation for the treatment of hemophilia A inhibitor patients and processes for producing such a preparation. Hemophilia A is a congenital disease which is due to lack of coagulation Factor VIII.C. This factor is present in blood plasma and can be partially purified from blood. Preparations containing this factor (AHF) can be admini¬ stered to hemophilia A patients so that the patients' blood will be able to coagulate. Production of this type of preparation is described e.g. in the US Patent Specification 3,652,530 and International Application WO 84/03628. In these preparations the Factor VIII.C _. protein typically amounts to 0.1?ό of the total protein "* amount. Factor VIII:C of greater purity can be obtained by affinity chromatography (Zimmerman et al., US Patent Specification 4,361,509, Fass et al. Blood 5__, 394, 1982). The Factor VIII:C protein has still not been fully charac¬ terized, but part of the structure is known (L.W. Hoyer, Blood _5___ > 1, 1981; M. Weinstein et al., Proc. Natl. Acad. Sci., USA ]___- 5137, 1981; G. Kuo et al., Thromb. Haemostas. 5__, 262, 1983). The molecular weight is about 300 kD. It is known that 10 to 20?ό of hemophilia A patients do not only lack Factor VIII:C, but also develop anti¬ bodies against Factor VIII.C. Such patients are called inhibitor patients, and the antibodies in these patients are called inhibitor antibodies because they inhibit the procoagulant activity of Factor VIII:C (H.R. Roberts & R. Cromartie, Progress in Clinical and Biological Research 150, 1, 1984). The presence of these antibodies causes administration of AHF preparation to have no effect since Factor VIII.C is neutralized, and AHF admini¬ stration induces increased antibody level. Inhibitor- antibodies can be used as reagents in immuno- assays to measure Factor VIII:C antigen (VIII.CAg) (B. Dinesen, C. Feddersen, Thromb. Res. Z_l , 707, 1983, 0. Nordfang et al., Thromb. Haemostas. 5TJ* 111, 1983). An VIII:CAg unit is defined as the content in 1 ml of normal human plasma. Inhibitor patients have till now been treated: a) By non-specific treatment with activated prothrombine complex preparations (FEIBA^, Autoplex ^ ). These preparations contain an unknown component (perhaps Factor Vila, see U. Hedner and W. Kisiel, 3 . Clin. Invest. ]_ _, 1837, 1983), which can make plasma coagu¬ late in spite of large inhibitor amounts. However, activated prothrombine complex preparations cannot help all inhibitor patients, and the patients are not cured of their inhibitor. Moreover, the use of this type of preparations involves a great risk of thrombosis . b) It has been found that the inhibitor formation can be suppressed by administration of very large doses of AHF (100 to 200 units/kg daily). This makes it possible to induce immunotolerance of Factor VIII (H.H. Brackman & J. Gormsen, Lancet, p. 933, 1977). After this type of treatment the patient can be treated with AHF preparations like other hemophiliacs. However, high dosis AHF treatment is also non-specific because very large doses of irrelevant protein are admini¬ stered, and the patients cannot utilize the procoagu¬ lant activity in AHF preparations during the treatment At the beginning of the treatment phase the inhibitor level increases, and is then gradually reduced to zero. Bleedings cannot be * prevented with AHF during the treatment, but bleadings can be partly stopped with the above-mentioned FEIBA ^ (factor eight inhibitor bypassing activity) preparations. This treatment is very expensive (typically US $ 600,000 per patient, see S. Stenbjerg et al., Thromb. Res. 3_4 > 533, 1984) and has accordingly found little application. The invention is based on the surprising .finding that a component from plasma fractions having Factor VIII:CAg reactivity and only little or no Factor VIII.C procoagu- lant activity is reactive to 7 of 7 antibodies from inhibitor patients. Therefore, the suppression treatment