RECOMBINANT ALPHA-GALACTOSIDASE, A THERAPY FOR FABRY DISEASE

Abstract: Fabry disease results from an X-linked deficiency in the enzyme alpha -galactosidase A. The present invention is directed to recombinant human alpha -galactosidase A and provides baculovirus expression vectors and recombinant virus that provide stable expression of extracellular and intracellular levels of this enzyme in an insect cell culture. The recombinant-derived enzyme can be used in enzyme replacement therapy to treat Fabry patients. Composition useful in therapeutic administration of alpha -galactosidase A are also provided.

Description: -i- RECOMBINANT ALPHA-GALACTOSIDASE A THERAPY FOR FABRY DISEASE Fabry disease results from an X-linked deficiency in the enzyme α-galactosidase A. The present invention is directed to recombinant human α-galactosidase A and provides baculovirus expression vectors and recombinant virus that provide stable expression of extracellular and intracellular levels of this enzyme in an insect cell culture. The recombinant-derived enzyme can be used in enzyme replacement therapy to treat Fabry patients. Compositions useful in therapeutic administration of α-galactosidase A are also provided. Fabry disease is an X-linked inborn error of metabolism resulting from a deficiency of the lysosomal enzyme, α-galactosidase A. Deficiency of α-galactosidase A results in the accumulation of its major glycosphingolipid substrate, globotriaosylceramide and related glycolipids with terminal α-galactosidic linkages. Progressive substrate deposition, especially in the plasma and vascular endothelium, leads to ischemia and infarction with early demise due to vascular disease of the heart and kidney. Fabry diseases is one of approximately 30 lysosomal storage diseases known to affect humans. Each of these diseases result from an inherited trait which affects the levels of enzymes in the lyosome. Tay-Sach's disease and Gauucher disease are members of this group of diseases. Since specific pathways for the uptake of these other lysosomal enzymes also exist, enzyme replacement therapy is expected to be effective in Fabry disease and could logically be expected to be successful in these other diseases as well. 1 Although these diseases are individually rare, (e.g., several thousand patients with Fabry disease are known to occur world wide, i.e., 1 to 40,000), as a group this class of diseases t- accounts for a significant fraction of all inherited diseases. Several lines of evidence suggest that enzyme replacement therapy may be beneficial for patients with Fabry disease. For example, it has been demonstrated in cell - j _o cultures of fibroblasts obtained from patients with this disease that enzyme present in the culture medium is specifically transported to lysosomes. Clinical trials of enzyme replacement therapy have been reported for patients with Fabry disease using infusions of normal plasma (Mapes 15 et al. , 1970, Science 169: 987-989); α-galactosidase A purified from placenta (Brady et al. , 1973, New Eng. J. Med. 279; 1163); or α-galactosidase A purified from spleen or plasma (Desnick et al. , 1979, Proc. Natl. Acad. Sci. USA 76; 5326-5330). In one study (Desnick e_t al. ) intravenous o injection of purified enzyme resulted in a transient reduction in the plasma levels of the substrate, globtriaosylceramide. However, due to the limited availability of the human enzyme obtained from human sources, insufficient quantities were available for further study. 25 The α-galactosidase A enzyme is a lysosomal enzyme which hydrolyzes globotriaosylceramide and related glycolipids which have terminal α-galactosidase linkages. It is a 45 kDa N-glycosylated protein encoded on the long arm of the X chromosome. The initial glycosylated forms (Mr = go 55,000 to 58,000) synthesized in human fibroblasts or Chang liver cells are processed to a mature glycosylated form (Mr = 35 1 50,000). The mature active enzyme as purified from human tissues and plasma is a homodi er. (Bishop et al. , 1986, Proc. Natl. Acad. Sci. USA 8_3: 4859-4863). c A human liver cDNA for α-galactosidase A was identified in a gtll expression library (Calhoun et al. , 1985, Proc. Natl. Acad. Sci. USA 8.2: 7364-7368), and its sequence reported by Bishop et al. The original cDNA isolated by Calhoun et al. encoded the mature amino acid 10 sequence of α-galactosidase A but did not contain the complete signal peptide sequence of the precursor form. The partial cDNA clone was used to construct an E. coli expression vector by placing the α-galactosidase A coding sequence under control of the trp promoter - c (Hantzopoulos et al. , 1987, Gene 57: 159-169). The level of expression of active enzyme was sufficient to support E. coli. growth using an α-galactoside substrate as the sole carbon source; however, increased levels of the 45 kDa protein were not detected in Coomassie blue-stained gels upon on trp promoter induction nor was biological activity detected in vitro. A genomic clone was later isolated which carried the promoter and first exon of the protein including the full signal peptide (Quinn et al. , 1987, Gene 58; 177-188). ry Clone of full length cDNAs of a precursor α-galactosidase A from human fibroblasts were reported (Tsuji et al. , 1987, Eur. J. Biochem. 165: 275-280) and used to obtain transient expression of the enzyme in monkey COS cells. The level of enzyme activity reported was only about 40% above background. 5Q Hence one long standing need of the prior art is to provide large quantities of active human α-galactosidase A, especially for use in enzyme replacement therapy. To achieve 35 this goal, a full length cDNA of human α-galactosidase A is needed which can be incorporated into an expression vector under control of a strong promoter. Furthermore, this vector r- should provide stable expression of the cDNA and use a host system in which the processing and glycosylation may occur. Finally, biologically active enzyme must be produced. One such expression vector is provided by the baculovirus expression system of the present invention. ^ Q Baculoviruses infect Lepidopterm insects and have proven useful as recombinant expression vectors (Smith et al. , 1983, Mol. Cell. Biol. J3 J 2156-2165; Luckow et al. , 1988, Bio./ Technology. 