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:
FIELD OF THE INVENTION  
         [0001]    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  
         [0002]    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).  
           [0003]    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  
         [0004]    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.  
           [0005]    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:  
           [0006]    (a) providing plasma from whole blood of a patient having a disorder associated with occurrence of anti-vWF-cp-abs;  
           [0007]    (b) subjecting said plasma to an affinity chromatography using the above-defined adsorbent; and  
           [0008]    (c) collecting the flow-through fraction which is essentially devoid of anti-vWF-cp abs. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0009]    100061 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.  
         [0010]    [0010]FIG. 2 depicts fragments of the recombinant hvWF-cp cloned into the T7 expression vector pRSET-B.  
         [0011]    [0011]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.  
         [0012]    [0012]FIG. 4 shows expression plasmids pICHvWFcp and pICH-SvWFcp containing recombinant hvWF-cp fragments for inducible expression in  Pichia pastoris.   
     
    
     DETAILED DESCRIPTION  
       [0013]    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).  
         [0014]    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.  
         [0015]    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.  
         [0016]    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.  
         [0017]    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).  
         [0018]    In one embodiment of the present invention, the fragments of rhvWF-cp are selected from the group consisting of SEQ ID NOs:1-5.  
         [0019]    In one preferred embodiment of the present invention, the recombinant fragments of vWF-cp can be prepared by expression in  E. coli  as follows:  
         [0020]    Fragments of rhvWF-cp are generated by PCR and cloned into a suitable expression vector such as the vector pRSET-B (Invitrogen).  
         [0021]    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                  
 
         [0022]    The PCR fragments are cut with suitable restriction enzymes such as EcoRI/Xho1 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 Nde1-Xho1 fragment is substituted by the synthetic oligonucleotides o.pRET-FPdHIS(1)-6929 and o.pRSET-FPdHIS(2)-6930 (FIG. 3).  
         [0023]    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.  
         [0024]    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).  
         [0025]    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 Sal1 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.  
         [0026]    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.  
         [0027]    The hvWF-cp or the fragments thereof which either have been prepared using recombinant gene technology, or have been purified form 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.  
         [0028]    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.  
         [0029]    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.  
         [0030]    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 .  
         [0031]    All chromatographic steps are preferably performed at a temperature of about ≦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.  
         [0032]    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-Schonlein purpura, preeclampsia, neonatal thrombocytopenia or hemolytic-uremic syndrome. This includes also medicament-associated formation of anit-vWF-cp-abs in man.  
         [0033]    The present invention will now be further illustrated in the following examples, without being limited thereto.  
       EXAMPLE 1  
     Ligand Coupling  
       [0034]    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  
       [0035]    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  
       [0036]    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.  
         [0037]    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.  
     
       
       
         1 
         
           
             5  
           
           
             1  
             175  
             PRT  
             human  
             
               misc_feature  
               Sequence name FP537 (SEQ ID No. 1)  
             
           
            1 

Arg Ala Ala Gly Gly Ile Leu His Leu Glu Leu Leu Val Ala Val Gly 
1               5                   10                  15 

Pro Asp Val Phe Gln Ala His Gln Glu Asp Thr Glu Arg Tyr Val Leu 
            20                  25                  30 

Thr Asn Leu Asn Ile Gly Ala Glu Leu Leu Arg Asp Pro Ser Leu Gly 
        35                  40                  45 

Ala Gln Phe Arg Val His Leu Val Lys Met Val Ile Leu Thr Glu Pro 
    50                  55                  60 

Glu Gly Ala Pro Asn Ile Thr Ala Asn Leu Thr Ser Ser Leu Leu Ser 
65                  70                  75                  80 

Val Cys Gly Trp Ser Gln Thr Ile Asn Pro Glu Asp Asp Thr Asp Pro 
                85                  90                  95 

Gly His Ala Asp Leu Val Leu Tyr Ile Thr Arg Phe Asp Leu Glu Leu 
            100                 105                 110 

Pro Asp Gly Asn Arg Gln Val Arg Gly Val Thr Gln Leu Gly Gly Ala 
        115                 120                 125 

