Abstract:
This invention relates to adsorbents and methods for highly selective removal of anti-von Willebrand Factor-cleaving protease antibodies (“anti-vWF-cp-abs”) from human plasma using human von Willebrand Factor-cleaving protease (“hvWF-cp”) or fragments thereof as affinity ligands. The adsorbents can be used for treating disorders associated with the occurrence of anti-vWF-cp-abs in patients, such as thromboembolic diseases.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a divisional application of U.S. application Ser. No. 10/395,900, filed Mar. 20, 2003, now abandoned, which application is herein incorporated by reference. 
    
    
     FIELD OF THE INVENTION 
     This invention relates to adsorbents and methods for highly selective removal of anti-von Willebrand Factor-cleaving protease antibodies (“anti-vWF-cp-abs”) from human plasma using human von Willebrand Factor-cleaving protease (“hvWF-cp”) or fragments thereof as affinity ligands. The adsorbents can be used for treating disorders associated with the occurrence of anti-vWF-cp-abs in patients, such as thromboembolic diseases. 
     BACKGROUND 
     The von Willebrand Factor-cleaving protease is involved in the limitation of platelet thrombus growth by proteolytic cleavage of von Willebrand Factor multimers in man (Furlan et al., Blood 1996, 87: 4223-4234). Recently, the molecular structure of von Willebrand Factor-cleaving protease and the corresponding gene have been described (WO 02/42441; Zheng et al., J. Biol. Chem. 2001, 276: 41059-41063). 
     A deficiency of von Willebrand Factor-cleaving protease could cause severe disorders such as acquired thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome. One reason for such a deficiency of von Willebrand Factor-cleaving protease is the occurrence of autoantibodies which inhibit the von Willebrand Factor-cleaving protease (Furlan et al., Blood 1998, 91: 2839-2846; Furlan et al., N Engl J Med 1998, 339: 1578-1584). So far, such autoantibodies can be removed from a patient&#39;s plasma by protein A immunadsorption. However, the main disadvantage of protein A immunadsorption is its low selectivity, i.e. essentially no discrimination against immunoglobulin isotypes. 
     SUMMARY 
     An object of the present invention is to provide an improved adsorbent for highly selective removal of anti-vWF-cp-abs from human plasma. The adsorbent comprises hvWF-cp or fragments thereof, coupled to an inert matrix material, said hvWF-cp or fragments thereof having at least one functionally active epitope directed to said anti-vWF-cp-abs under physiological conditions. 
     Another object of the present invention is to provide a method for highly selective removal of anti-vWF-cp-abs from human plasma using the above-defined adsorbent. In particular, the method comprises the steps of:
         (a) providing plasma from whole blood of a patient having a disorder associated with occurrence of anti-vWF-cp-abs;   (b) subjecting said plasma to an affinity chromatography using the above-defined adsorbent; and   (c) collecting the flow-through fraction which is essentially devoid of anti-vWF-cp abs.       

    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic illustration of the immunadsorption procedure of anti-vWF-cp-abs from patient&#39;s plasma using hvWF-cp or fragments thereof as an affinity ligand coupled to a matrix. 
         FIG. 2  depicts fragments of the recombinant hvWF-cp cloned into the T7 expression vector pRSET-B. 
         FIG. 3  shows plasmids for inducible expression of recombinant hvWF-cp in  E. coli . Proteins containing a His 6 tag can be purified on Ni columns. 
         FIG. 4  shows expression plasmids pICHvWFcp and pICH-SvWFcp containing recombinant hvWF-cp fragments for inducible expression in  Pichia pastoris.    
     
    
    