6. :47-55). The latter of these two references provides a detailed description of the available c - baculovirus vectors, methodology for their use and a list of proteins which have been expressed in this system. The particular advantage of baculovirus expression systems are very high levels of production (1 mg to 500 g of protein per liter of culture have been reported) , glycosylation and 2o processing of the so-produced proteins. The early baculovirus expression vectors employ a strong promoter for a nonessential gene, the polyhedrin gene. To facilitate cloning, a DNA sequence encoding several restriction endonuclease sites had been inserted into the polyhedrin 2 c promoter (Luckow, et al. , 1988, Bio/Technology :47-55). Subsequently, it was discovered that this genetically engineered promoter was less effective (2-1000 fold) than the wild type polyhedrin promoter (Page, 1987, Nuc. Acids Res. 17 454; Ooi et al. , 1989, J. Molec. Biol. 210 " 721-736). The go present invention thus employs both the genetically altered polyhedrin promoter and the wild type polyhedrin promoter to generate expression systems providing large amounts of active α-galactosidase A. 35 The present invention is directed to replicable expression vectors that express the human α-galactosidase A enzyme. These vectors are baculovirus derivatives which have c been genetically engineered to contain a full length cDNA or recombinant DNA encoding the precursor form of α-galactosidase A. The cDNA, for example, is operably linked to -the baculovirus polyhedrin promoter so that the promoter directs expression of biologically active, human 0 α-galactosidase A under the appropriate conditions. This promoter can be either a genetically engineered polyhedrin promoter with an insertion of DNA encoding several restriction endonuclease sites or the wild type polyhedrin promoter. Transformant microorganisms or cell cultures j c; carrying these vectors are also provided by the present invention. Another aspect of the present invention provides recombinant baculoviruses having a gene sequence for the precursor form of a human α-galactosidase A enzyme and which 2o express high levels of said enzyme. A further aspect of this invention provides homogenous recombinant human α-galactosidase A and antibodies directed against this enzyme or directed against any active fragment or derivative of the enzyme. 25 A still further aspect of the present invention provides pharmaceutical compositions comprising an effective amount of recombinant human α-galactosidase A, or an active derivative thereof, and a pharmaceutically acceptable carrier. These compositions are useful in treating diseases go associated with a deficiency of this enzyme, especially Fabry disease. 35 Fig. 1 depicts the human α-galactosidase cDNA features encoded on M13.L21.1 (top), the restriction enzyme recognition sites in the polylinker region of pSPRl (middle) t - and a partial restriction map and some of the features of pAc373, a baculovirus transfer vector (bottom) . Fig. 2 depicts the differences in DNA sequence between transfer vectors pCC4 (top) and pCC14 (bottom). Elimination of 93 bp from the promoter sequences of pCC4 0 (underlined) and replacement with 7bp (underlined) in pCC14 increases α-galactosidase A protein expression at least 2-10 fold. The ATG start codon (box) is indicated in both sequences. The present invention is directed to replicable p expression vectors which are used to express human α-galactosidase A. These vectors are recombinant baculoviruses having an α-galactosidase A cDNA under control of the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter or any other strong baculovirus o promoter capable of expressing foreign genes. An insect cell culture, for example Spodoptera frugiperda line Sf9, infected with this recombinant virus expresses α-galactosidase A both intracellularly and extracellularly at unexpected levels. Baculovirus expression vectors and their use are reviewed in Luckow et al. The baculoviruses have double-stranded circular DNA genomes approximately 130 kb in length. These viruses have a wide host range infecting about 30 species of Lepidopterm insects. In particular, AcMNPV infection of Spodoptera frugiperda cell cultures produce very o high levels of a protein known as polyhedrin which may account for 50% of the cellular protein. Polyhedrin is a 5 1 nonessential viral gene which can be replaced by a foreign gene in order to express that gene under control of the strong polyhedrin gene promoter. The recombinant viruses are r- then used to infect a S . frugiperda cell culture to produce the desired protein. A particular advantage of this system is its similarity to higher eukaryotes with regard to protein modification, processing and transport. Thus, recombinant- derived eukaryotic proteins will be processed and 0 glycosylated in a manner important for biological activity. To produce a recombinant baculovirus which expresses a foreign gene, that gene is inserted into a transfer vector downstream of the polyhedrin promoter. The transfer vector is a plasmid which has been genetically -^ r- engineered to contain DNA flanking the polyhedrin gene as well as convenient restriction enzyme recognition sites adjacent to the polyhedrin promoter or the polyhedrin gene initiation codon. Alternatively, the engineered polyhedrin promoter can be mainpulated, e.g. its sequences changed to 20 the original wild type or other expression-effecting sequence, to further increase expression levels. The transfer vector and viral DNA are used to co-transfect insect cell cultures. Homologous recombination will occur between the transfer vector and viral DNA during transfection and the 25 native polyhedrin gene is replaced by the engineered polyhedrin promoter and foreign gene on the transfer vector. The viral progeny are then screened for occlusion negative (Occ~) virus, the phenotypic characteristic for loss of the polyhedrin gene. Occlusions are particles that appear in the o Q cytoplasm of an infected cell and consist of virus particles embedded in a crystalline lattice of the polyhedrin protein. 35 1 The recombinant baculovirus expression vectors of the present invention are made by inserting a cDNA or a recombinant DNA for the precursor form of human α-galactosidase A downstream of the polyhedrin promoter in an baculovirus transfer vector, for example the AcMNPV transfer vector pAc373. The precursor form consists of the signal peptide and the mature α-galactosidase A. This transfer vector and many other suitable vectors are described in 10 Luckow et al. In pAc373 there is a unique BamHI site between the polyhedrin promoter and coding region into which a DNA fragment containing the α-galactosidase A gene may be cloned. A DNA fragment encoding the full α-galactosidase A coding sequence including the complete signal peptide is -, -- found on the EcoRI fragment from M13.L21.1. Construction of this M13 derivative is described in Hantzopoulos (1987, Ph.D. Thesis). To put BamHI ends on this EcoRI fragment, the fragment is cloned into the polylinker of pSPRl (Krystal et al. (1986) Proc. Natl. Acad. Sci. USA 83.: 2709-2713). 