Title:  Method for increasing the serum half-life of a biologically active molecule

United States Patent:  6,423,685

Inventors:  Drummond; Robert J. (Richmond, CA); Rosenberg; Steve (Oakland, CA)

Assignee:  Chiron Corporation (Emeryville, CA)

Appl. No.:  263117

Filed:  March 5, 1999

A method is provided for preparing a biologically active molecule having an increased serum half-life. The method involves conjugating a polymer such as polyethylene glycol to the biologically active molecule. Also provided are polypeptide drugs having an increased serum half-life, e.g., human urokinase plasminogen activator (human "uPA" or "hUPA") or a fragment or derivative thereof. Pharmaceutical compositions containing such molecules and methods of using them to treat uPA-mediated and uPA receptor-mediated disorders are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Overview and Definitions:

Before describing the present invention in detail, it is to be understood that this invention is not limited to specific compositions, components or process steps, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a molecule" includes a plurality of molecules and/or a mixture of different molecules, reference to a "polypeptide conjugate" includes a plurality of polypeptide conjugates and/or a mixture of different such conjugates, and the like.

In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

The terms "uPA" and "huPA" refer specifically to human urokinase-type plasminogen activator. Urokinase plasminogen activator ("uPA") is a multidomain protein which binds to a cell surface receptor and cleaves plasminogen to plasmin. uPA is involved in clot resolution, wound healing, inflammation, tissue restructuring and cancer. Variants of uPA such as uPA_1-48 have previously been found useful for treating inappropriate angiogenesis, inflammatory disorders and cancer. uPA_1-48 is a catalytically inactive protein comprising the first 48 amino acids of uPA, and still retains the binding domain for the uPA receptor. uPA_1-48 thus acts by competing with native uPA for its receptor, and thus inhibiting plasminogen activation. Prior to this invention, nothing was known of the serum half-life of uPA_1-48, and consequently there was no reason to modify uPA_1-48 to increase its serum half-life.

The term "uPA_1-48 " refers to a polypeptide having a sequence identical to the EGF-like domain of uPA (residues 1-48), or an active portion thereof. An "active portion" is one which lacks up to 10 amino acids, from either the N-terminal or C-terminal ends, or from both ends, of the uPA_1-48 polypeptide, and exhibits a Kd less than or equal to about 5 nM with uPAR. The term "active analog" refers to a polypeptide differing. from the sequence of the EGF-like domain of uPA_1-48, or an active portion thereof by 1-7 amino acids, but which still exhibits a Kd less than or equal to about 5 nM with uPAR. The differences are preferably conservative amino acid substitutions, in which an amino acid is replaced with another naturally occurring amino acid of similar character. For examnple, the following substitutions are considered "conservative": Gly - Ala; Val - Ile - Leu; Asp - Glu; Lys - Arg; Asn - Gln; and Phe - Trp - Tyr. Nonconservative changes are generally substitutions of one of the above amino acids with an amino acid from a different group (e.g., substituting Asn for Glu), or substituting Cys, Met, His, or Pro for any of the above amino acids. The uPA_1-48 polypeptides should be substantially free of peptides derived from other portions of the uPA protein. For example, a uPA_1-48 composition should contain less than about 20 wt % uPA B domain ("uPA-B", dry weight, absent excipients), preferably less than about 1.0 wt % uPA-B, more preferably less than about 5 wt % uPA-B, most preferably no amount detectable by conventional methods well known in the art. The uPA_1-48 polypeptides also preferably exclude the kringle region of uPA.

The "EGF-like domain" of uPA is that portion of the uPA molecule responsible for mediating uPA binding to its receptor ("uPAR"). The EGF-like domain, sometimes called the growth factor-like domain ("GFD"), is located within the first 48 amino acid residues of uPA. The residues essential for receptor binding activity have been localized to positions 12-32, although a peptide containing only those residues does not exhibit a binding affinity high enough to serve as a useful receptor antagonist.

The terms "uPA-disorder" and "uPA receptor-disorder" refer to a disease state or malady which is caused or exacerbated by a biological activity of uPA. The primary biological activity exhibited is plasminogen activation; other activities are related to cell migration and invasiveness. Disorders by plasminogen activation include, without limitation, inappropriate angiogenesis (e.g., diabetic retinopathy, corneal angiogenesis, Kaposi's sarcoma, and the like), metastasis and invasion by tumor cells, and chronic inflammation (e.g, rheumatoid arthritis, emphysema, and the like). Fucosylated uPA is also mitogenic for some tumor cells (e.g., SaOS-2 osteosarcoma cells), which sometimes self-activate in an autocrine mechanism. Accordingly, uPA_1-48 is effective in inhibiting the proliferation of uPA-activated tumor cells.

The term "effective amount" refers to an amount of a biologically active molecule or conjugate thereof sufficient to exhibit a detectable therapeutic effect. The therapeutic effect may include, for example, without limitation, inhibiting the growth of undesired tissue or malignant cells, inhibiting inappropriate angiogenesis, limiting tissue damage caused by chronic inflammation, and the like. The effective amount for a subject will depend upon the subject's size and health, the nature and severity of the condition to be treated, and the like. Thus, it is not possible to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by one of skill in the art using routine experimentation based on the information provided herein.

The term "pharmaceutically acceptable" refers to compounds and compositions which may be administered to mammals without undue toxicity. Exemplary pharmaceutically acceptable salts include mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like, and the salts of organic acids such as acetates, prdpionates, malonates, benzoates, and the like.