     DETAILED DESCRIPTION 
     A variety of matrix materials can be used in the adsorbent of the present invention as long as it is inert. In this respect the term “inert” means that the matrix material does not have any negative impact on the different fluids used in affinity chromatography, such as human plasma, binding buffers, regeneration buffers, or storage buffers. Specific examples of suitable matrix materials include, but are not limited to, carbohydrates such as cross-linked modified agarose, silicates, glasses, and organic polyreaction products including polymers or copolymers. The matrix material may be present in any form such as spherical, planar or fibrous, and may be porous or non-porous. In certain embodiments of the present invention, the matrix material is present in the form of porous beads. Further, the matrix material is typically biocompatible, and additionally, exhibits substantially no leakage. Examples of solid matrix material for ligand immobilisation include polymers such as agarose (e.g., NHS-, CNBr-, Epoxy-activated Sepherose®, EAH Sepharose®, commercially available from Amersham Pharmacia); Affi-Gel (commercially available form Bio-rad); cellulose (e.g., Cellufine, commercially available from Millipore); polystyrene (e.g., activated Poros Media, commercially available from Perseptive Biosystems); Mono Q; Source; and activated affinity resins (e.g., TSK gel ABA-, Boronate-, Chelate-, and Tresyl-5PW, commercially available from Tosoh Biosep). 
     For coupling the affinity ligand to the matrix material, the affinity ligand can be directly coupled onto the matrix material via a chemical reaction or indirectly coupled onto the matrix material using a spacer. In certain embodiments of the present invention, the affinity ligand is coupled to CnBr-activated Sepharose® 4B as the matrix material. 
     The hvWF-cp or fragments thereof can be obtained using: (i) recombinant gene technology, or (ii) purification procedures starting from e.g., plasma of whole blood, optionally combined with cleaving steps of the purified hvWF-cp from plasma, yielding desired fragments. Preferably, recombinant hvWF-cp (“rhvWF-cp”) or the fragments thereof are used in the adsorbent of the present invention. 
     A number of different vectors can be used for the preparation of rhvWF-cp, including eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein the promoters used in prokaryotic expression vectors include lac, trc, trp, recA, araBAD etc. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET etc., using promoters such as AOX1, GAP, GAL1, AUG1 etc; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc., and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived form viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses, retroviruses etc., using promoters such as CMV, SV40, EF-1α, UbC, RSV, ADV, BPV, β-Ac, etc. 
     The host cells, i.e., prokaryotic or eukaryotic systems, used for expression of rhvWF-cp or fragments thereof depend on the expression vector being introduced into the host cells, and may be bacteria cells (e.g.,  E. coli, B. subtilis ); or eukaryotic cells such as yeast cells, (e.g.,  Pichia  strains); insect cells (e.g. Sf9, Sf 21, High Five, and S2); and mammalian cells (e.g., Vero, MRC5, CHO, COS, 3T3, HEK 293, BHK, SK-Hep, HepG2, CV-1, and Hela). 
     In one embodiment of the present invention, the fragments of rhvWF-cp are selected from the group consisting of SEQ ID NOs:1-5. 
     In one preferred embodiment of the present invention, the recombinant fragments of vWF-cp can be prepared by expression in  E. coli  as follows: 
     Fragments of rhvWF-cp are generated by PCR and cloned into a suitable expression vector such as the vector pRSET-B (Invitrogen). 
     Using the full length cDNA clone disclosed in WO 02/42442 as a template, fragments containing different fragments of the gene of the mature protein are generated by PCR using the following primer combinations: 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 SEQ ID 
                   
               
               
                 Name of primer 
                 Sequence 
                 Orientation 
                 NO: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 pPCR.FURLAN-6699 
                 GCCTTACTCGAGGGCTGCAGGCGGCGGCATCC 
                 DIR 
                 6 
                   
               
               
                   
               
               
                 pPCR.FURLAN-6700 
                 CGAGGAATTCACACGTGTCCGCTGGGGCCG 
                 REV 
                 7 
               
               
                   
               
               
                 pPCR.FURLAN-6740 
                 GCCTTACTCGAGCAGCGGACACGTGATGGCTTC 
                 DIR 
                 8 
               
               
                   
               
               
                 pPCR.FURLAN-6741 
                 CGAGGAATTCAATAGCGCCCTCCGATCCTCAC 
                 REV 
                 9 
               
               
                   
               
               
                 pPCR.FURLAN-6742 
                 GCCTTACTCGATGTCTACATTGCCAACCACA 
                 DIR 
                 10 
               
               
                   
               
               
                 pPCR.FURLAN-6743 
                 CGAGGAATTCACTCTTCCTGGACAGGCACC 
                 REV 
                 11 
               
               
                   
               
               
                 pPCR.FURLAN-6744 
                 GCCTTACTCGAGGGTGCCTGTCCAGGAAGAG 
                 DIR 
                 12 
               
               
                   