will be just as effective if a preparation is administered which contains a suitably high amount of VIIIrCAg without significant VIII.C procoagulant activity. Accordingly, the preparation of the invention contains a protein or peptide having a specific Factor VIII:CAg activity of at least 0.5 VIII:CAg U/mg, preferably at least 1 VIIIrCAg U/mg, and is characterized in that the ratio between the Factor VIII:CAg activity and the Factor VIII:C activity is greater than 5:1, preferably greater than 10:1. The preparation is preferably essen¬ tially free of Factor VIIIzC procoagulant activity. In the formulation of the preparation to an injectable solution, the VIII:CAg concentration is usually adjusted to more than 10 U/ml, preferably more than 50 U/ml. Admittedly, it is known that prothrombine complex pre- parations, such as FEIBA (^R-) ** , which can be administered concurrently with the Factor VIII treatment of inhibitor patients, also contains VIII:CAg, cf. Allain et al., Progress in Clinical and Biological Research 150, 99, 1984, but the content is generally of the order of only 4.5 VIII:CAg U/ml. However, the FEIBA preparations also contain significant amounts of Factor VIII:C procoagulant activity (Barrowcliffe et al., Thromb. Res. 2___, 181-186, 1981), which is believed to play an important role for the effect. Barrowcliffe has measured an VIIIrCAg concen¬ tration of 2 U/ml and a Factor VIII.C activity of 1.3 U/ml, corresponding to a ratio of 1.5:1 Prothrombine complex preparations also contain a number of other coagulation factors, in particular Factor II, VII, IX and X as well as possibly Factor Vila, IXa, and Xa (Aronson, Progress in Clinical and Biological Research 150, 243, 1984) which conceivably promote coagu¬ lation of hemophilia A plasma and are therefore also used for stopping bleedings in hemophilia A patients. As stated, the preparation of the invention exhibits a VIII:CAg to VIII:C ratio of at least 5:1, preferably at least 10:1, and is, as mentioned, preferably essen¬ tially free of VIII:C activity. Usually, the preparation is free of other coagulation factors and do not promote coagulation of hemophilia A plasma, cf. Table 1 below. If desired, coagulation factors, such as those mentioned above, can be added to obtain the known additional effects of the factors. The protein with VIIIrCAg reactivity, which is used as an active ingredient in the preparation, may e.g be a Factor VIII:C fragment which in SDS-PAGE exhibits a doublet of an approximate molecular weight of 80/77 kD, or a fragment thereof which can be obtained by throm- bine activation and exhibits a doublet of an approximate molecular weight of 70/67 kD, cf. Kuo et al. loc. cit. It appears from Kuo et al. that the 80/77 kD fragment exhibits VIII:CAg activity against a specific inhibitor antibody (zHI) and no procoagulant activity. However, it could not be expected from this that the fragment, as demonstrated below, is capable of blocking the inhibi¬ tion activity of 7 of 7 inhibitor antibodies. According to the invention, recovery of the 80/77 kD fragment with VIII:CAg reactivity from plasma fractions can take place in several ways, e.g. by affinity chroma¬ tography, hydrophobic interaction chromatography ' or by cation exchange. The starting material can in principle be any VIII :CAg-containing fraction, but is expediently a cryosupernatant or a plasma fraction obtained by preci¬ pitation of redissolved cryoprecipitate with 2 to 6% by weight, preferably about 4?