ENHANCEMENT OF THE TERAPEUTIC PROPERTIES OF GLYCOPROTEIN Background of the Invention Many proteins being developed for pharmaceutical applications have oligosaccharides attached to their polypeptide backbone when they are isolated from natural sources. The sugar chains of these glycoproteins may be attached by an N-glycosidic bond to the amide group of asparagine residues (Asn-linked oligosaccharides) or by an O-glycosidic bond to the hydroxyl group of serine or threonine residues. Examples of glycoproteins with Asn-linked oligosaccharides include tissue plasminogen activator (tPA), prourokinase, granulocyte- macrophage colony stimulating factor, Factor VIII, Factor IX, glucocerebrosidase, erythropoietin (EPO), alpha-l-antitrypsin, various monoclonal antibodies, and human chorionic gonadotropin. When proteins such as these are produced by means of recombinant DNA technology, they will be glycosylated if secreted from the host cell unless the cell is of bacterial origin. Thus, yeast, other fungi, insect cells, plant cells, and animal cells are capable of attaching oligosaccharide chains to a protein containing the Asn-glycosylation signal Asn-X-Ser/Thr where X is any amino acid except proline. The initial Asn-glycosylation event in all systems examined to date involves transfer of a preassembled tetradecylsaccharide (GlcNAc ₂ Man _g Glc ₃ ) from a dolichol pyrophosphate carrier to appropriate Asn residues during protein synthesis and translocation across the rough endoplasmic reticulum (RER) membrane. As the newly synthesized protein is transported through the cell from the RER to the cis, medial, and trans Golgi and to the cell

surface, processing of the oligosaccharide chains occurs. Exoglycosidases remove certain sugars and glycosyltransferases add other sugars.

This processing takes place by mass action, i.e. without the aid of a template. As a consequence, it is common for considerable heterogeneity to exist in the carbohydrate chains of a glycoprotein. This heterogeneity is observed at each individual glycosylation site and across different glycosylatiσn sites within a glycoprotein.

The fully mature structures of the Asn-oligosaccharides on an isolated glycoprotein depend upon the cell employed to produce the glycoprotein. It has been observed that each cell type produces a unique spectrum of oligosaccharide chains. Typical classes of structures found on proteins isolated from different sources are illustrated in Figure 1.

Considerable variation in these basic structures is common. For example, mammalian high mannose chains may have from five to nine mannose units and structural isomers are possible for each of these except an _g . Some possible structural variations of the complex chains are given in Figure 2. A more complete description of the structures of Asn-oligosaccharides can be found in the review of Kornfeld and Kornfeld ((1985) Ann. Rev. Biochem. 54, - 631)-, _-._._:,_. _ - The glycosylation pattern of a glycoprotein is both cell and protein dependent. Two different glycoproteins produced in the same cell line will usually have different glycosylation patterns. (Some of the oligosaccharides may be identical, but the overall pattern will vary.) Likewise, the same protein isolated from two different cell lines will very likely have a

different array of oligosaccharides attached. Thus it can be expected that even though a glycoprotein, for example, erythropoietin, may have the same polypeptide backbone when produced in two different animal cells, it will likely have a different pattern of Asn-linked carbohydrate chains. Likewise, "natural" erythropoietin will probably possess its own unique set of oligosaccharide structures.

A major mechanism for the clearance of serum glycoproteins is through carbohydrate-specific uptake by liver cells. The asialoglycoprotein receptor of hepatocytes binds glycoproteins with exposed galactose-terminated Asn-oligosaccharide chains. After binding, the proteins are usually internalized and degraded. Likewise, the mannose/N-acetylglucosamine receptor of macrophages binds exposed Asn-oligosaccharides with terminal mannose or N-acetylglucosamine residues. Therapeutic proteins with sugar chains that are recognized by these cell surface receptors will be rapidly cleared from the circulation and degraded. One consequence of these carbohydrate - specific uptake systems is that the distribution of a glycoprotein between different organs and physiological compartments of an individual depends in part on the structures of the attached oligosaccharides.

Several carbohydrate structures are known to be ^" antigenic in humans. These include structures that give rise to the ABO blood groups, complex-type chains with terminal alpha-l,3-galactose units (Figure 2), and the mannan chains of yeast. In general, it is undesirable to treat a patient with a protein that will generate an immune response. Adverse consequences range from the formation of circulating antibodies that can neutralize the activity of the protein to anaphalactic shock.

Thus there are at least three problems associated with non-optimal glycosylation - shortened serum half-life, ineffective biodistribution, and increased antigenicity. An additional complication is that each species of a heterogeneous population of glycoprotein molecules may have a different άj vivo biological activity.

There have been several reports of attempts to modify the carbohydrate chains of glycoproteins to increase serum lifetime. U.S. Patent No. 4 326 033 (Abbott Laboratories) discloses a urokinase derivative in which the carbohydrate chain was modified in an undetermined manner by treatment with periodate. The modified urokinase retained 20% of its biological activity and was reported to have an increased serum half-life. In a similar vein J. Robinson (PCT Patent Application GB83/00273) claims a tPA derivative in which an undetermined part of the carbohydrate portion of the glycoprotein was degraded with periodate treatment; Robinson claims retention of at least 50% biological activity and an increased serum lifetime.