               
               
                 pPCR.FURLAN-6745 
                 CGAGGAATTCAAAGCAGCAACATGTCCC 
                 REV 
                 13 
               
               
                   
               
               
                 pPCR.FURLAN-6746 
                 GCCTTACTCGAGGGACATGTTGCTGCTTTGG 
                 DIR 
                 14 
               
               
                   
               
               
                 pPCR.FURLAN-6760 
                 GGCGAATTCTCAGGTTCCTTCCTTTCCCTTC 
                 REV 
                 15 
               
               
                   
               
             
          
         
       
     
     The PCR fragments are cut with suitable restriction enzymes such as EcoRI/Xhol and cloned into the vector such as pRSET-B, cleaved with the same enzymes resulting in the desired plasmids such as pRSET-FP537, pRSET-FP539, pRSETFP541, pRSET-FP543, pRSET-FP545 ( FIG. 2 ). The constructs are cloned in two variants, with or without a HIS-6 tag. In the constructs without a HIS-6 tag, the Ndel-Xhol fragment is substituted by the synthetic oligonucleotides o.pRET-FPdHIS(1)-6929 and o.pRSET-FPdHIS(2)-6930 ( FIG. 3 ). 
     All these plasmids contain the gene of interest under the control of the T7 promoter. The plasmids are transformed into a suitable  E. coli  strain such as JM109. Expression of the protein is induced by infection with, e.g., the M13 mp18/T7 coding for the T7 polymerase under the control of the pLac promoter. The advantage of this system is the extremely tight regulation of expression which is important if the protein is toxic to the cells. 
     Another embodiment is the generation of recombinant rhvWF-cp or fragments thereof by expression in  Pichia pastoris . Here, the complete gene of the rhvWFcp (without propeptide) or fragments thereof are cloned into suitable  Pichia pastoris  expression vectors (e.g., pICHJ-43839), as a Csp451-EcoR1 fragment. Alternatively the gene or fragments are cloned behind the yeast alpha-secretion factor signal of the vector pICH-S 43465 as EcoRI cassettes. The signal sequence allows secretion of the protein into the supernatant ( FIG. 4 ). 
     The plasmids are grown under standard conditions in  E. coli  and transformed into the  Pichia  strain GS115 by electroporation. The plasmids are either integrated into the His-4 locus after linearization with Sall or into the AOX locus after removal of the  E. coli  specific cassette with Not1. Amplification of the inserts is possible by G418 selection. The gene of interest is expressed after induction of the AOX promoter by methanol. 
     In another embodiment of the present invention, the fragments to be used as affinity ligands are peptides having amino acid sequences contained in the hvWF-cp and preferably having a length in the range of from about 6 to 30 amino acids, more preferably from about 6 to about 20 amino acids. One advantage of using such peptides as affinity ligands in the adsorbent of the present invention is the increase in the capacity of the adsorbent. This results in a reduction of time for a patient being subjected a selective removal of anti-vWF-cp-abs from whole blood. 
     The hvWF-cp or the fragments thereof which either have been prepared using recombinant gene technology, or have been purified from plasma of whole blood, have at least one functionally active epitope directed to anti-vWF-cp-abs under physiological conditions. The term “physiological conditions” means an artificial environment which mimics on the one hand the natural environment of the blood circulating system in man, and on the other hand provides an environment suitable for antibody-antigen-binding. In a preferred embodiment, the term “physiological conditions” includes buffer-systems such as PBS (pH 7.3) for carrying out such binding. The term “functionally active epitope” means an antigenic determinant capable of binding one anti-vWF-cp-ab under the above-defined physiological conditions. 
     In the method for selective removal of anti-vWF-cp-abs from human plasma, the provision of plasma from whole blood of a patient having a disorder associated with occurrence of anti-vWF-cp-abs can be carried out by any method known in the art. Examples include plasmaphareses. 