ό by weight of PEG. The latter is particularly expedient because this precipitate is normally not used during the further plasma fractiona- tion . ' The invention is not limited to the use of the 80/77 kD fragments of Factor VlllrC. As further explained below it is shown on Fig. lb that also smaller Factor VlllrC fragments are reactive against inhibitor antibodies. Likewise it may be expected that " other inhibitor anti¬ bodies are reactive against other parts of the Factor VIII:C molecule. Thus Factor VIII.C isolated from blood plasma contains a 92 kD fragment (Zimmermann et al. U.S. Patent No. 4,361,509). To the extent such fragments are capable of blocking inhibitor antibodies such as shown in Table 5 below they are part of the invention. Neither is the invention limited to the use of Factor VlllrC fragments isolated from blood plasma. Based on the DNA sequence of Factor VlllrC partial fragments of the Factor VlllrC gene may be produced. These partial fragments may be inserted in suitable vectors (e.g. plasmids or vira). These vectors may be inserted in suitable host cells (e.g. E. Coli or yeast or CHO, COS or other mammalian cells), rendering the cells capable of producing partial fragments of Factor VlllrC. It may be expected that such fragments are capable of block¬ ing inhibitor antibodies such as shown in table 5 without containing essential amounts of Factor VlllrC procoagulant activity, since they have the same molecular structure as the corresponding peptide sequences isolated from blood plasma. Consequently, such fragments may also be used in preparations according to the- invention. Reactivity of antibodies Several authors (H.P. Muller et al., Blood 5_8, 1000, 1982; B. Sola et al., Proc. Natl. Acad. Sci., USA 79, 1983, 1982) have described production of monoclonal antibodies against Factor VIII.C. These antibodies can be obtained after immunization of mice with Factor VIII by fusing spleen cells with tumor cells, as described by Kohler & Milstein (Nature !____, 495, 1985). Use of this technique has resulted in the production of mono- clonal antibodies against Factor VIII:C, called 42 IgG, 47 IgG and 56 IgG, respectively. Fig. la shows a solid phase immunoisolation with mono¬ clonal antibodies. Plastic beads were coated with antibody against mouse immunoglobulin/monoclonal antibody. After washing the beads were incubated with 125 I labelled Factor VIII:C. Factor VlllrC was produced as described by E. Tuddenham et al., J. Lab. Clin. Med. ____, 40, 1979. After washing the beads were extracted with a SDS sample buffer, and the eluates were run on a SDS gel. The figure shows an autoradiogram of SDS gel: Lane 1: Tracer. ( 125I labelled Factor VlllrC). Lane 2r Normal mouse IgG; Lane 3r 42 IgG; Lane 4r 47 IgG; Lane 5: 56 IgG. It appears from Fig. la that these antibodies bind the 80/77 kD component of Factor VIIIxC (lanes 3, 4, 5). This is the same component as is bound by a human inhi¬ bitor antibody, zHI. Fig. lb shows a solid phase immunoisolation performed like the test in Fig. la with zHI bound to the bead and with a decomposed VIII:C sample. It appears from Fig. lb that other peptides than 80/77 kD doublet with VIII:CAg activity against zHI (70/67 kD and other decom¬ position products) may occur in a decomposed sample. Fig. 2 shows that 7 of 7 inhibitor antibodies are capable of blocking the binding of zHI in an VIII:CAg inhibition assay. Since the zHI antibody binds the 80/77 kD doublet, the other inhibitor antibodies must also have reactivity against this fragment of Factor VlllrC. The assay was carried out as follows: Wells in microtiter plates were coated with zHI immunoglobulin . After washing Factor VlllrC containing plasma was added, and after another washing inhibitor immunoglobulin was added together with peroxidase labelled zHI antibody. Complete binding of peroxidase labelled zHI is obtained by addition of buffer instead of inhibitor immunoglobulin. Affinity chromatography of VIII:CAq It has been found in adsorption of AHF with monoclonal 47 IgG coupled to Sepharose that only the VIII:CAg is absorbed and not the coagulation active Factor VIII:C (Table 1). This is surprising because the antigen is present both on Factor VIII:C and VIII:CAg. However, the antigen appears to be more accessible on the coagula¬ tion inactive VIII:CAg than on VIII:C. TABLE 1 Specific