Bergh et al. (PCT Patent Application US86/00495) discloses the conversion of Asn-oligosaccharides, preferably high mannose oligosaccharides, to the tri- or tetrasaccharide sequences-Asn-GlcNAc-Gal-NeuAc or

Asn-GlcNAc-GlcNAc-Gal-NeuAc, respectively. It is stated that these modified Asn-oligosaccharide chains cause less rapid clearance of glycoproteins. The toxin ricin has been coupled to monoclonal antibodies to prepare immunotoxins that are under investigation as anti-tumor agents. One difficulty in the use of such an immunotoxin is uptake of the ricin-antibody conjugate by cells of the

reticuloendothelial system (RES) because of the carbohydrate chains present on ricin. J. Thorpe and coworkers have described the use of periodate ((1985) Eu . J. Bioche . 147, 197-206) and alpha-mannosidase and

5 endoglycosidases H, F and D ((1985) Biochem. Biophys■

Acta 840, 193-203) to degrade the carbohydrate chains of ricin to reduce uptake by the cells of the RES ((1987) Cancer Res■ 47, 947-952). Limited digestion of native ricin by Endo F was observed (one chain) . More

10 extensive cleavage was obtained if the protein was first denatured with SDS.

Furbish et al. ((1981) Biochem. Biophys. Acta 673, 425-434) describes a method for altering the biodistribution of the enzyme glucocerebrosidase to

15 enhance its efficacy in enzyme replacement therapy of Gaucher' s disease. Specifically, human placental glucocerebrosidase, which has three complex and one high mannose chain (Takasaki, S. et a_l. (1984) J. Biol. Chem. 259, 10112-10117) is treated sequentially with a

20 neuraminidase, galactosidase and hexosaminidase to expose the alpha-linked mannose units of the pentasaccharide core of the complex chains. The modified enzyme is more effectively taken up by macrophage cells, the site which the enzyme must reach

25 to be effective.

4

The use of the enzyme peptides-N - ^{' " ""} asparagine amidase (EC

3.5.1.52; isolated, for example, from Flavobacterium meningosepticum, and available from Genzyme Corporation, 30 Boston, MA under the trademark N-GLYCANASE to remove the carbohydrate chains from glycoproteins in their native, non-denatured state has been reported in the

scientific literature (Tarentino, A. et a_l. (1985) Biochemistry, 24, 4665; Chu, F.K. (1986) J. Biol. Chem. , 261, 172) . The enzyme will be referred to hereafter as "amidase".) These authors describe the deglycosylation of ribonuclease β, alpha.-acid glycoprotein, fetuin, human transferrin, lipase β, ovomucoid, and an Fab fragment under native and denaturing conditions. In the reported cases, a significantly higher concentration of Amidase was required to deglycosylate the native protein than the denatured form. In the case of native alpha,-acid glycoprotein only very incomplete digestion was observed at the highest concentration of Amidase employed.

Dordal and co-workers ((1985) Endocrinology 116, 2293-2299) reported the removal of sugars from the carbohydrate chains of erythropoietin (EPO) by treatment with exoglycosidases from Streptococcus pneumonia and by treatment with a partially purified sample of "Endoglycosidase F" from Flavobacterium meninqosepticum■ The latter enzyme cleaves the O-glycosidic bond connecting the innermost oligosaccharide chains of high mannose, hybrid, and bi-antennary complex chains. It is likely that the sample employed by the authors was contaminated with Amidase and that in fact this activity, although not recognized by the authors, was responsible for the deglycosylation of EPO. This conclusion can be drawn because it is now known that the carbohydrates of EPO are primarily tri- and tetraantennary complex chains and that Amidase, but not Endo F, will cleave such structures. The authors reported that their deglycosylated EPO retained 50-60% of its in vitro activity, but that it had no in vivo activity.

Treatment of therapeutic glycoproteins according to the present invention can for (1) increase serum lifetime by removing carbohydrate chains recognized by the carbohydrate-specific receptors of hepatocytes or macrophages, (2) obtain more desirable biodistribution of the product, (3) reduce the antigenicity of the glycoprotein, and (4) increase the homogeneity of the final protein.

Summary of the Invention In general, the invention features treating a mammal in need of administration of a glycoprotein, by providing the glycoprotein; treating the glycoprotein with a peptide-N -(N-acetyl-β-glucosaminyl) asparagine amidase to form a modified glycoprotein; admixing the modified glycoprotein with a pharmaceutically acceptable carrier substance to form a therapeutic composition, and administering a pharmaceutically effective amount of the therepeutiσ composition to the said mammal.

In preferred embodiments, the amidase is derived from Flavobacterium meningosepticum.

Preferably, the amidase treatment involves dissolving the glycoprotein, at 0.5 - 2.0 mg/mL, in a buffer solution of pH between 6.0 and 9.0 and treating the glycoprotein solution with 1-100 U/mL of the amidase. In a related aspect, the invention features modified human tissue plasminogen activator prepared by the process of providing human tissue plasminogen activator and treating the human tissue plasminogen activator with a peptide-N ⁴ -(N-acetyl-β-glucosaminyl) asparagine amidase to convert the asparagine residue at position 448 to an aspartate residue, without completely deglycosylating the asparagine residue at position 117. In this modified human tissue plasminogen activator, the asparagine residue at position 184 may or may not be

completely deglycosylated; where deglycosylation is complete at position 184, the asparagine residue at that position is converted to an aspartate residue.