     In step (b) of the method of the present invention, the plasma is typically diluted in binding buffer such as PBS (pH 7.3), and applied to the above-defined adsorbent which is already equilibrated in binding buffer. 
     After step (c), the adsorbent can be regenerated by diluting the bound anti-vWF-cp-abs using a regeneration buffer such as 50% (v/v) ethylene glycol followed by 50 mM glycine/150 mM NaCl (pH 3.0). The adsorbent is then re-equilibrated in storage buffer such as binding buffer containing 0.05% NaN 3 . 
     All chromatographic steps are preferably performed at a temperature of about &lt;room temperature, preferably at about 4° C., with e.g., a linear flow rate ranging from e.g., about 10 to about 60 cm/hrs. 
     The method of the present invention can be used for treating a patient having a disorder associated with occurrence of anti-vWF-cp-abs such as a thromboembolic disease. Examples of thromboembolic diseases include thrombotic thrombocytopenic purpura, Henoch-Schönlein purpura, preeclampsia, neonatal thrombocytopenia or hemolytic-uremic syndrome. This includes also medicament-associated formation of anti-vWF-cp-abs in man. 
     The present invention will now be further illustrated in the following examples, without being limited thereto. 
     Example 1 
     Ligand Coupling 
     The fragment pRSET-FP 537 (SEQ ID NO:1) of rhvWF-CP is recombinantly expressed in  E. coli , IM 109 purified and coupled as affinity ligand to CNBr-activated Sepharose® 4B (Amersham Pharmacia Biotech, Uppsala, Sweden) following the procedure of the suppliers protocol. The resulting affinity matrix (fragment FP 537 (SEQ ID NO:1) of rhvWF-cp/Sepharose® 4B) is used to specifically capture and remove anti-vWF-cp-abs out of human patient plasma, which inhibit endogenous vWF-cp activity. 
     Example 2 
     Affinity Chromatography 
     Human plasma is diluted in binding buffer (PBS pH 7.3) and applied to the equilibrated fragment FP 537 (SEQ ID NO:1) of rhvWF-cp/Sepharose® 4B as affinity matrix to allow selective immunadsorption of vWF-cp-abs to the fragment FP 537 (SEQ ID NO:1) of rhvWF-cp as affinity ligand. The flow-through consisting of the unadsorbed plasma fraction is collected and analyzed for its selective depletion of anti-vWF-cp-abs. Captured antibodies are recovered by 50% ethylene glycol elution following 50 mM glycine/150 mM NaCl, pH 3.0 for complete regeneration of the affinity matrix. After re-equilibration with binding buffer the column is stored in binding buffer containing 0.05% NaN 3 . All chromatographic steps are performed at 4° C. with a linear flow rate of 10-60 cm/hrs. The collected fractions are analyzed for vWF-cp activity inhibiting antibodies to monitor the efficiency of the immunadsorption. 
     Example 3 
     Assay of vWF-cp Activity in the Presence of Inhibiting Antibodies 
     The activity of vWF-cp after incubation with samples containing vWF-cp activity inhibiting antibodies and fractions thereof collected following the immunadsorption is determined at low ionic strength and in the presence of urea as described in Furlan et al., 1996, supra. A pool of normal human plasma (NHP, Baxter AG) is incubated with the samples for 10 minutes at 37° C. in various ratios in a total volume of 10 μl. The mixture is then diluted to 100 μl in 0.15 M NaCl, 10 mM Tris, pH 7.4, 1 mM Pefabloc SC containing 10 mM BaCl 2  and incubated for 5 minutes at 37° C. The incubation mixture is immediately added to 50 μl of plasma-derived vWF (50 μg/ml) (Stago, Parsippany, N.J.) and transferred onto circular dialysis membranes (VSWP, 25 mm diameter, Millipore) floating on 50 ml 1.5 M urea/5 mM Tris, pH 8.0. Overnight incubation at 37° C. is stopped by the addition of 10 μl 0.2 M EDTA, pH 7.4. Dilutions of NHP without previous incubation with inhibiting antibodies is used for vWF-cp activity calibration. For the analysis of vWF multimers approximately 25 ng vWF per lane was subjected to non-reducing SDS-1% agarose gels and detected with rabbit anti-vWF antibodies (Dakopatts, Copenhagen, Denmark) and alkaline phosphatase conjugated goat anti-rabbit IgG antibodies (Promega, Madison, Wis.). The procedural steps of this example are illustrated in  FIG. 1 . 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application were specifically and individually indicated to be so incorporated by reference.