VIIIrCAg adsorption to 47 IgG coupled to Sepharose AHF Unbound EG/NaCl eluate Immunosorbefit VIII rC VIIIrCAg VIII.C VIII :CAq - Vπi:C VIII :CAq 47 IgG/Cl 2B 88 270 90 90 0.32 138 Control Cl 2B 88 270 77 248 0.14 0.09 Table 2 shows that a number of salts can be used together with ethylene glycol (EG) to elute VIII:CAg from immuno- sorbent with monoclonal antibody. l-1gCl 2 , which is sparing- ly soluble in EG, can be used for elution without EG present . TABLE 2 Effect of different salts on ethyleneglycol elution from 56 IgG Sample VIII :CAq units AHF (Nordiocto ® ) 210 Flow through 140 Eluate, 50?. EG/saturated NaCl 22 , 50?. EG/saturated KC1 19 , 50?,. EG/2 M KI 3. ,6 , 50?. EG/2 1-1 CaCl„ 2. . 2 21-1 MgCl 2 22 50?o' EG/saturated NaAc 24 With 56 IgG coupled to sepharose it is possible to purify VIIIrCAg from cryosupernatant.. Cryosupernatant contains 0.4 VIII:CAg U/ml, and less than 1 ppm of the total protein is VIIIrCAg. Table 3 shows that 8000-fold puri¬ fication can be obtained in one step, enabling production of a preparation with a specific VIIIrCAg activity of 61 units VIII:CAg/mg protein. This is a significantly higher specific activity than is found in existing AHF preparations. In the production of high purity (HP) preparations from less purified AHF, i.a. a 4?ό PEG precipitate may occur (U.S. Patent No. 3,652,330, International Application No. W0 84/03628). This precipitate contains VIIIrCAg, and, as appears from Table 3, it is possible to use this precipitate as a starting material for VIII:CAg purification. With just 0.25 ml of im unosorbent it is possible to purify 920 VIIIrCAg units from 140 ml of redissolved 4?ό PEG precipitate. The resulting pre¬ paration has a specific VIIIrCAg activity of 6,900 units V111 :CAg/mg protein. With human inhibitor IgG coupled to Sepharose Cl 2B it is also possible to purify VIIIrCAg from redissolved 4?. PEG precipitate (Table 3). The yield of VIIIrCAg, however, is smaller than in the use of monoclonal anti¬ body, since 50?ό ethylene glycol with 2.51-1 NaCl is not able to quantitatively elute the bound VIIIrCAg. The above tests are described more fully in the following examples 2-4. TABLE 3 Purification of VIIIrCAg from various starting materials by means of 56 IgG and human inhibitor IgG Fraction VIIIrCAg Protein spec . act . mg, 280 units/ g (Ex. 2) Cryosup. , 500 ml 200 30,000 0.007 Cryosup. flow through, 500 ml 100 30,000 0.003 EG/NaCl eluate from 56 IgG, 2 ml 39 0.64 61 (Ex. 3) 4?. PEG prec. , 140 ml 1500 5,200 0.29 458 PEG prec. flow through, 140 ml 340 5,200 0,07 EG/NaCl eluate from 56 IgG, 1 ml 920 0.131 6900 (Ex. 4) 4?. PEG prec. , 110 ml 1000 3,400 0.29 4?_ PEG prec. flow through, 110 ml 340 3,400 0.10 EG/NaCl eluate from human inhibitor, 1 ml 68 0.082 830 The invention also concerns a process for producing a Factor VIIIrCAg preparation for the treatment of hemophilia A inhibitor patients, which is characterized by treating a Factor V111 :CAg-containing solution, e.g. a plasma fraction with an im unosorbent comprising anti¬ bodies which are specific to Factor VIIIrCAg, bound to solid particles, and then desorbing the bound VIIIrCAg by elution with a buffer and processing it to a prepara¬ tion. Processing usually requires change to a physio- logical buffer. This can e.g. be done by drying, dialysis, gel filtration or ion exchange. Human inhibitor antibodies or monoclonal antibodies, preferably 56 IgG or 47 IgG, can be used as the irnmuno- sorbent. Hydrophobic interaction chromatography of VIIIrCAg and cation exchang—e _, » VIIIrCAg can be purified from VIIIrCAg containing solu¬ tions, e.g. plasma fractions without using affinity chromatography with antibodies. Thus, it has been found that 77/80 kD Factor VIIIrCAg is extremely basic and hydrophobic. These properties can be utilized -for recovery of Factor VIIIrCAg from plasma fractions by hydrophobic interaction chromatography and cation exchange, cf. also the following example 5. Hydrophobic chromatography may be performed according to the invention at a pH from 6 to 9.5, but the binding is strongest at a high pH, e.g. pH 8.5. A hydrophobic gel is used, such as phenyl sepharose (Pharmacia). The binding to phenyl sepharose can take place without addition of a salt, but is improved significantly by addition of NaCl. 0.31-1 NaCl addition to the cryosupernatant is suitable since it will not interfere with the later recovery of other plasma proteins, such as albumin and immunoglobulin G. When using a 4?