In one preferred embodiment, following amidase treatment, the process further involves treating the tissue plasminogen activator with endoglycosidase H or alpha-mannosidase to modify the oligosaccharide attached to the asparagine residue at position 117; preferably, this additional step converts the oligosaccharide at position 117 to GlcNac.

In another related aspect, the invention features modified prourokinase prepared by the process of providing prourokinase, and treating the prourokinase with a peptide-N -(N-acetyl -β-glucosaminyl) asparagine amidase to convert the asparagine residue at position 302 to an aspartate residue.

The amidase treatment of the invention advantageously is capable of effecting cleavage of all known classes of enzyme-accessible Asn-linked oligosaccharides. The reaction results in the cleavage of the N-glycosidic bond between the oligosaccharide chain and the amide group of the asparagine residue, and forms the reaction products aspartate (within the primary amino acid structure of the protein), ammonia, and the free reducing oligosaccharide. _._ Arnidase treatment of therapeutic protein according to the invention, unlike some other treatments (e.g., periodate) is capable of completely removing certain N-linked oligosaccharides without leaving residual attached sugar residues, which might be detrimental insofar as they promote rapid clearance of the protein, like the parent sugar.

Other features and advantages of the invention will be apparent from the following description of the preferred embodiment thereof and from the claims. Brief Description of the Figures Figure 1 shows structures of common classes of

Asn-oligosaccharides isolated from different sources. N=N-acetylglucosamine, M=Mannose, G=galactose, SA=sialic acid.

Figure 2 shows examples of heterogeneity found in the structures of Asn-oligosaccharides of the complex type. N=N-acetylglucosamine, M=mannose, G=galactose, SA=sialic acid, F=fucose.

Figure 3 shows the electrophoretic mobility through an 11% SDS-polyacrylamide gel of tPA (lane 1) and tPA treated with Amidase at a concentration of 15 U/ml (lane 2) . After electrophoresis the gel was stained with silver (panel A) or the proteins were transferred to nitrocellulose and probed for ConA (panel B) or ricin (panel C) reactivity. Figure 4 shows the electrophoretic mobility through an 11% SDS-polyacrylamide gel of tPA (lane 1) and tPA treated with Amidase at a concentration of 2.5 U/ml (lane 2) . After electrophoresis the gel was stained with silver (panel A) or the proteins were transferred to nitrocellulose and probed for ConA (panel B.X reactivity-.

Figure 5 shows the electrophoretic mobility through an 11% SDS-polyacrylamide gel of tPA (lane 1) and tPA treated with Amidase and endoglucosaminidase H (lane 2). After electrophoresis the gel was stained with silver (panel A) or the proteins were transferred to nitrocellulose and probed for ConA (panel B) or ricin (panel C) reactivity.

Figure 6 shows SDS-polyacrylamide gels of natural and recombinant prourokinase. Samples (5 ug) of prourokinase with and without Amidase treatment were analyzed on a 10% SDS-polyacrylamide slab gel Lanes 2 and 4 are natural prourokinase without Amidase; lane 3 is natural prourokinase with Amidase; lanes 6 and 8 are recombinant pruorokinase with Amidase; lane 7 is recombinant prourokinase with Amidase; lanes 1, 5 and 9 are protein standards (phosphorylase β, 95,500; BSA, 66,200; ovalbumin 45,000; carbonic anhydrase, 31,000; and soybean trypsin inhibitor, 21,500.

Detailed Description of the Invention Structural Analysis of Asn-Oligosaccharides

The starting point for the present invention was the analysis of the Asn-oligosaccharides of several recombinant glycoproteins. Analysis of the oligosaccharides generally followed the procedure described by Hirani, S., et ^L.(1987) Anal. Biochem. 162, 485-492, and can be summarized as follows. The Asn-oligosaccharides were released from SDS-denatured glycoproteins by Amidase treatment, the isolated oligosaccharides radiolabeled at the reducing end by treatment with sodium borotritide, and the labeled oligosaccharides were then analyzed by a combination of high performance liquid chromatography (hplc) and exoglycosidase digestion. Two points of particular interest for the present invention relate to methods for determining the degree of sialylation of the oligosaccharides and the nature of the asialo-oligosaccharide structures.

The degree of sialylation was determined by analyzing the tritium-labeled oligosaccharides by hplc on a MicroPak AX-10 column that was pre-equilibrated in

25mM KH ₂ PO. titrated to pH4 with phosphoric acid. The column was eluted with the same buffer for 15 minutes and then for 30 minutes using a linear gradient of 25mM KH ₂ P0., pH 4.0, to a final concentration of 500mM KHPO., pH 4.0. Oligosaccharides elute from the column in characteristic positions depending upon the number of attached sialic acid residues.

The size of each neutral and/or desialylated oligosaccharide was analyzed by hplc using a MicroPak AX-5 column. The column was pre-equilibrated with acetonitrile: water (65:35) and elution performed by a 60-minute gradient in which the water content of the solvent increased at the rate of 0.5%/minute. The column was calibrated with oligosaccharide standards of known structures.

Analysis of the Asn-oligosaccharides obtained from samples of tPA (non-recombinant, from melanoma cells), prourokinase (non-recombinant, from a human cell line derived from a benign tumor), and prourokinase (recombinant, from CHO cells) indicated that the oligosaccharides from these proteins were heterogeneous, undersialylated and/or large, structures that are potentially antigenic. Detailed results of the analyses are described later in this section under the headings of the individual glycoproteins.