2o Because the pSPRl polylinker is composed of two smaller polylinkers cloned head to head at a unique EcoRI site, it provides a convenient way to introduce a variety of restriction enzyme sites on the ends of any EcoRI fragment. The BamHI fragment of the pSPRl derivative containing the 25 α-galactosidase A gene is then cloned into the unique BamHI site in pAc373. The resulting transfer vector, designated pCC4, contains the complete α-galactosidase A cDNA in the proper orientation relative to the polyhedrin promoter. To increase expression of α-galactosidase A the g Q genetically engineered polyhedrin promoter in pCC4 was replaced by the wild type polyhedrin promoter (Ooi, e_t al. , 35 1989, J. Molec. Biol. 210; 721-736). pCC4 DNA was cut with EcoRV and BssHII restriction endonucleases and the 213 bp EcoRV/BssHII fragment was discarded. This deletes 178 bp c upstream- and 35 bp downstream of the α-galactosidase A ATG start codon of pCC4 (See Figure 2). To reconstruct the wild type polyhedrin promoter, and to replace the portion of the coding region lost by this deletion, 4 overlapping oligonucleotides were synthesized, annealed together and 10 ligated into the EcoRV/BssHII cut pCC4 DNA. This new α-galactosidase A transfer vector is called pCC14. The pCC14 DNA sequences which replace the deleted DNA sequences in pCC4 are shown in Fig. 2 (differences in DNA sequence are underlined). These manipulations do not alter the ATG start , c codon or the coding region. The only alteration is in the length an sequence of DNA lying between the transcription start signals and the ATG start codon (7 bp in pCC14 replace 93 bp in pCC4). The DNA sequence of the pCC14 promoter was confirmed by DNA sequencing. Protein expression from pCC14 20 can increase up to 1000-fold for α-galactosidase A, relative to expression from pCC4. To prepare a recombinant baculovirus derivative carrying the human α-galactosidase A gene as a cDNA, a transfer vector carrying that DNA, for example pCC4 or pCC14, -y e - and AcMNPV DNA, are used to cotransfect an insect cell culture line, preferably £. frugiperda cell line Sf9. The transfer vector and viral DNA undergo recombination between the homologous baculovirus sequences which flank the α-galactosidase A gene and thereby produce the desired go recombinant baculovirus. To identify this recombinant baculovirus, the viral progeny from the transfection are 35 1 plated on Sf9 cells and the plaques are screened to find Occ " viruses. Methods for distinguishing Occ plaques from Occ plaques are described in Summers et al . (1987) A Manual of r- Methods for Baculovirus Vectors and Insect Cell Culture D — Procedure, Texas Agricultural Experiment Station, Bulletin No. 1555. In addition, this publication provides protocols for all of the above described manipulations and accordingly is incorporated herein by reference, 0 The human α-galactosidase A cDNA encoding the precursor form of this polypeptide has the following nucleotide sequence and amino acid sequence: 0 5 0 5 ATG Met CAG CTG AGG AAC CCA GAA CTA CAT CTG GGC TGC GCG CTT GCG CTT -45 Glu Leu Arg Lys Pro Glu Leu His Leu Gly Lys Ala Leu Ala Leu -15 CGC TTC CTG GCC CTC GTT TCC TGG GAC ATC CCT GGG GCT AGA GCA -1 Arg Phe Leu Ala Leu Val Gly Trp Asp lie Pro Gly Ala Arg Ala -1 CTG GAC AAT GGA TTG GCA AGG ACG CCT ACC ATG GGC TGG CTG CAC 45 Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His 15 TGG GAG CGC TTC ATG TGC AAC CTT GAC TGC CAG CAA GAG CCA GAT 90 Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp 30 Ser Arg TCC TGC ATC AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG 135 Ser Cys lie Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met 45 Ser GTC TCA GAA GGC TGG AAG GAT GCA GGT TAT GAG TAC CTC TGC ATT 180 Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys lie 60 GAT GAC TGT TGG ATG GCT CCC CAA AGA GAT TCA GAA GGC AGA CTT 225 Asp Asp Cys Trp Met Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu 75 CAG GCA GAC CCT CAG CGC TTT CCT CAT GGG ATT CGC CAG CTA GCT 270 Gin Ala Asp Pro Gin Arg Phe Pro His Gly lie Arg Gin Leu Ala 90 AAT TAT GTT CAC AGC AAA GGA CTG AAG CTA GGG ATT TAT GCA GAT 315 Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly lie Tyr Ala Asp 105 GTT GGA AAT AAA ACC TGC GCA GGC TTC CCT GGG AGT TTT GGA TAC 360 Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr 120 TAC GAC ATT GAT GCC CAG ACC TTT GCT GAC TGG GGA GTA GAT CTG 405 Tyr Asp lie Asp Ala Gin Thr Phe Ala Asp Trp Gly Val Asp Leu 135 CTA AAA TTT GAT GGT TGT TAC TGT GAC AGT TTG GAA AAT TTG GCA 450 Leu Lys Phe Asp Gly Cys yr Cys Asp Ser Leu Glu Asn Leu Ala 150 GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AGG ACT GGC AGA 495 Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg 165 AGC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT ATG TGG CCC TTT 540 Ser lie Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe 180 CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TGC AAT CAC TGG 585 Gin Lys Pro Asn Tyr Thr Glu lie Arg Gin Tyr Cys Asn His Trp 195 CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA AAG AGT 630 Arg Asn Phe Ala Asp lie Asp Asp Ser Trp Lys Ser lie Lys Ser 210 Asn SUBSTITUTE SHEET -ll/I- ATC TTG GAC TGG He Leu Asp Trp A C A T C T TTT AAC CAG G A G A G A Thr ser Phe Asn Gin Glu Arg ATT GTT GAT GTT 675 He Val Asp Val 225 GCT GGA CCA GGG Ala Gly Pro Gly GG T T G G AAT GAC CCA G AT AT G G ly Trp Asn Asp Pro Asp Met TTA GTG ATT GGC 720 Leu Val He Gly 240 AAC TTT GGC CTC Asn Phe A G C T G G AAT CAG CAA GTA ACT Gly Leu S er Trp Asn Gin Gin Val Thr C AG ATG GCC CTC 765 G in Met Ala Leu 255 TGG GCT ATC ATG Trp Ala G CT G CT CCT TTA TTC AT G T C T He Met Ala Ala Pro Leu Phe Met S er AAT GAC CTC CGA 810 Asn Asp Leu Arg 270 Ala CAC ATC AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA 855 His He Ser Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val 285 Arg ATT GCC ATC AAT CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT 900 He Ala He Asn Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu 300 Glu AGA CAG GGA GAC AAC TTT GAA GTG TGG GAA CGA CCT CTC TCA GGC 945 Arg