ό PEG precipitate from redissolved cryoprecipitate, higher NaCl concentrations may be employ¬ ed. The bound Factor VIIIrCAg is eluted with a buffer, e.g. under the conditions stated in example 5 below. Cation exchange may be performed according to the invention below 8.0 since this gives the best binding. A suitable value is pH 5.5 since pH in the cryosupernatant can be reduced to this value without impairing VIIIrCAg. A strong cation exchanger may be used, such as "Whatman ®SE53". The bound Factor VIIIrCAg is eluted with a buffer, e.g. under the conditions stated in example 5 below. Both of these methods result in very selective purifica¬ tion of Factor VIIIrCAg, and give a very pure eluate. A particularly expedient method of additional concentra¬ tion of the VIIIrCAg activity consists in combining a hydrophobic chromatography and cation exchange, as described in the foregoing and illustrated in examples 5 and 6 below. In this case a weaker cation exchanger is preferred, e.g. "CM fast flow sepharose". As appears from example 5 and Table 4 these two purifica¬ tion methods utilize various properties in VIIIrCAg and give in combination an 1100-fold purification from the cryosupernatant. Like affinity chromatography, these purification methods can be applied to all VIIIrCAg- containing solutions, e.g. plasma fractions, cf. Table 4, which both shows purification of cryosupernatant and the precipitate from 4?ό PEG precipitation of redis¬ solved cryoprecipitate. TABLE 4 Purification of VIIIrCAg by hydrophobic interaction chromatography and cation exchange Fraction VIIIrCAg Protein spec, act units mg, E 280 units/mg (Ex. 5) Cryosup 1100 160,000 0.007 Flow-through from phenyl-Sepharose 390 160,000 0.002 Eluate from phenyl-Sepharose 440 2,100. 0.21 Flow-through from SE 53 50 2,000 0.04 Eluate from SE 53 300 37.5 8.0 4?. PEG prec. 798 3,000 0.26 Flow-through from phenyl-Sepharose 36 2,400 0.02 Eluate from phenyl-Sepharose 560 83 6.7 Flow-through from SE 53 64 83 0.77 Eluate from SE 53 380 1.83 210 Effect of VIIIrCAg according to the invention In vitro coagulation inhibition experiments indicate that the purified VIIIrCAg according to the invention will also have effect in vivo. In coagulation inhibition assay, the coagulation inhibiting effect of inhibitor IgG on normal plasma is measured. The dilution giving 50?ό inhibition is stated in Bethesda units (C.K. Kasper et al. Thromb. Diath. Haemorr. ____, 860, 1972). Table 5 shows a comparative experiment in which normal plasma was incubated with about 3 Bethesda units of inhibitor IgG from 6 different inhibitor patients and as a control with the zHI IgG previously described. The experiments were conducted with and without presence of a preparation according to the invention containing 100 VIIIrCAg U/ml. It appears from the table that the purified VIII:CAg of the invention has a distinct inhibitor suppressing effect on all the antibodies with respect to the experi¬ ments without VIIIrCAg. Thus, the Bethesda titer of all the antibodies is reduced by more than 25?ό. This shows that the inhibitor effect of all the 7 tested antibodies is due to reactivity against the 80/77 kD ingredient of Factor VlllrC. TABLE 5 Suppression of coagulation inhibition with 80/77-VIII:CAg Inhibitor IgG ?