Deglycosylation of Glycoproteins with Amidase

The reaction conditions required to release a particular Asn-oligosaccharide from a glycoprotein with a peptide-N -(N-acetyl-β-glucosaminyl) asparagine amidase depend upon the accessibility of the sugar chain(s) and must be determined empirically. In certain cases it is possible to cleave an oligosaccharide easily from a native glycoprotein. In other cases, the glycoprotein must be first partially unfolded by the use

of mild detergents (for example, cholate, Nonidet P-40, Triton X-100, or octylglucoside) or chaotropic agents (urea or thiocyanate) , by manipulation of pH or choice of buffer, or in extreme cases, by the use of denaturating agents such as β-mercaptoethanol and sodium dodecyl sulfate (SDS). The choice of reaction conditions also depends upon the physiochemical and biological properties of the glycoprotein undergoing treatment. For therapeutic proteins, it is desirable to employ reaction conditions that maintain, to the maximum extent possible, the protein in its native, soluble state.

₄

Peptide-N -(N-acetyl-β-glucosaminyl) asparagine amidase activity has been detected in a variety of seed extracts and in the supernatant of Flavobacterium meningosepticum (Plummer, Jr., T.H. et al. (1987) Eur. J. Biochem. 163, 167-173). For purposes of the present invention, it is advantageous to have a highly purified source of enzyme substantially free of contaminating activities such as proteases, phosphotases, and glycosidases. The amidases from almond emulsin and Flavobacterium meningosepticum have been purified and are commercially available (Seikagaku and Genzyme Corp., respectively). In most cases the preferred enzyme for the present invention is the one from Flavobacterium meningosepticum because of its higher activity toward the Asn-oligosaccharides of native glycoproteins. In a few instances, the amidase from almond emulsin may be preferred because of its lower pH optimum (pH 7.0 vs. 8.6).

A wide range of buffers may be employed for the deglycosylation with Amidase. Examples of acceptable buffers include sodium phosphate, ammonium bicarbonate, HEPES, Tris.HCl and sodium acetate. Citrate buffer

generally should not be used because it has been found to inhibit the action of Amidase. Salts such as sodium chloride may be used in the reaction mixture.

Amidase can be employed over a wide pH range (approximately 6-9 depending to some extent, as stated above, on source), but the preferred reaction pH, (not considering factors related to the glycoprotein undergoing deglycosylation) is 7.5-8.5.

A variety of nondenaturing detergents may be employed in the incubation mixture. These include octylglucoside (1%), cholate (0.5%), Nonidet P-40 (1-2%) and Triton X-100 (1-2%). Amidase is inactivated by SDS unless Nonidet P-40 or a chemical equivalent is present in at least a 7-fold excess by weight. The preferred reaction conditions do not employ detergents or chaotropic reagents unless they are required to obtain the desired degree of deglycosylation or maintain the solubility of the treated protein. The concentration of glycoprotein to be deglycosylated in the reaction mixture may be very low up to the limits of its solubility. A preferred concentration for many glycoproteins is 0.5-2.0 mg/mL.

The concentration of Amidase required to obtain the desired degree of deglycosylation varies, depending upon the treated glycoprotein. In most cases a concentration of 10-60 U/mL of reaction mixture is preferred. - (One unit is defined as the amount of enzyme required to hydrolyze one nanomole of dansyl fetuin glycopeptide per minute at 37°C.) In exceptional cases it was found that as little as 1 U/mL was sufficient. In difficult cases higher concentrations of Amidase (up to 100 U/mL) were required and even then, certain chains may not be cleaved. It may be

advantageous to treat highly sialylated glycoproteins with neuraminidase prior to incubation with Amidase.

Incubations may be performed conveniently between 15-40°C, the preferred temperature being 37 . The incubation may be performed for up to seven days, but generally an incubation time of not more than 18 hours is preferred. The reaction time can often be shortened by increasing the amount of Amidase employed. This may be desirable if the treated glycoprotein is sensitive to the reaction conditions.

The progress of the deglycosylation reaction can be monitored by the change in the glycoprotei 's electrophoretic mobility and by its reactivity with lectins (Hirani, S., et al. (1987) Anal. Biochem. 162, 485-492). Alternatively, compositional analysis can be performed on the deglycosylated protein after isolation to determine the residual sugar content.

At the conclusion of the deglycosylation reaction the protein sample can be separated from the Amidase by any one of a number of standard purification techniques including ion-exchange chromatography, gel-filtration chromatography, hydrophobic chromatography and affinity chromatography. Derivatives of tPA with Enhanced Therapeutic Properties Tissue plasminogen activator (tPA) is a serine protease that converts plasminoger__-to_plasmin. Plasmin in turn is capable of degrading blood clots (fibrin). tPA is being developed as a fibrinolytic agent for the treatment of myocardial infarction. Other applications, such as deep-vein thrombosis, are also being considered. Small quantities of tPA can be extracted from tissues (Astrup, T., Permin, P. M. (1947) Nature 159, 681-682) and highly purified preparations have been isolated from pig heart (Wallen, P., Bersdorf, N. ,

Ranby, B. (1982) BBA 719, 318-328) and human uterus (Rijken, D. C, Wijngaards, G. , Zaal-de Jong, M. , Welbergen, J. (1979) BBA 580, 140-153).