Gin Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly 315 Leu Gly Ser Lys TTA GCC TGG GCT GTA GCT ATG ATA AAC CGG CAG GAG ATT GGT GGA 990 Leu Ala Trp Ala Val Ala Met He Asn Arg Gin Glu He Gly Gly 330 CCT CGC TCT TAT ACC ATC GCA GTT GCT TCC CTG GGT AAA GGA GTG 1035 pro Arg Ser Tyr Thr He Ala Val Ala Ser Leu Gly Lys Gly Val 345 GCC TGT AAT CCT GCC TGC TTC ATC ACA CAG CTC CTC CCT GTG AAA 1080 Ala Cys Asn Pro Ala Cys Phe He Thr Gin Leu Leu Pro Val Lys 360 AGG AAG CTA GGG TTC TAT GAA TGG ACT TCA AGG TTA AGA AGT CAC 1125 Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His 375 ATA AAT CCC ACA GGC ACT GTT TTG CTT CAG CTA GAA AAT ACA ATG 1170 He Asn Pro Thr Gly Thr Val Leu Leu Gin Leu Glu Asn Thr Met 390 CAG ATG TCA TTA AAA GAC TTA CTT TAAAAAAAAAAAAAA 1209 Gin Met Ser Leu Lys Asp Leu Leu Ter 398 Hence, the recombinant baculoviruses of the present invention provide high level, stable expression of biologically active human α-galactosidase A. The stable expression of high levels of biologically active human SUBSTITUTE SHEET -12- 1 α-galactosidase A is unique to the present invention. Such expression and activity is particularly high in insect cell cultures infected with these recombinant baculoviruses in ,_ this preferred expression system, especially Sf9 cells. Recombinant α-galactosidase A activity is present in the culture medium as well as the cells, with the 'majority of the activity found in the culture medium. Extracellular, recombinant enzyme activity is thus achieved by the present - 0 invention. Furthermore, the expression system of the present invention provides glycosylated α-galactosidase A which is biologically active. The α-gaiactosidase A activity can be measured using a fluorescent substrate, 4-methylumbelliferyl- α-D-galactopyranoside, for example. ,- The following cultures have been deposited with the American Type Culture Collection located at 12301 Parklawn Drive, Rockvilie, Maryland, United States of America: Strain Deposit Date Accession No. 20 E. coli. containing pCC14 March 21, 1990 ATCC No. E. coli. containing pCC14 March 21, 1990 ATCC No. AcCCl in SF9 cells March 21, 1990 ATCC No. AcCC2 in SF9 ceils March 21, 1990 ATCC No. 25 AcCC4 in SF9 cells March 21, 1990 The deposits were made in accordance with the provisions of the Budapest Treaty. Said cultures will remain permanently available for a term of at least five years after 30 the most recent request for the furnishing of a sample and in any case 30 years after the date of the deposit. 35 - 13- 1 Another aspect of the present invention provides homogeneous recombinant human α-galactosidase A. Homogeneous preparations are particularly useful for enzyme replacement r- therapy of Fabry disease and any other disease resulting from a deficiency of α-galactosidase A. Recombinant production of α-galactosidase A provides a plentiful source of active enzyme which was heretofore unavailable from natural sources or other recombinant sources. Apparently, the increased ιn activity relative to the E. coli expression system already described is due to glycosylation and processing of the enzyme neither of which occurred when the α-galactosidase was expressed in E. coli. Purification of recombinant α-galactosidase A from l -,p r- the culture medium or the intact cells, if desired, is achieved by conventional purification means such is ammonium sulfate precipitation, column chromatography and the like by following the enzymatic activity of the recombinant α-galactosidase A by the assay described herein. 0 One preferred scheme to purify recombinant α-galactosidase A produced by a baculovirus expression vector of the present invention involves harvesting the culture supernatent when the α-galactosidase A activity is at a peak, typically about 48 to 72 hours after viral infection of Sf9 cells. The proteins in the supernatent are precipitated by ammonium sulfate, dialyzed into an appropriate buffer and applied to a Concanavalin A-Sepharose (Pharmacia trademark) chromatography resin. The resin is eluted with 0.1 M α-methγlglucoside to remove contaminants and then with 1 M Q α-methylglucoside to release the bound α-galactosidase A activity. After concentrating and dialyzing the eluate which 35 -14- 1 contains the activity, it is applied to a Sephadex G-200 or DEAE Sephadex (Pharmacia trademark) gel filtration column. The resulting active fractions consist of 70-80% pure .. recombinant α-galactosidaεe A. The remaining contaminants are removed by conventional purification means, including FPLC (Pharmacia trademark) or HPLC techniques. The present invention also provides polyclonal and monoclonal antibodies to recombinant α-galactosidase A, _ r especially from a human source. These antibodies are prepared by methods well known in the art. Extensive protocols for preparing, purifying, identifying, and use of monoclonal antibodies are found in Harlowe et jil. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor __ Laboratory, Cold Spring Harbor, NY, 726p. , which is ID incorporated herein by reference. Monoclonal antibodies are conveniently prepared by immunizing mice with homogeneous or partially purified recombinant α-galactosidase A. Alternatively, fragments or n active derivatives of α-galactosidase A may be used for immunization. These fragments may be made by proteolytic digestion and purified by conventional means. The enzyme derivatives may be made by chemical modification or site-directed mutagenesis of the cloned α-galactosidase A _ ( _ gene or cDNA. Likewise, any of these α-galactosidase A preparation are used to prepare polyclonal antibodies in rabbits or other animals such as goat, sheep, rats or the like. Methods of identifying the desired antibody include ELISA assay using any purified α-galactosidase A as the test _ 0 material, Western or immunoblotting against pure or partially pure α-galactosidase A, and other methods as described in 35 - 15- 1 Harlowe et al. The antibodies are useful to affinity purify large quantities of α-galactosidase A, including the recombinant enzyme. .. - In a related embodiment, the present invention also 5 contemplates the gene fusion of α-galactosidase A to homologous human proteins including, but not limited to, albumin, insulin and apoprotein B. Fusions may be constructed to specific fragments of these genes that stabilize the α-galactosidase A and retain specific receptors for endocytosis. This technique also contemplates the genetically altered versions of the proteins employed where, for example, it