ό VIII:C after incubation % VIII:C after incubation without VIII :CAg with VIII :CAg HZ (zHI) 0 82 AAJJ 66 89 KB 17 61 E 0 71 THL 10 95 KH 5 80 BBEE 66 76 Normal IgG 100 102 The above-mentioned 7 antibodies were deposited on October 30, 1984 at Statens Seruminstitut (the State Serum Institute) of Copenhagen under No. 50-KR-306. Use of VIII:CAg as an immunosorbent It is possible to remove Factor IX inhibitor antibodies from the blood of a Factor IX inhibitor patient by cir¬ culating blood through a column to which Factor IX is coupled, C. Freiburghaus, Thromb. Haemostas. 50, 208, 1983. On the other hand, it is not possible specifically to remove Factor VIII inhibitor antibodies correspondingly This is due to several reasons: 1) The molecular weight of Factor VIII in AHF preparations is usually 1,000 to 20,000 kD, since the preparation also contains Factor VIII:RAg. Therefore, Factor VIII couples very poorly to gels. 2) In coupling a Factor VIII complex, VIII:CAg can be 5 coupled via VIII:RAg or other parts of Factor VIII. Accordingly, VIII:CAg will easily be washed from the coupled gel during use. These circumstances do not apply to VIII:CAg purified 10 as described here. Therefore, VIII:CAg according to the invention bound to a suitably solid matrix, such as a sepharose gel, will be suitable as an immuπosorbent, e.g. in extra- 15 corporeal specific adsorption treatment of Factor VIII inhibitor patients. The production of preparations according to the invention _, ' by the various methods described above* is further illu- 20 strated in the following examples, from which some of the results obtained have already been summarized in the preceding tables. Example 1 25 5 mg of 47 IgG are coupled to 5 ml of Sepharose 4B acti¬ vated with 0.5 g of CNBr. After blocking with 1 M glycine, pH 8.5 and washing with elution buffer cyclus, the gel is incubated overnight with 100 ml of AHF (Nordioctα ^~ * 30 containing 400 VIII:CAg U/ml. The gel is washed on a column with 20 ml of buffer A (20 M i idazole, 10 mM CaCl £ , 0.15 M NaCl, 0.02?.. NaN 3 , pH = 7.35) and 100 ml of a buffer A with 0.5 M NaCl. The gel is eluted with buffer A with 0.5 M NaCl in 50?_ EG. 6 ml of eluate contain 35 7200 units of VIII.CAg. Fig. 3 shows SDS-PAGE of eluted fractions. Evaluated by the colour intensities more 2 % of the protein in the eluate is 80/77 kD protein. Example 2 0.5 mg of 56 IgG are coupled to 0.5 ml of Sepharose 2B/C1. After blocking and washing with elution buffer cyclus the gel is incubated overnight with 500 ml of cryosupernatant (plasma after cryoprecipitation) contain¬ ing 200 VIII:CAg units (Spec, act.: 0.007 VIIIrCAg U/mg). The immunosorbent is isolated from the cryosupernatant by passing the incubation mixture through a column, produced from a 2 ml disposable syringe. The flow through contains 100 VIIIrCAg units. After washing with 2 ml of buffer B (50 mM imidazole, 0.15 M NaCl, 0.02?. NaN 3 , pH = 7.35) and 100 ml of buffer B with 2.5 M NaCl, elution is effected with 2.5 ml of buffer B with 2.5 M NaCl in 50?ό EG. The elua.te contains 39 VIIIrCAg units (Spec. Act. 61 U/mg) _^nd 0.5 unit VlllrC procoagulant activity, cf. also Table 3 above. Example 3 Redissolved cryoprecipitate is absorbed with A1„0, and is precipitated with 4?ό PEG, as described in the Inter- national Application W0 84/03628. The precipitate is redissolved by stirring for 45 min. with 1/4 cryovolume of buffer B with 0.5 M NaCl and 10 M EDTA. Cloudiness is removed by centrifugation , and the redissolved preci¬ pitate containing 1,500 VIIIrCAg units (Spec, act.: 0.29 U/mg) is incubated overnight with 0.25 ml "Sepharose 4B" gel coupled with 5 mg of 56 IgG/ml. The gel is col¬ lected, as described in example 2, and the flow through contains 340 VIII:CAg units. The gel is washed with 3 ml of buffer B and 2 ml of buffer B with. 2.5 M NaCl. Elution is performed with 1.1 ml of buffer B with 2.5M NaCl in 50?