Larger quantities of purified tPA have been isolated from the culture media of a human melanoma cell line (Rijken, D.C. , Collen, D. , J. Biol. Chem. (1981) 256, 7035-7041) and by using recombinant DNA techniques (Pennica, D. et aJL. (1983) Nature 301, 214-221 ; Browne, J. J. et al. , (1985) Gene 33, 279-284; Kaufman, R. J. et al. (1985) Mol. Cell. Biol. 5, 1750-1759).

Human tPA has an approximate molecular weight of 67,000 daltons and is originally synthesized as a single polypeptide. The protein has 35 cysteine residues and thus has the potential to form 17 disulfide bonds. During isolation or fibrinolysis tPA is readily cleaved to a two-chain form consisting of a heavy chain of approximatly 38,000 daltons and a light chain of approximately 34,000 daltons connected by a disulfide bond. The light chain contains the active site, which is homologous to other serine proteases. The heavy chain consists of several regions: the finger domain, the epidermal growth factor domain, and two kringle structures. The kringle regions are believed to be essential for the fibrin-binding properties of tPA. on the basis of the cDNA-derived amino acid sequence tPA has four potential sites for Asn-linked glycosylation, i-e. at asparagine residues 117; 184, 218 and 448 (using the numbering for the melanoma derived tPA) . Asn _21g , however, is not likely to be glycosylated because of an adjacent proline. Two variants of tPA, which differ by 2,000-3,000 daltons in molecular weight, have been described (Rijken, D. C, Emeis, J. J., Gerwig, G. J. (1985) Thromb. Haemost■ 54, 788-791). These two forms differ in the extent of

glycosylation: the lower molecular weight variant lacks a carbohydrate chain at Asn.,-. (Pohl, G. et al. (1984) Biochemistry 23, 3701-3707). Asn..- and Asn .„ appear to carry a oligosaccharide chain in each variant. Analysis of the carbohydrate chains has been performed by us as well as others (e.g., Carr, S. et a_l, (1987) Proceedings 9th Intern. Symp. Glycoconjugates, Montreuil, J. et al. (eds.) Abstr. #12). The studies by the inventors were of several lots of melanoma-derived tPA (BioResponse), and the studies by Carr et al . were of recombinant CHO-derived tPA. The results were generally similar. Asn ₁₁₇ is glycosylated with a high mannose chain that consists of Man ₅ (48%), Man _g (38%), Man _? (12%) and Man- (2%). Asn _lg . and Asn ₄₄ „ have complex chains attached. The complex chains of the melanoma-derived sample appeared to be primarily of the biantennary type while the CHO-derived material was a mixture of bi-,tri-and tetra-antennary chains. In each case the tPA samples appeared to be undersialylated (no or one sialic acid residue). tPA has been demonstrated to be an effective thrombolytic agent in humans (TIMI Study Group, N. Engl. J. Med. (1985) 312, 932-936), but its use has several significant drawbacks including (1) systemic fibrinolysis which leads to excessive bleeding in about 5% of the. atients; (2) the necessity of large-doses (50-100 mg); (3) the need for intravenous perfusion of the patient; and (4) the relative high cost of treatment. A proposed solution to these problems is to employ a form of tPA that has a longer circulatory half-life than the six minutes reported for melanoma-derived and recombinant CHO-derived tPA (Verstraete, M. et al., (1985) J. Pharmacol. Exp. Ther. 235, 506-512).

Our analyses indicate that the carbohydrates present on tPA have the potential to promote rapid uptake of tPA by the carbohydrate-specific receptors of the liver. However the role of the Asn-oligosaccharides of tPA in its clearance from the circulation is controversial.

Organ distribution studies have demonstrated that the primary site of uptake of tPA is the liver. Although carbohydrate-specific receptors in the liver are known to be involved in the clearance of many serum glycoproteins (Ashwell, G. and Harford, J., Ann. Rev. Biochem. (1982) 51_, 531-554), it has been claimed that tPA is taken up by a previously unidentified receptor of unknown specificity (Bakhit, C. et al.., (1987) J. Biol. Chem. 262, 8716-8720 ). This claim is largely based on the observation that glycoproteins such as asialo-fetuin do not interfere with the uptake of tPA. It was therefore suggested that clearance of tPA from the circulation is mediated by a polypeptide-specific receptor system.

Several other reports in the literature support the conclusion that carbohydrates are not involved in the clearance of tPA. In one example, endoglycosidase H treatment of tPA did not affect its clearance time in rabbits (Little, S. P. et al. (1984) Biochemistry 23, &191-6195).- . This glycosidase treatment removes the high-mannose chain from Asn- ₁₇ , which might be - expected, if involved in clearance, to mediate endocytosis of tPA by macrophages or Kupffer cells via their mannose/GlcNAc receptor system. In another experiment, tPA produced in the presence of tunicamycin (which completely blocks any Asn-linked protein glycosylation) was also found to have an equally short circulatory half-life as the unmodified protein (Rijken,

D. C. , Emeis, J. J., and Gerwig, G. J. , Thromb. Haemostas. (1985) 54 _. , 788-791).

The conclusion from each of these independent approaches is that the clearance of tPA from the circulation is not a carbohydrate-mediated event.

On the other hand, J. Robinson (PCT Patent Application GB83/00273) describes a tPA derivative in which part of the carbohydrate portion of the glycoprotein was removed or degraded. This derivative was claimed to retain 50% biological activity coupled with an increased half-life. The method used for the partial degradation chain(s) was periodate oxidation.