Cys Ser Pro Thr Trp Ser Cys Leu Ile Thr Glu Asp Thr Gly Phe Asp 
    130                 135                 140 

Leu Gly Val Thr Ile Ala His Glu Ile Gly His Ser Phe Gly Leu Glu 
145                 150                 155                 160 

His Asp Gly Ala Pro Gly Ser Gly Cys Gly Pro Ser Gly His Val 
                165                 170                 175 

 
           
             2  
             359  
             PRT  
             human  
             
               misc_feature  
               Sequence name FP539 (SEQ ID No. 2)  
             
           
            2 

Ser Gly His Val Met Ala Ser Asp Gly Ala Ala Pro Arg Ala Gly Leu 
1               5                   10                  15 

Ala Trp Ser Pro Cys Ser Arg Arg Gln Leu Leu Ser Leu Leu Ser Ala 
            20                  25                  30 

Gly Arg Ala Arg Cys Val Trp Asp Pro Pro Arg Pro Gln Pro Gly Ser 
        35                  40                  45 

Ala Gly His Pro Pro Asp Ala Gln Pro Gly Leu Tyr Tyr Ser Ala Asn 
    50                  55                  60 

Glu Gln Cys Arg Val Ala Phe Gly Pro Lys Ala Val Ala Cys Thr Phe 
65                  70                  75                  80 

Ala Arg Glu His Leu Asp Met Cys Gln Ala Leu Ser Cys His Thr Asp 
                85                  90                  95 

Pro Leu Asp Gln Ser Ser Cys Ser Arg Leu Leu Val Pro Leu Leu Asp 
            100                 105                 110 

Gly Thr Glu Cys Gly Val Glu Lys Trp Cys Ser Lys Gly Arg Cys Arg 
        115                 120                 125 

Ser Leu Val Glu Leu Thr Pro Ile Ala Ala Val His Gly Arg Trp Ser 
    130                 135                 140 

Ser Trp Gly Pro Arg Ser Pro Cys Ser Arg Ser Cys Gly Gly Gly Val 
145                 150                 155                 160 

Val Thr Arg Arg Arg Gln Cys Asn Asn Pro Arg Pro Ala Phe Gly Gly 
                165                 170                 175 

Arg Ala Cys Val Gly Ala Asp Leu Gln Ala Glu Met Cys Asn Thr Gln 
            180                 185                 190 

Ala Cys Glu Lys Thr Gln Leu Glu Phe Met Ser Gln Gln Cys Ala Arg 
        195                 200                 205 

Thr Asp Gly Gln Pro Leu Arg Ser Ser Pro Gly Gly Ala Ser Phe Tyr 
    210                 215                 220 

His Trp Gly Ala Ala Val Pro His Ser Gln Gly Asp Ala Leu Cys Arg 
225                 230                 235                 240 

His Met Cys Arg Ala Ile Gly Glu Ser Phe Ile Met Lys Arg Gly Asp 
                245                 250                 255 

Ser Phe Leu Asp Gly Thr Arg Cys Met Pro Ser Gly Pro Arg Glu Asp 
            260                 265                 270 

Gly Thr Leu Ser Leu Cys Val Ser Gly Ser Cys Arg Thr Phe Gly Cys 
        275                 280                 285 

Asp Gly Arg Met Asp Ser Gln Gln Val Trp Asp Arg Cys Gln Val Cys 
    290                 295                 300 

Gly Gly Asp Asn Ser Thr Cys Ser Pro Arg Lys Gly Ser Phe Thr Ala 
305                 310                 315                 320 

Gly Arg Ala Arg Glu Tyr Val Thr Phe Leu Thr Val Thr Pro Asn Leu 
                325                 330                 335 

Thr Ser Val Tyr Ile Ala Asn His Arg Pro Leu Phe Thr His Leu Ala 
            340                 345                 350 

Val Arg Ile Gly Gly Arg Tyr 
        355 

 
           
             3  
             350  
             PRT  
             human  
             
               misc_feature  
               Sequence name FP541 (SEQ ID No. 3)  
             