may be advantageous to eliminate the biological activity of the protein, e.g., insulin, while .,„ maintaining its intended efficacy in this context. --D Another aspect of the present invention provides recombinant human α-galactosidase A as a valuable therapeutic agent for treating diseases resulting from deficiencies of this enzyme, especially Fabry disease, in a mammal by „ administering to said mammal an effective amount of recombinant α-galactosidase A or an active derivative or fragment thereof for a time and under conditions sufficient to treat the deficiency by increasing enzyme level. A therapeutically effective amount of recombinant „ α-galactosidase A may range from about 50 to about 10,000 units enzyme activity per kg body weight per day. A unit of α-galactosidase A activity is as defined in Calhoun et al. , with one unit corresponding to one nanomole of 4-methylumbelliferyl-α-D-galactopyranoside hydrolyzed per _- hour at 37°C. 30 35 - 16- 1 Accordingly, the subject invention contemplates treating Fabry disease or other diseases resulting from a deficiency of α-galactosidase A in mammals by administering a r- pharmaceutical composition containing a pharmaceutically effective amount of recombinant α-galactosidase A or an active fragment or derivative thereof. Additionally, a method for treating Fabry disease (or other diseases characterized by this enzyme deficiency) in a mammal is P contemplated in which a nucleic acid molecule encoding α-galactosidase A is introduced into a cell in such a manner that said nucleic acid molecule is expressed intracellularly. By active fragments is meant any part of the enzyme which is derived from the intact whole enzyme and still retains ι c - biological activity. Likewise, derivatives of α-galactosidase mean enzymes which have been chemically modified or genetically engineered to effect minor changes, for example amino acid substitutions, which maintain biological activity. Such expression may be extrachromosomal 2o i- n said cell or occur following integration into the genome of said cell. In this case where the enzyme is introduced into the cell via a nucleic acid, the nucleic acid molecule may be carried to the cell and transferred into said cell by a second nucleic acid molecule (e.g., various viruses). The first nucleic acid molecule is manipulated such that it contains the appropriate signals for expression in the target cell. That is, in accordance with the present invention, a method of treating Fabry disease in a mammal is contemplated by administering a first nucleic acid molecule encoding go α-galactosidase A. This nucleic acid is contained in a pharmacologically acceptable second nucleic acid carrier such 35 -17- that said first nucleic acid enters a target cell and is either maintained extrachromosomally or integrated into the genome of said target. Thus, expression of the first nucleic acid produces an ' effective amount of human α-galactosidase A. The active ingredients of the pharmaceutical compositions comprising recombinant α-galactosidase A are contemplated to exhibit excellent and effective therapeutic activity in replacing the enzymatic deficiency found in Fabry disease or other conditions resulting from this deficiency. Thus, the active ingredients of the therapeutic compositions including recombinant α-galactosidase A exhibit enzymatic activity when administered in therapeutic amounts from about 0.1 ug to about 2000 ug per kg of body weight per day. The dosage regimen may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A decided practical advantage is that the active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intra¬ muscular, intravenous, intranasal, intradermal, subcutaneous, or suppository routes. Depending on the route of administration, the active ingredients of a recombinant α-galactosidase A-containing pharmaceutical composition may be required to be coated in a material to protect said ingredients from the action of enzymes, acids or other natural conditions. The active compounds may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of - 18- storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or -dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. Coatings for α-galactosidase A preparations are useful to reduce degradation of the enzyme when administered as a therapeutic agent. Coatings also reduce the - 19- 1 immunogenicity of the enzyme to help prevent undesirable side effects of administering such a therapeutic agent. A particularly useful coating to provide these characteristics -- is polyethylene glycol. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter , 0 sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the reguired other ingredients from those enumerated above. In the case of sterile powders for the preparation of -r- sterile injectable solutions, the preferred methods of 15 preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof. 0 When recombinant α-galactosidase A is suitably protected as described above, the active compound may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed c - into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. ■ OQ Such compositions and preparation should contain at least 1% of active compound. The percentage of the compositions and 35 -20- 1 preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful _ compositions is such that a suitable dosage is obtained. D Preferred compositions or preparations according to the present invention are prepared so that an oral unit dosage form contains between about 10 ug and 1000 ug of active compound. The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum agragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant __ such as magnesium stearate; a sweetening agent such as 15 sucrose, lactose or saccharin or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other p materials may be present as coatings or to otherwise modify the physical form of the unit dosage. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as pj . preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release _ n preparations and formulations. 35 -21- 1 It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form as _ used herein refers to physically discrete units suited as D unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The n specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active _,_ material for the treatment of disease in living subjects having a diseased condition in which bodily health impaired as herein disclosed in detail. The principal active ingredient, especially, recombinant α-galactσsidase A, is compounded for convenient n and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 10 ug to about 1000 ug. Expressed in r _ proportions, the active compound is generally present in from about 10 ug to about 1000 ug/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. 