ό ethylene glycol. The eluate contains 920 VIII:CAg units, and the specific activity is 6900 U/mg, cf. also Table 3 above. Example 4 A 4?. PEG precipitate from an AHF process containing 1000 VIII:CAg units is redissolved in buffer B with 0.5 M NaCl and 10 mM EDTA. After redissolutioπ , incubation is effected overnight with 0.25 ml of "Sepharose Cl 2B" gel coupled with 10 mg of human inhibitor IgG/ml. The gel is collected, as described in example 2, and the flow through contained 340 VIII:CAg units. The gel is washed and eluted, as described in example 3. The eluate contains 68 VIII:CAg units, and the specific activity is 830 U/mg, cf. also Table 3 above. Example 5 (see table 4J 2.6 1 of cryosupernatant containing 1100 VIII:CAg units are admixed' with'5 mM ethylene glycol-bis-(beta-amiήo- ethylether)-N,N'-tetra acetic acid (EGTA)/0,3 M NaCl, and pH is adjusted to 8.5. 180 πτl of phenyl Sepharose (Pharmacia) are added and incubated for 1 hour. The gel is collected on a column and washed with 400 ml of 5 mM imidazole/0.45 M NaCl, pH 7.4 and eluted with 250 ml of 50?8 ethylene glycol/5 mM imidazole/pH 7.4. The eluate contains 440 units of VIII:CAg with a specific activity of 0.21 U/mg protein. The pH of the eluate is adjusted to 5.5, and 25 ml of cation exchanger of the "Whatman (^R SE 53" type are added and incubated for 30 minutes. The ion exchanger is col¬ lected on a column and washed with 50 ml of 50 mM phos¬ phate/5 mM EGTA/pH 7.4. VIII.CAg is eluted with 35 ml of 1 M NaCl/50 M phosphate/5 mM EGTA/pH 7.4. The eluate contains 300 units of VIII:CAg with a specific activity of 8.0 U/mg protein. Example 6 500 g of 4/°ό PEG precipitate from an AHF process accord¬ ing to International Application WO 84/03628 (from 300 1 of plasma) are redissolved in 3.7 1 of 50 M phosphate/ 0,75 M NaCl/5 M EDTA/pH 8.5. pH is adjusted to 8.5 with 0.5 M NaOH, and, after filtration through filter paper, the solution contains 17,500 VIII:CAg units and 190,000 mg of protein (specific activity' 0.09 U/mg pro¬ tein). The redissolved precipitate is passed through a column with 250 ml of phenyl Sepharose gel with a flow of 3.7 1/h. The phenyl Sepharose is washed with 1.3 1 of 25 M phosphate/5?ά ethylene glycol/pH 7.4 with a flow of 3.7 1/h. 13.500 VIIIrCAg units with 2,000 mg of protein (specific activity 6.8 U/mg protein) are eluted in 1.3 1 of 25 mM phosphate/65?_ ethylene glycol/pH 7.4. The eluate is admixed with NaCl to a concentration of 50 M, and pH is adjusted to 7.0. The eluate is passed through a cation exchanger column with 6.25 ml "CM fast flow Sepharose" (Pharmacia) with a flow rate of 500 ml/h. The column is washed with 60 ml of 10 mM phos¬ phate/50 M NaCl/pH 7.3 at a flow of 500 ml/h. 10,800 VIIIrCAg units with 4.9 mg of protein (specific activity 2,200 U/mg) are eluted in 18 ml of 5 M phosphate/0.5 M NaCl/7 1/2 ?_ saccharose, pH 7.3 (600 VIIIrCAg U/ml). The eluate is admixed with human albumin to a concentra- tion of 0.5?ό, is sterile filtrated and dispensed into 3 bottles containing 6 ml each. After freeze drying the preparation is heat treated at 68°C for 72 hours. Each bottle is dissolved in 18 mg of sterile H„0 and contains 190 VIIIrCAg U/ml. E xamp le 7 VIIIrCAg is produced as described in example 6, except for the use of 50 mM NaHC0 3 0.5 M NaCl, pH 7.3 to elute the CM ion exchanger. pH of the eluate is adjusted to 8.5, and 4800 VIIIrCAg units in 3 ml of eluate are coupled to 1 ml of CNBr activated "Sepharose 4B". The coupled gel is blocked with 1 M glycine pH 8.5. 200 ,ul of VIIIrCAg Sepharose 4B gel was incubated for 2 hours at 37°C with 6.4 ml of plasma from a Factor VIII inhibitor patient. Before incubation, the plasma contained inhibitor antibodies in an amount of 22 BU/ml. After incubation the inhibitor amount was reduced to 1.8 BU/ml. After reactivation with 3 M NH 4 SC the 200 ,ul of VIIIrCAg Sepharose 4B gel were reincubated for 2 hours at 37°C with 6.4 ml of plasma from the same Factor VIII inhibitor patient. In this incubation the inhibitor titer was reduced to 3.5 Bll/ml.