Experimental evidence was presented that the periodate treatment extended the biological half-life of tPA. Although periodate will degrade susceptible carbohydrate structures, it also is a strong oxidizing agent that can modify amino acid residues such as methionine and cysteine. Thus, it is not possible to conclude whether it is protein- or carbohydrate-related modifications that give rise to a prolonged half-life of periodate-treated tPA. In the above mentioned patent application claims are also made for longer-lived forms of tPA obtained by treatment with exo- and/or endoglycosidases, or tPA produced in the presence of tunicamycin. No experimental evidence is presented to substantiate these claims of increased lifetimes.

^~~ Another report in whicl_ it ^~ as suggested that oligosaccharide chains of tPA participate in its clearance from the circulation was recently published by D. Lau et al. ((1987) Biotechnology 5, 953-958). These authors describe an altered form of uterine tPA, obtained through site-specific mutagenesis, in which Asn. ₅ - , . was converted to glutamine. This modification destroys the acceptor properties of that

site for glycosylation (the enzyme involved in the attachment of oligosaccharide chains does not recognize glutamine residues), and the resulting protein carries one carbohydrate chain less. The modified protein was demonstrated to have an extended in vivo half-life. This result suggests that the oligosaccharide chain at ^Asn _45l(448) mediates uptake of tPA from the circulation, although other interpretations (such as altered protein conformation) are possible. Finally, it was reported by Spell an et al.

((1987) Proc. Soc Compl. Carbohydrates, 77) that a modified version of tPA, in which the high-mannose carbohydrate chain at Asn..- was absent, had a longer ______ vivo half-life. Deletion of the high-mannose chain was accomplished either by endo H treatment, or by site-specific mutagenesis, converting Asn ₁₁₇ to Gln ₁₁₇ . These authors also reported that periodate treatment of tPA had a similar effect.

The present invention provides new methods for altering and/or removing the Asn-oligosaccharide chains of tPA have been developed. In one derivative, the two complex-type oligosaccharides attached to Asn's 184 and 448 of tPA are cleaved by treatment with Amidase under nondenaturing conditions. Preferred reaction conditions involve incubation of tPA (200 ug) in 200 ul of a buffer solution adjusted to pH 8.5 at 37°C_for 16h.with at least- 3 units of Amidase. tPA treated in this manner was analyzed for biological activity using the amidolytic substrate S2288. The extent of deglycosylation was determined by examining the alteration of protein mobility on SDS polyacrylamide gels (Figure 3a) and the protein's reactivity towards lectins following transfer of the protein to nitrocellulose paper (Lectin blots) (Figures 3b and 3c ⁾ .

When assayed for amidolytic activity (Little, S.P. et al. (1984) Biochemistry 23, 6191-6195), it was observed that a sample of amidase-treated tPA retained 80% of its starting biological activity following treatment with Amidase for 16 hours. Analysis of a portion of the sample by SDS-PAGE showed an increase in electrophoretic mobility of the protein corresponding to a decrease in molecular weight of 4-5 kilodaltons. Also, whereas the untreated tPA migrated as a doublet, the modified protein migrates as a single band (Figure 3a). Finally, lectin blots of modified tPA were probed with biotinylated Concanavalin A (strong binding to high mannose chains, weak binding with biantennary complex chains, little or no binding to tri- and tetraantennary chains) and biotinylated ricin (strong binding to exposed terminal galactose residues on complex type chains. The binding of tPA oligosaccharide chains by each biotinylated lectin was visualized with alkaline phosphatase-conjugated avidin. Treatment with Amidase under the aforementioned conditions completely abolishes the reactivity of tPA with ricin (Figure 3c) . The reactivity with Con A is virtually unchanged (Figure 3b). Taken together these results indicate that treatment with Amidase removed the complex-type chains at Asn's 184 and 448 (converting the asparagine to aspartate residues), but not the high mannose chain at Asn 118. It is also possible to selectively remove the complex chain at Asn 448 while leaving wholly or partially intact the sugar chain at Asn 184 by reducing the amount of Amidase used to digest tPA. In this case tPA is treated as described before, but with only 0.5U of Amidase. After this digestion tPA was observed to have 75% of the starting amidolytic activity. The shift in electrophoretic mobility of each band of the doublet

of the starting material was 2-3 kD, with a doublet still observed after the digestion (Figure 4a) . This material exhibited the same Con A reactivity as the starting material (Figure 4b) . Either of the Amidase treatments described above can be combined with Endo H digestion to also remove the high mannose chain. The preferred method involves sequential digestion with the enzymes so that the buffer composition can be optimized for each digestion. For example, following digestion with Amidase to remove both complex chains as described above, the buffer conditions were changed to 600 microliters PBS/1.6 M KSCN/0.1% Tween 80 by ultraflltration (centπfugation in a Centπcon TM

[Amicon]; gel filtration or dialysis may also be employed). Endo H (obtained from Genzyme Corporation,

Boston, MA) was added (1.5 U) and the mixture was incubated for 4 h at 37°C. Analysis of the resulting protein was then carried out as described above.

On SDS-polyacrylamide gel electrophoresis the protein migrated as a single band with an apparent molecular weight of approximately 57kD (Figure 5a) . On lectin blots reactivity with Con A (Figure 5b) was completely absent. in this case tPA has been converted to a derivative that bears a single N-acetylglucosamine residue ^" at A_*n _118. and that has no.remaining sugar residues at Asn _lg4 and Asn _448< In a similar manner, a derivative can be prepared in which there is a single GlcNAc at Asn. ₁₈ , no remaining sugar residues at Asn ₄₄₈ and a complex-type carbohydrate chain at Asn_ ₈₄ .