30 35 -22- As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and _ absorption delaying agents, and the like. The use of such 5 media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or .agent is incompatible with the active ingredient, its use in the-therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The following examples further illustrate the invention. 5 0 5 0 5 -23- EXAMPLE 1 Construction of a Baculovirus Derivative to ExDress Human α-Galactosidase A: The clone that contains the complete coding region for the precursor form of human α-galactosidase A, M13.L21.1 (Hanztopoulos) was digested with EcoRI. All restriction enzyme digestions were performed according to manufacturers instructions, New England Biolabs. This EcoRI digestion was ethanol precipitated and mixed with EcoRI-digested pSPRl in ligation buffer. After ligation with T4 DNA ligase as recommended by the manufacturer (New England Biolab) , recombinants were selected for ampicillin resistance and restriction mapped to identify a pSPRl recombinant with the EcoRI fragment having the human α-galactosidase A coding sequence. The pSPRl derivative was then digested with BamHI and mixed with BamHI-digested pAc373, the baculovirus transfer vector which has a unique Bam HI site 8 bp upstream from the ATG codon of the polyhedrin gene coding region. After ligation, recombinants were selected for ampicillin resistance, and restriction mapped to identify two clones having the pSPRl-derived BamHI fragment inserted into the BamHI site of pAc373 in opposite orientations. These clones are the transfer vectors pCC4 which has the α-galactosidase A cDNA in the orientation properly aligned the polyhedrin promoter and pCC5 which has the cDNA in the opposite orientation. The vector pCC5 is a control plasmid. Transfer vector pCC4 or pCC5 were co-transfected with AcMNPV DNA into S. frugiperda Sf9 cells by the methods described in Summers et al. About 3% of the plaques were Occ~. Several recombinant viral clones were plaque purified -24- three times from the Occ clones. The resulting recombinant baculoviruses from transfer vector pCC4 are designated AcCCl and AcCC2. The control recombinant virus from transfer vector pCC5 is designated AcCC3. Restriction maps of M13.L21.1 and pAc373 are shown in Fig. 1 (top and bottom). The center portion of Fig. 1 depicts the polylinker region of pSRPl. -25- 1 EXAMPLE 2 Expression of Human α-Galactosidase A by Recombinant Baculovirus Vectors: To test for r- α-galactosidase A production, the recombinant viruses were D used to infect Sf9 cells. Cells were cultured and infections carried out as described in Summers et al. Exponential phase infected cells were harvested, separated from the culture medium, and both the cells and the culture medium tested for _ 0 α-galactosidase A activity. The wild type virus, AcMNPV and mock infected cells were included as controls. The activity of α-galactosidase A was measured using the fluorescent substrate, 4-methylumbelliferyl-α-D-galactopyrassoside and is expressed as units (nanomoles per hour at 37°C) present in 2 m r- the cells or culture medium in a 25 cm flask containing 5 ml of medium and approximately 3 x 10 cells. Table 1 indicates that recombinant viruses AcCCl and AcCC2 express high levels of active recombinant human α-galactosidase A in the culture medium and significant 20 levels in the cells. In contrast, the control viruses AcCC3, and AcMNPV as well as mock infections have relatively little enzyme activity. The recombinant viruses AcCCl and AcCC2 express about 1000-fold higher levels of enzyme activity than the recombinant control virus, AcCC3. Further, since α-galactosidase A activity is found 25 in the culture medium, it suggests that the precursor form is properly processed to the mature form. 30 35 -26- EXAMPLE 3 Construction and Expression of Human α-Galactosidase A in a Modified Baculovirus Vector: Transfer vector pCC4, containing the human α-galactosidase A cDNA cloned in proper orientation to the modified polyhedrin promoter derived from pAc373, was digested with EcoRV and BssHII (enzymes and used as recommended by the manufacturer, New England Biolabs). The 213 bp EcoRV/BssHII fragment was discarded. To construct the wild type polyhedrin promoter, 4 overlapping oligonucleotides were synthesized, annealed together and mixed with the large fragment of EcoRV/BssHII-cut pCC4 DNA in ligation buffer. After ligation with T4 DNA ligase recombinants were transformed into E^_ coli and selected on ampicillin plates. Recombinants with the wild type polyhedrin promoter were identified by their inability to hybridize (Sambrook e al. , 1989, Molecular Cloning, a Laboratory Manual 2 pp. 8.1-15.173) to a 32P labeled oligσnucliotide 'probe derived from the 93 bp region of DNA deleted from pCC4 (Fig. 2, top). To confirm that the selected clone had the proper DNA sequence of the wild type promoter, double stranded recombinant DNA was sequenced using unique oligonucliotide primers homologous to sequences lying outside the altered region. Two clones were identified with the correct sequence (Fig. 2 bottom) . The modified pCC4 transfer vector was named pCC14. After co-transfection of pCC14 and viral DNA into SF9 cells as described in Example 2, virally infected cloned cells were tested for α-galactosidase A activity. The recombinant virus which has incorporated pCC14 sequences is called AcCC4 and produce at least 2-10 fold more -27- α-galactosidase A activity than does the pCC4 containing virus AcCCl after on round of plague purification. After three rounds of plaque purification to yield a homogeneous preparation of AcCC4, the α-galactosidase A levels can increase up to about 1000 fold relative to AcCCl-derived enzyme levels. -28- TABLE 1 Enzyme Units Virus Transfer Vector Culture Media Cells AcCCl pCC4 2,301 830 AcCC2 pCC4 1,959 530 AcCC3 pCC5 29 2 AcMNPV None 2 0.2 Mock Infection None 3 0.4