           
            3 

Ser Val Tyr Ile Ala Asn His Arg Pro Leu Phe Thr His Leu Ala Val 
1               5                   10                  15 

Arg Ile Gly Gly Arg Tyr Val Val Ala Gly Lys Met Ser Ile Ser Pro 
            20                  25                  30 

Asn Thr Thr Tyr Pro Ser Leu Leu Glu Asp Gly Arg Val Glu Tyr Arg 
        35                  40                  45 

Val Ala Leu Thr Glu Asp Arg Leu Pro Arg Leu Glu Glu Ile Arg Ile 
    50                  55                  60 

Trp Gly Pro Leu Gln Glu Asp Ala Asp Ile Gln Val Tyr Arg Arg Tyr 
65                  70                  75                  80 

Gly Glu Glu Tyr Gly Asn Leu Thr Arg Pro Asp Ile Thr Phe Thr Tyr 
                85                  90                  95 

Phe Gln Pro Lys Pro Arg Gln Ala Trp Val Trp Ala Ala Val Arg Gly 
            100                 105                 110 

Pro Cys Ser Val Ser Cys Gly Ala Gly Leu Arg Trp Val Asn Tyr Ser 
        115                 120                 125 

Cys Leu Asp Gln Ala Arg Lys Glu Leu Val Glu Thr Val Gln Cys Gln 
    130                 135                 140 

Gly Ser Gln Gln Pro Pro Ala Trp Pro Glu Ala Cys Val Leu Glu Pro 
145                 150                 155                 160 

Cys Pro Pro Tyr Trp Ala Val Gly Asp Phe Gly Pro Cys Ser Ala Ser 
                165                 170                 175 

Cys Gly Gly Gly Leu Arg Glu Arg Pro Val Arg Cys Val Glu Ala Gln 
            180                 185                 190 

Gly Ser Leu Leu Lys Thr Leu Pro Pro Ala Arg Cys Arg Ala Gly Ala 
        195                 200                 205 

Gln Gln Pro Ala Val Ala Leu Glu Thr Cys Asn Pro Gln Pro Cys Pro 
    210                 215                 220 

Ala Arg Trp Glu Val Ser Glu Pro Ser Ser Cys Thr Ser Ala Gly Gly 
225                 230                 235                 240 

Ala Gly Leu Ala Leu Glu Asn Glu Thr Cys Val Pro Gly Ala Asp Gly 
                245                 250                 255 

Leu Glu Ala Pro Val Thr Glu Gly Pro Gly Ser Val Asp Glu Lys Leu 
            260                 265                 270 

Pro Ala Pro Glu Pro Cys Val Gly Met Ser Cys Pro Pro Gly Trp Gly 
        275                 280                 285 

His Leu Asp Ala Thr Ser Ala Gly Glu Lys Ala Pro Ser Pro Trp Gly 
    290                 295                 300 

Ser Ile Arg Thr Gly Ala Gln Ala Ala His Val Trp Thr Pro Ala Ala 
305                 310                 315                 320 

Gly Ser Cys Ser Val Ser Cys Gly Arg Gly Leu Met Glu Leu Arg Phe 
                325                 330                 335 

Leu Cys Met Asp Ser Ala Leu Arg Val Pro Val Gln Glu Glu 
            340                 345                 350 

 
           
             4  
             320  
             PRT  
             human  
             
               misc_feature  
               Sequence FP543 (SEQ ID No. 4)  
             
           
            4 

Arg Val Pro Val Gln Glu Glu Leu Cys Gly Leu Ala Ser Lys Pro Gly 
1               5                   10                  15 

Ser Arg Arg Glu Val Cys Gln Ala Val Pro Cys Pro Ala Arg Trp Gln 
            20                  25                  30 

Tyr Lys Leu Ala Ala Cys Ser Val Ser Cys Gly Arg Gly Val Val Arg 
        35                  40                  45 

Arg Ile Leu Tyr Cys Ala Arg Ala His Gly Glu Asp Asp Gly Glu Glu 
    50                  55                  60 

Ile Leu Leu Asp Thr Gln Cys Gln Gly Leu Pro Arg Pro Glu Pro Gln 
65                  70                  75                  80 