An alternative to Endo H treatment to remove exposed mannose residues at Asn ₁₁₇ is to treat the partially deglycosylated tPA sample with alpha-mannosidase (from jack bean for example).

Although these derivatives have been prepared from tPA produced in human melanoma cells, they could equally well be derived from tPA produced by recombinant DNA technology in animal, insect or yeast cells.

Derivatives of Prourokinase

Prourokinase (prourokinase) is a single chain, precursor form of the serine protease urokinase, which converts plasminogen to plas in. Prourokinase is currently in clinical trials as a thrombolytic agent for treatment of heart attack victims. Prourokinase has been isolated from the culture fluid of a number of cells including human kidney cells (Kasai, S., et al. (1986) J. Biol. Chem., 260, 12377-12381), human epidermoid carcinoma cells (Corti, A. et a_l. (1986) Thromb■ Haemost■ 56, 219-224), human glyoblastoma cells (Nieben, L.S. et al. (1982)

Biochemistry 21, 6410-6415), human fibroblasts (Eaton, D.L. et al. (1984) J. Biol. Chem. 259, 6241-6247) and from urine (Husain, S.S. et al. (1983) Arch. Biochem. Biophys 220, 31-38) . Recombinant prourokinase has also been produced in E. coli (Holmes, W.E. et al. (1985) Biotechnology,3y_923-929) , yeast and CHO cells.

Human prourokinase has an approximate molecular weight of 54,000 daltons. The single-chain form has a high affinity for fibrin and a low amidolytic activity. Cleavage at the Lys-Ile bond between amino acids 158 and 159 converts prourokinase to its catalytically active form (urokinase) and at the same time reduces the protein's affinity for fibrin, Prourokinase is believed to have 12 disulfide bonds, one of which Cys, ₄₈ -

Cys _27g ) holds together the activated, two-chain form of prourokinase. The prourokinase possesses three characteristic domains. The N-terminal sequence bears homology to the N-terminal region of epidermal growth factor. Next there is a single kringle, which is believed to be responsible for the fibrin affinity of prourokinase. Finally the C-terminal region bears the active site and has similarities with other serine proteases. On the basis of the cDNA-derived amino acid sequence, prourokinase has a single N-glycosylation site at Asn _3Q2 . There have been no literature reports of the structure of this Asn-oligosaccharide. We have examined the structure of the Asn-oligosaccharides derived from recombinant prourokinase isolated from CHO cells and natural prourokinase isolated from a human kidney cell line.

In each case the oligosaccharides appeared to be large, complex-type sugar chains that are undersialylated and that could promote premature clearance and, potentially, an antigenic response.

Oligosaccharides from human kidney cell prourokinase consisted of non-sialylated (51%), monosialylated (43%), and disialylated (6%) species. These oligosaccharides were treated with neuraminidase and sized on the MicroPak AX-5 column. The oligosaccharides α_isplayed-a~great- dea±-of size-- -- heterogeneity. Approximately 50% of the oligosaccharides appeared to be larger than a tetraantennary sugar chain. The large size and heterogeneity of these chains is believed to be caused by the presence of N-acetyllactosaminyl repeats or additional N-acetyl lactosaminyl branches.

Approximately 25% of the oligosaccharides were of the tetra—antennary type, and the remainder consisted of bi-and tri-antennary chains.

The sugar chains of recombinant prourokinase from CHO cells possessed zero (33%), one (38%), two (18%) or three (10%) sialic acid residues. These oligosaccharides were desialylated with neuraminidase and sized on the MicroPak AX-5 column. Bi-(37%), tri- (13%) and tetra-(20%) antennary oligosaccharide chains _were found to be present. The remainder of the oligosaccharides were larger and likely contain N-acetyllactosaminyl repeats. Like tPA, prourokinase appears to be an effective thrombolytic agent that has a short serum half-life. To enhance the therapeutic efficacy of prourokinase, we have developed a procedure to cleave the Asn-linked oligosaccharide of native prourokinase by treatment with Amidase. Preferred reaction conditions involve incubation of natural or recombinant prourokinase (200 ug) in 250 ul of a buffer solution (for example, 0.13 M sodium phosphate) adjusted to pH 8.6 at 37°c for 18 hours with 2.5 units of Amidase. The oligosaccharide chain is unexpectedly sensitive to cleavage by Amidase. Deglycosylation was confirmed by examining the alteration of protein mobility on SDS polyacrylamide gels (Figure 6)- and the-reactivity of the sample on lectin blots.

Analysis of the Amidase-treated sample by SDS-PAGE showed an increase in electrophoretic mobility of the protein corresponding to a decrease in molecular weight-of 3-4 kilodaltons. A lectin blot of Amidase-treated prourokinase probed with wheat germ

agglutinin confirmed that no complex chains remained on the protein sample. Both the recombinant and natural prourokinase were completely deglycosylated (of Asn-oligosaccharides) under the preferred reaction conditions.

When assayed for amidolytic activity, using the synthetic substrate S2244 (Wijngaards, G. et al _. . (1986) Thromb. Res. 42, 749-760), it was observed that a sample of recombinant prourokinase retained 100% of its amidolytic activity.