Claims: -29- WHAT IS CLAIMED IS: 1. A replicable expression vector comprising a recombinant DNA or a cDNA encoding a precursor form of an j - α-galactosidase A polypeptide which is operably linked to a baculovirus nucleic acid sequence capable of effecting expression of said polypeptide. 2. The vector of Claim 1 wherein said precursor form comprises a signal peptide and the mature π α-galactosidase A polypeptide. 3. The vector of Claims 1 or 2 wherein said DNA comprises a nucleotide sequence encoding the amino acid sequence of a precursor form of α-galactosidase A. 4. The vector of any one of Claims 1 to 3 wherein -- said amino acid sequence comprises: 0 5 0 5 -30- Met Glu Leu Arg Lys Pro Glu Leu His Leu Gly Lys Ala Leu Ala Leu -15 Arg Phe Leu Ala Leu Val Gly Trp Asp He Pro Gly Ala Arg Ala -1 Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His 15 Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp 30 Ser Cys He Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met 45 Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys He 60 Asp Asp Cys Trp Met Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu 75 Gin Ala Asp Pro Gin Arg Phe Pro His Gly He Arg Gin Leu Ala 90 Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly He Tyr Ala Asp 105 Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr 120 Tyr Asp He Asp Ala Gin Thr Phe Ala Asp Trp Gly Val Asp Leu 135 Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala 150 Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg 165 Ser He Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe 180 Gin Lys Pro Asn Tyr Thr Glu He Arg Gin Tyr Cys Asn His Trp 195 Arg Asn Phe Ala Asp He Asp Asp Ser Trp Lys Ser He Lys Ser 210 He Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg He Val Asp Val 225 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val He Gly 240 Asn Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu 255 Trp Ala He Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg 270 His He Ser Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val 285 He Ala He Asn Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu 300 Arg Gin Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly 315 Leu Ala Trp Ala Val Ala Met He Asn Arg Gin Glu He Gly Gly 330 SUBSTITUTE SHEET -30/ 1- Pro Arg Ser Tyr Thr He Ala Val Ala Ser Leu Gly Lys Gly Val 345 Ala Cys Asn Pro Ala Cys Phe He Thr Gin Leu Leu Pro Val Lys 360 Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His 375 He Asn Pro Thr Gly Thr Val Leu Leu Gin Leu Glu Asn Thr Met 390 Gin Met Ser Leu Lys Asp Leu Leu Ter 398 SUBSTITUTESHEET -3 1- 5. The vector of any one of Claims 1 to 3 wherein said DNA has a nucleotide seguence comprising: ATG CAG CTG AGG AAC CCA GAA CTA CAT CTG GGC TGC GCG CTT GCG CTT -45 CGC TTC CTG GCC CTC GTT TCC TGG GAC ATC CCT GGG GCT AGA GCA -1 CTG GAC AAT GGA TTG GCA AGG ACG CCT ACC ATG GGC TGG CTG CAC 45 TGG GAG CGC TTC ATG TGC AAC CTT GAC TGC CAG CAA GAG CCA GAT 90 TCC TGC ATC AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG 135 GTC TCA GAA GGC TGG AAG GAT GCA GGT TAT GAG TAC CTC TGC ATT 180 GAT GAC TGT TGG ATG GCT CCC CAA AGA GAT TCA GAA GGC AGA CTT 225 CAG GCA GAC CCT CAG CGC TTT CCT CAT GGG ATT CGC CAG CTA GCT 270 AAT TAT GTT CAC AGC AAA GGA CTG AAG CTA GGG ATT TAT GCA GAT 315 GTT GGA AAT AAA ACC TGC GCA GGC TTC CCT GGG AGT TTT GGA TAC 360 TAC GAC ATT GAT GCC CAG ACC TTT GCT GAC TGG GGA GTA GAT CTG 405 CTA AAA TTT GAT GGT TGT TAC TGT GAC AGT TTG GAA AAT TTG GCA 450 GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AGG ACT GGC AGA 495 AGC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT ATG TGG CCC TTT 540 CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TGC AAT CAC TGG 585 CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA AAG AGT 630 ATC TTG GAC TGG ACA TCT TTT AAC CAG GAG AGA ATT GTT GAT GTT 675 GCT GGA CCA GGG GGT TGG AAT GAC CCA GAT ATG TTA GTG ATT GGC 720 AAC TTT GGC CTC AGC TGG AAT CAG CAA GTA ACT CAG ATG GCC CTC 765 TGG GCT ATC ATG GCT GCT CCT TTA TTC ATG TCT AAT GAC CTC CGA 810 CAC ATC AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA 855 ATT GCC ATC AAT CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT 900 SUBSTITUTE SHEET -3 1 / 1— AGA CAG GGA GAC AAC TTT GAA GTG TGG GAA CGA CCT CTC TCA GGC 945 TTA GCC TGG GCT GTA GCT ATG ATA AAC CGG CAG GAG ATT GGT GGA 990 CCT CGC TCT TAT ACC ATC GCA GTT GCT TCC CTG GGT AAA GGA GTG 1035 GCC TGT AAT CCT GCC TGC TTC ATC ACA CAG CTC CTC CCT GTG AAA 1080 AGG AAG CTA GGG TTC TAT GAA TGG ACT TCA AGG TTA AGA AGT CAC 1125 ATA AAT CCC ACA GGC ACT GTT TTG CTT CAG CTA GAA AAT ACA ATG 1170 CAG ATG TCA TTA AAA GAC TTA CTT TAAAAAAAAAAAAAA 1209 6. T e vector of any one of Claims 1 to 5 wherein said vector is a baculovirus transfer vector or a recombinant baculovirus. SUBSTITUTE SHEET -32- 7. The vector of Claim 6 wherein said baculovirus is Autographa California nuclear polyhedrosis virus. 8. The vector of any one of Claims 1 to 7 wherein said baculovirus nucleic acid sequence is the polyhedrin gene promoter. 9. The vector of Claim 8 wherein the source of said promoter is the Autographa californica nuclear polyhedrosis virus. 10. A vector of any one of Claims 1 to 9 comprising pCC4, pCC14, AcCCl, AcCC2 or AcCC4. 11. The vector of any one of Claims 1 to 10 wherein said α-galactosidase A is human α-galactosidase A. 12. A recombinant baculovirus comprising a nucleic acid encoding the precursor or mature form of α-galactosidase A. 13. The baculovirus of Claim 12 wherein the source of said α-galactosidase is a human. -33- 14. The recombinant baculovirus of Claim 12 or 13 comprising a nucleic acid encoding a polypeptide having an amino acid sequence of Met Glu Leu Arg Lys Pro Glu Leu His Leu Gly Lys Ala Leu Ala Leu -15 Arg Phe Leu Ala Leu Val Gly Trp Asp He Pro Gly Ala Arg Ala -1 Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His 15 Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp 30 Ser Cys He Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met 45 Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys He 60 Asp Asp Cys Trp Met Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu 75 Gin Ala Asp Pro Gin Arg Phe Pro His Gly He Arg Gin Leu Ala 90 Asn yr Val His Ser Lys Gly Leu Lys Leu Gly He Tyr Ala Asp 105 Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr 120 Tyr Asp He Asp Ala Gin Thr Phe Ala Asp Trp Gly Val Asp Leu 135 Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala 150 Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg 165 Ser He Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe 180 Gin Lys Pro Asn Tyr Thr Glu He Arg Gin Tyr Cys Asn His Trp 195 Arg Asn Phe Ala Asp He Asp Asp Ser Trp Lys Ser He Lys Ser 210 He Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg He Val Asp Val 225 