Claims: P A T E N T C L A I M S 1. Preparation for the treatment of hemophilia A inhibitor patients, containing a protein or peptide having a speci- fie Factor VIIIrCAg activity of at least 0.5 U/mg protein, c h a r a c t e r i z e d in that the ratio between the Factor VIIIrCAg activity and the Factor VlllrC pro¬ coagulant activity is greater than 5:1, preferably greater than 10:1. 2. Preparation according to claim 1, c h a r a c t e r i z e d by being essentially free of Factor VlllrC procoagulant activity. 3. Preparation according to claim 1 or ' 2, c h a r a c t e r i z e d by being essentially free of other coagulation factors, e.g. Factor II, VII, IX or X or their activated forms. 4. Preparation according to claim 1 or 2, c h a r a c t e r i z e d in that the protein with Factor VIIIrCAg activity is a fraction of the Factor VlllrC molecule having a molecular weight of 80/77 kD or 70/67 kD, determined by SDS-PAGE. 5. Preparation according to claim 1 or 2, c h a r a c t e r i z e d in that the protein with Factor VIIIrCAg activity is a biosynthetically produced peptide sequence containing the antigenic site(s), the protein being different from Factor VlllrC. 6. Preparation according to any of claims 1 - 5, c h a r a c t e r i z e d in that its Factor VIIIrCAg activity is at least 50 U/ml. 7. A process for producing a preparation according to claim 1 , c h a r a c t e r i z e d by treating a Factor VIIIrCAg- containing solution with an immunosorbent comprising antibodies which are specific to Factor VIIIrCAg, bound to solid particles, and then desorbing the bound VIII:CAg by elution with a buffer and processing it to a prepara¬ tion . 8. A process according to claim 7, c h a r a c t e r i z e d in that the immunosorbent comprises human inhibitor antibodies or monoclonal anti¬ bodies, preferably 56 IgG or 47 IgG. 9. A process for producing a preparation according to claim 1, c h a r a c t e r i z e d by subjecting a Factor VIII rCAg- * containing solution to cation exchange chromatography, followed by processing to a preparation. ** 10. A process according to claim 9, c h a r a c t e r i z e d by using a Factor VIIIrCAg- containing solution which has been purified previously by chromatography on a hydrophobic gel. 11. A process according to any of the claims 7-10, c h a r a c t e r i z e d by using a cryosupernatant plasma fraction as the Factor VIII :CAg-con ' taining solu¬ tion. 12. A process according to any of the claims 7-10, c h a r a c t e r i z e d by using as the Factor VIIIrCAg- containing solution a plasma fraction obtained by preci¬ pitation of a redissolved cryoprecipitate with 2 to 6?_ by weight, preferably about 4?ό by weight of PEG. 13. Use of a protein or peptide with reactivity against Factor VlllrC inhibitor antibodies and having Factor VIIIrCAg activity above about 10 units per ml as the active component in the production of preparations for treating hemophilia A inhibitor patients. 14. Use of a protein with reactivity against Factor VIII inhibitor antibodies and Factor VIIIrCAg activity and exhibiting in SDS PAGE a doublet of an approximate molecular weight of 80/77 kD as the active component in the production of preparations for treating hemophilia A inhibitor patients. 15. Use of a protein or peptide according to claims 1 to 6 or produced according to claims 7 to 12 as immuno- sorbent.

Inventors: NORDFANG OLE, RASMUSSEN MIRELLA EZBAN, NORDFANG, OLE, RASMUSSEN, MIRELLA, EZBAN
Applicants: NORDISK GENTOFTE, NORDISK GENTOFTE A/S
Application year: 1985
Main classification: A61K37
IPC codes: A61K38, C07K14