The reaction conditions described for prourokinase can be equally well applied to urokinase to remove the Asn-oligosaccharide from that glycoprotein. Another therapeutic protein that has been successfully deglycosylated in its native state by Amidase treatment is erythropoietin.

The modified therapeutic glycoproteins of the invention are admixed with conventional pharmaceutically acceptable carrier substances, e.g., saline, and administered to human patients or other mammals according to the same techniques by which the parent proteins are administered, e.g., intravenously, the only difference being the lower dosage which in many instances can be used because of the longer in vivo half-lives of the proteins or the invention. The following are examples of the deglycosylation reactions described in the preceding sections:

Example 1: Removal of the carbohydrate chain at Asn _44Q of tPA

Amidase (0.5 U in a volume of 2 microliters) was added to 200 microliters 0.75 M NH ₄ HCO ₃ /50 mM TrisHCl pH 8.5/50 mM EDTA/0.05% NaN ₃ containing 200 micrograms tPA. After an incubation of 16 h at 37°C,

part of the incubation mixture was subjected to SDS-polyacrylamide gel electrophoresis (11% gel). On one portion of the gel protein was visualized using silver-staining. Another portion was subjected to electrophoretic protein transfer to nitrocellulose, in order to analyze the transferred proteins for reactivity with Concanavalin A. The nitrocellulose filter was incubated in 25 mM TrisHCl pH 7.5/150 mM NaCl/0.1% Tween 20/1% BSA for 30 minutes, changing the buffer at 10 minute intervals. The filter was then incubated for one hour in 10 ml of the same buffer to which was added biotinylated ConA (Vector) at a concentration of 5 micrograms/ml. The filter was then washed for 30 minutes (3 washes at 10 min intervals) with 10 mM Tris HCl pH 8.0/150 mM NaCl/0.05% Tween 20, after which the filter was incubated for 30 minutes with alkaline phosphatase-conjugated avidin (Boehringer Mannheim) at a concentration of 1.25 microgram/ml in the same buffer system. The filter was washed for three 10 minute periods in the same buffer and then incubated in 0.1 M TrisHCl pH 9.5/0.1 M NaCl/5 mM gCl ₂ containing nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl- phosphate (Promega Biotech) at a concentration of 0.33 and 0.16 microgram/ml, respectively. The incubation was continued until sufficient color development, and washed with distilled water. Figure 4 is a photograph of the resulting~geϊτ--' - ^~

Example 2: Removal of carbohydrate chains at

Asn ₄ . _Q and As^ ₈₄ of_tPA

Amidase (3 U in a volume of 12 microliters) was added' to 200 microliters 0.75 M NH ₄ HCO ₃ /50 mM TrisHCl pH 8.5/50 mM EDTA/0.5% Na ₃ , containing 200 micrograms tPA. After an incubation of 16 h at 37°C, part of the incubation mixture was subjected to

SDS-polycrylamide gel electrophoresis (11%). On one portion of the gel protein was visualized using silver-staining. Another portion was subjected to electrophoretic protein transfer to nitrocellulose, in order to analyze the transferred proteins for reactivity with Conσanavalin A and ricin. Treatment of the nitrocellulose filter was exactly as described above. For analyzing reactivity with ricin the Tris/NaCl/Tween/BSA buffer system contained Ricinus Communis agglutinin I (Vector) in a concentration of 1 microgram/ml. Figure 3 is a photograph of the resulting gel.

Example 3: Removal of all Asn-linked carbohydrate chains of tPA After treating 200 microgram tPA with 3 U Amidase as described above, the incubation mixture was diluted to 2 ml with PBS (Dulbecco's phosphate buffered saline, pH 7.2), containing 1.6 M KSCN and 0.1% Tween 80. This solution was passed through a Centricon filter with a MW cut-off of 10,000 daltons (Amicon) by centrifugation until the volume was < 80 microliters. An additional 2 ml of PBS buffer with KSCN and Tween 80 was added and the solution concentration a final volume of 600 microliters. Endoglucosaminidase H was added (ι.5 U) and the mixture was incubated for 4 h at 37°C. Analysis,of the -resulting protein.(gel electrophoresis mobility and ConA and ricin reactivity) was as described above. Figure 5 is a photograph of the resulting gel. .. .Example 4: Removal of the carbohydrate chain at Asn_ _Q2 of prourokinase. Human tumor cell-derived or recombinant CHO cell-derived prourokinase (200 micrograms in 150 microliters of 20 mM sodium acetate, pH 4.8) was mixed

with 81 microliters of 0.4 M sodium phosphate buffer, pH 8.6, and 19 microliters of Amidase (2.5U). The reaction mixture was incubated for 18 h at 37°C. At the conclusion of the reaction the deglycosylated protein was analyzed by SDS polyacrylamide gel electrophoresis and lectin reactivity. Figure 6 is a photograph of the resulting gel.

Example 5: Removal of the Asn-Carbohydrate chains from Erythropoietin. Recombinant CHO cell-derived EPO (10 micrograms in 22 microliters of saline) was mixed with 16 microliters of 0.4M sodium phosphate buffer, pH 8.6, and 12 microliters of Amidase (3U). The reaction mixture was incubated for 18 h at 37°C. At the conclusion of the reaction the deglycosylated protein was analyzed by SDS polyacrylamide gel electrophoresis and lectin reactivity. Approximately 90% of the rEPO was fully deglycosylated by Amidase under these conditions.