Glu Ala Cys Ser Leu Glu Pro Cys Pro Pro Arg Trp Lys Val Met Ser 
                85                  90                  95 

Leu Gly Pro Cys Ser Ala Ser Cys Gly Leu Gly Thr Ala Arg Arg Ser 
            100                 105                 110 

Val Ala Cys Val Gln Leu Asp Gln Gly Gln Asp Val Glu Val Asp Glu 
        115                 120                 125 

Ala Ala Cys Ala Ala Leu Val Arg Pro Glu Ala Ser Val Pro Cys Leu 
    130                 135                 140 

Ile Ala Asp Cys Thr Tyr Arg Trp His Val Gly Thr Trp Met Glu Cys 
145                 150                 155                 160 

Ser Val Ser Cys Gly Asp Gly Ile Gln Arg Arg Arg Asp Thr Cys Leu 
                165                 170                 175 

Gly Pro Gln Ala Gln Ala Pro Val Pro Ala Asp Phe Cys Gln His Leu 
            180                 185                 190 

Pro Lys Pro Val Thr Val Arg Gly Cys Trp Ala Gly Pro Cys Val Gly 
        195                 200                 205 

Gln Gly Thr Pro Ser Leu Val Pro His Glu Glu Ala Ala Ala Pro Gly 
    210                 215                 220 

Arg Thr Thr Ala Thr Pro Ala Gly Ala Ser Leu Glu Trp Ser Gln Ala 
225                 230                 235                 240 

Arg Gly Leu Leu Phe Ser Pro Ala Pro Gln Pro Arg Arg Leu Leu Pro 
                245                 250                 255 

Gly Pro Gln Glu Asn Ser Val Gln Ser Ser Ala Cys Gly Arg Gln His 
            260                 265                 270 

Leu Glu Pro Thr Gly Thr Ile Asp Met Arg Gly Pro Gly Gln Ala Asp 
        275                 280                 285 

Cys Ala Val Ala Ile Gly Arg Pro Leu Gly Glu Val Val Thr Leu Arg 
    290                 295                 300 

Val Leu Glu Ser Ser Leu Asn Cys Ser Ala Gly Asp Met Leu Leu Leu 
305                 310                 315                 320 

 
           
             5  
             188  
             PRT  
             human  
             
               misc_feature  
               Sequence name FP545 (SEQ ID No. 5)  
             
           
            5 

Asp Met Leu Leu Leu Trp Gly Arg Leu Thr Trp Arg Lys Met Cys Arg 
1               5                   10                  15 

Lys Leu Leu Asp Met Ala Phe Ser Ser Lys Thr Asn Thr Leu Val Val 
            20                  25                  30 

Arg Gln Arg Cys Gly Arg Pro Gly Gly Gly Val Leu Leu Arg Tyr Gly 
        35                  40                  45 

Ser Gln Leu Ala Pro Glu Thr Phe Tyr Arg Glu Cys Asp Met Gln Leu 
    50                  55                  60 

Phe Gly Pro Trp Gly Glu Ile Val Ser Pro Ser Leu Ser Pro Ala Thr 
65                  70                  75                  80 

Ser Asn Ala Gly Gly Cys Arg Leu Phe Ile Asn Val Ala Pro His Ala 
                85                  90                  95 

Arg Ile Ala Ile His Ala Leu Ala Thr Asn Met Gly Ala Gly Thr Glu 
            100                 105                 110 

Gly Ala Asn Ala Ser Tyr Ile Leu Ile Arg Asp Thr His Ser Leu Arg 
        115                 120                 125 

Thr Thr Ala Phe His Gly Gln Gln Val Leu Tyr Trp Glu Ser Glu Ser 
    130                 135                 140 

Ser Gln Ala Glu Met Glu Phe Ser Glu Gly Phe Leu Lys Ala Gln Ala 
145                 150                 155                 160 

Ser Leu Arg Gly Gln Tyr Trp Thr Leu Gln Ser Trp Val Pro Glu Met 
                165                 170                 175 

Gln Asp Pro Gln Ser Trp Lys Gly Lys Glu Gly Thr 
            180                 185