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val He Gly 240 Asn Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu 255 Trp Ala He Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg 270 His He Ser Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val 285 He Ala He Asn Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu 300 SUBSTITUTE SHEET -33/ 1- Arg Gin Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly 315 Leu Ala Trp Ala Val Ala Met He Asn Arg Gin Glu He Gly Gly 330 Pro Arg Ser Tyr Thr He Ala Val Ala Ser Leu Gly Lys Gly Val 345 Ala Cys Asn Pro Ala Cys Phe He Thr Gin Leu Leu Pro Val Lys 360 Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His 375 He Asn Pro Thr Gly Thr Val Leu Leu Gin Leu Glu Asn Thr Met 390 Gin Met Ser Leu Lys Asp Leu Leu Ter 398 SUBSTITUTE SHEET -34- 15. The recombinant baculovirus of Claim 12 or 13 comprising a nucleic acid having a nucleotide seguence of ATG CAG CTG AGG AAC CCA GAA CTA CAT CTG GGC TGC GCG CTT GCG CTT -45 CGC TTC CTG GCC CTC GTT TCC TGG GAC ATC CCT GGG GCT AGA GCA -1 CTG GAC AAT GGA TTG GCA AGG ACG CCT ACC ATG GGC TGG CTG CAC 45 TGG GAG CGC TTC ATG TGC AAC CTT GAC TGC CAG CAA GAG CCA GAT 90 TCC TGC ATC AGT GAG AAG CTC TTC ATG GAG ATG GCA GAG CTC ATG 135 GTC TCA GAA GGC TGG AAG GAT GCA GGT TAT GAG TAC CTC TGC ATT 180 GAT GAC TGT TGG ATG GCT CCC CAA AGA GAT TCA GAA GGC AGA CTT 225 CAG GCA GAC CCT CAG CGC TTT CCT CAT GGG ATT CGC CAG CTA GCT 270 AAT TAT GTT CAC AGC AAA GGA CTG AAG CTA GGG ATT TAT GCA GAT 315 GTT GGA AAT AAA ACC TGC GCA GGC TTC CCT GGG AGT TTT GGA TAC 360 TAC GAC ATT GAT GCC CAG ACC TTT GCT GAC TGG GGA GTA GAT CTG 405 CTA AAA TTT GAT GGT TGT TAC TGT GAC AGT TTG GAA AAT TTG GCA 450 GAT GGT TAT AAG CAC ATG TCC TTG GCC CTG AAT AGG ACT GGC AGA 495 AGC ATT GTG TAC TCC TGT GAG TGG CCT CTT TAT ATG TGG CCC TTT 540 CAA AAG CCC AAT TAT ACA GAA ATC CGA CAG TAC TGC AAT CAC TGG 585 CGA AAT TTT GCT GAC ATT GAT GAT TCC TGG AAA AGT ATA AAG AGT 630 ATC TTG GAC TGG ACA TCT TTT AAC CAG GAG AGA ATT GTT GAT GTT 675 GCT GGA CCA GGG GGT TGG AAT GAC CCA GAT ATG TTA GTG ATT GGC 720 AAC TTT GGC CTC AGC TGG AAT CAG CAA GTA ACT CAG ATG GCC CTC 765 TGG GCT ATC ATG GCT GCT CCT TTA TTC ATG TCT AAT GAC CTC CGA 810 CAC ATC AGC CCT CAA GCC AAA GCT CTC CTT CAG GAT AAG GAC GTA 855 ATT GCC ATC AAT CAG GAC CCC TTG GGC AAG CAA GGG TAC CAG CTT 900 SUBSTITUTE SH? 1 ; -34/ 1- AGA CAG GGA GAC AAC TTT GAA GTG TGG GAA CGA CCT CTC TCA GGC 945 TTA GCC TGG GCT GTA GCT ATG ATA AAC CGG CAG GAG ATT GGT GGA 990 CCT CGC TCT TAT ACC ATC GCA GTT GCT TCC CTG GGT AAA GGA GTG 1035 GCC TGT AAT CCT GCC TGC TTC ATC ACA CAG CTC CTC CCT GTG AAA 1080 AGG AAG CTA GGG TTC TAT GAA TGG ACT TCA AGG TTA AGA AGT CAC 1125 ATA AAT CCC ACA GGC ACT GTT TTG CTT CAG CTA GAA AAT ACA ATG 1170 CAG ATG TCA TTA AAA GAC TTA CTT TAAAAAAAAAAAAAA 1209 SUBSTITUTE SHEET -35- 2 16. The baculovirus of any one of Claims 12 to 15 wherein said baculovirus is AcCCl, AcCC2 or AcCC4. 17. A transfor ant microorganism or cell comprising the replicable expression vector of any one of Claims 1 to 11. c 18. A cultured cell line comprising the recombinant baculovirus of any one of Claims 12 to 16. 19. Homogeneous recombinant human α-galactosidase A. 20. The α-galactosidase A of Claim 19 comprising the amino acid sequence: 0 Met Glu Leu Arg Lys Pro Glu Leu His Leu Gly Lys Ala Leu Ala Leu -15 Arg Phe Leu Ala Leu Val Gly Trp Asp He Pro Gly Ala Arg Ala -1 Leu Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His 15 5 Trp Glu Arg Phe Met Cys Asn Leu Asp Cys Gin Glu Glu Pro Asp 30 Ser Cys He Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met 45 Val Ser Glu Gly Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys He 60 Asp Asp Cys Trp Met Ala Pro Gin Arg Asp Ser Glu Gly Arg Leu 75 0 Gin Ala Asp Pro Gin Arg Phe Pro His Gly He Arg Gin Leu Ala 90 Asn Tyr Val His Ser Lys Gly Leu Lys Leu Gly He Tyr Ala Asp 105 Val Gly Asn Lys Thr Cys Ala Gly Phe Pro Gly Ser Phe Gly Tyr 120 Tyr Asp He Asp Ala Gin Thr Phe Ala Asp Trp Gly Val Asp Leu 135 Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Leu Glu Asn Leu Ala 150 Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Arg Thr Gly Arg 165 Ser He Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met Trp Pro Phe 180 o Gin Lys Pro Asn Tyr Thr Glu He Arg Gin Tyr Cys Asn His Trp 195 Arg Asn Phe Ala Asp He Asp Asp Ser Trp Lys Ser He Lys Ser 210 He Leu Asp Trp Thr Ser Phe Asn Gin Glu Arg He Val Asp Val 225 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val He Gly 240 5 SUBSTITUTESHEET -35/1- Asn Phe Gly Leu Ser Trp Asn Gin Gin Val Thr Gin Met Ala Leu 255 Trp Ala He Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg 270 His He Ser Pro Gin Ala Lys Ala Leu Leu Gin Asp Lys Asp Val 285 He Ala He Asn Gin Asp Pro Leu Gly Lys Gin Gly Tyr Gin Leu 300 Arg Gin Gly Asp Asn Phe Glu Val Trp Glu Arg Pro Leu Ser Gly 315 Leu Ala Trp Ala Val Ala Met He Asn Arg Gin Glu He Gly Gly 330 Pro Arg Ser Tyr Thr He Ala Val Ala Ser Leu Gly Lys Gly Val 345 Ala Cys Asn Pro Ala Cys Phe He Thr Gin Leu Leu Pro Val Lys 360 Arg Lys Leu Gly Phe Tyr Glu Trp Thr Ser Arg Leu Arg Ser His 375 He Asn Pro Thr Gly Thr Val Leu Leu Gin Leu Glu Asn Thr Met 390 Gin Met Ser Leu Lys Asp Leu Leu Ter 398 SUBSTITUTE SHEET -36- 21. The α-galactosidase A of Claim 19 or 20 comprising des-MQLRNPELHLGCALALRFLALVSWDIPGARA α-galactosidase A. 22. A monoclonal or polyclonal antibody to the recombinant α-galactosidase A of any one of Claims 19 to 21. 23. A monoclonal or polyclonal antibody to a derivative or fragment of the recombinant α-galactosidase A of any one of Claims 19-21. 24. A recombinant human α-galactosidase A, a derivative thereof, or an active fragment thereof, for use in preparing a composition for treating diseases associated with deficiency of α-galactosidase A in mammals. 25. A pharmaceutical composition comprising a pharmaceutically effective amount of recombinant α-galactosidase A, a derivative thereof, or an active fragment thereof and a pharmacologically acceptable carrier. 26. The pharmaceutical composition of Claim 25 having a unit dosage form containing about 10 ug to about 1000 ug of recombinant α-galactosidase A. 27. The pharmaceutical composition of Claim 25 wherein said effective amount comprises from about 0.1 ug of about 2000 ug per kilogram body weight per day.

Inventors: CALHOUN DAVID H, COPPOLA GEORGE, CALHOUN, DAVID, H., COPPOLA, GEORGE
Applicants: RES CORP TECHNOLOGIES INC, RESEARCH CORPORATION TECHNOLOGIES, INC.
Application year: 1990
Main classification: A61K37
IPC codes: A61K38, A61K48, C07K16, C12N9, C12N15, C12P21