By "polypeptide" is meant a molecule comprising a series of amino acids linked through amide linkages along the alpha carbon backbone. Modifications of the peptide side chains may be present, along with glycosylations, hydroxylations and:,the like. Additionally, other nonpeptide molecules, including lipids and small molecule agents, may be attached to the polypeptide.

As used herein, the term "amino acid" is intended to include not only the L-, D- and nonchiral forms of naturally occurring amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine), but also modified amino acids, amino acid analogs, and other chemical compounds which can be incorporated in conventional oligopeptide synthesis, e.g., 4-nitrophenylalanine, isoglutamic acid, isoglutamine, .epsilon.-nicotinoyl-lysine, isonipecotic acid, tetrahydroisoquinoleic acid, .alpha.-aminoisobutyric acid, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine, 4-aminobutyric acid, and the like.

By "biologically active" is meant the ability to modify the physiological system of an organism. A molecule can be biologically active through:its own functionalities, or may be biologically active based on its ability to activate or inhibit molecules having their own biological activity. "Biologically active molecules" include, but are not limited to, small organic compounds, nucleic acids and nucleic acid derivatives, saccharides or oligosaccharides, peptide mimetics including peptides, proteins, and derivatives thereof, such as peptides containing nonpeptide organic moieties, synthetic peptides which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a peptide ligand, and peptoids and oligopeptoids which are molecules comprising N-substituted glycine, such as those described by Simon et al., Proc. Natl. Acad Sci. USA 89:9367 (1992), and antibodies, including anti-idiotype antibodies.

A "peptoid" is a polymer made up, at least in part, of monomer units of "amino acid substitutes", which are any molecule other than an amino acid, but which serve in the peptoid polymer to mimic an amino acid. Particularly preferred monomer units are N-alkylated derivatives of glycine. Peptoids are produced by linking the "amino acid substitutes" into a linear chain or cyclic structure with amino acids and/or other amino acid substitutes. The links may include, peptide bonds, esters, ethers, amines, phosphates, sulfates, sulfites, thioethers, thioesters, aliphatic bonds, carbamates and the like. Examples of amino acid substitutes include N-substituted glycine, N-alkylated glycines, N-substituted alanine, N-substituted D-alanine, urethanes, substituted hydroxy acids, such as hydroxyacetic acid, 2-hydroxypropanoic acid, 3-hydroxypropanoic acid, 3-phenyl-2-hydroxypropanoic acid, and the like. A peptoid may comprise amino acid substitutes using more than one type of link provided the chemistry for the reaction schemes are compatible and encompassed genera.lly by the reactions described herein. Other examples of amino acid substitutes and peptoids are described in Bartlett et al., PCT WO91/19735 and Zuckermann et al., PCT WO94/06451.

The terms "conventional" and "naturally occurring" as applied to peptides herein refer to polypeptides, also referred to as proteins, constructed only from the naturally occurring amino acids: Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp and Tyr.

By "conjugated" is meant the covalent linkage of at least two molecules. As described herein, a biologically active molecule is conjugated to a pharmaceutically acceptable polymer to increase its serum half-life. The polymer may or may not have its own biological activity. The suitable polymers include, for example, polyethylene glycol (PEG), polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinylether maleic anhydride, N-(2-Hydroxypropyl)-methacrylamide, dextran, dextran derivatives including dextran sulfate, polypropylene glycol, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, cellulose and cellulose derivatives, including methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and .alpha.,.beta.-Poly[(2-hydroxyethyl)-DL-aspartamide, and the like, or mixtures thereof. In a preferred embodiment, the polymer is PEG.

By "PEGylated" is meant the covalent attachment of at least one molecule of polyethylene glycol to a biologically active molecule. The average molecular weight of the reactant PEG is preferably between about 5,000 and about 50,000 daltons, more preferably between about 10,000 and about 40,000 daltons, and most preferably between about 15,000 and about 30,000 daltons. Particularly preferred are PEGs having nominal average sizes of about 20,000 and about 25,000 daltons. The method of attachment is not critical, but preferably does not alter, or only minimally alters, the activity of the biologically active molecule. Preferably the increase in half-life is greater than any decrease in biological activity. A preferred method of attachment is via N-terminal linkage to a polypeptide. PEGylated uPA_1-48 is sometimes referred to herein as PEG hu1-48.

By "increase in serumn half-life" is meant the positive change in circulating half-life of a modified biologically active molecule relative to its non-modified form. Serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. The increase is desirably at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. Preferably the increase is at least about three-fold, more preferably at least about five-fold, and most preferably at least about ten-fold, and most preferably at least about fifteen-fold. Increases of up to 28.8-fold in serum half-life are demonstrated herein.

The increase in serum half-life preferably occurs through a method that at least preserves biological activity, measured, for example, in a binding assay. In some instances, the method may even increase biological activity. However, where the method does provide a decrease in biological activity, it is preferable that the proportionate increase in serum half-life is at least as great as the proportionate decrease in biological activity. More preferably, the increase in serum half-life is greater than the decrease in biological activity, proportionately. This is not an absolute requirement, and depends, for example, on the pharmacokinetics and toxicity of the specific derivative. The percentage of biological activity which is retained is preferably about 10 to about 100%, more preferably about 15 to about 100%, and most preferably about 20 to about 100%. In an especially preferred embodiment, about 25 to about 100% of the biological activity is retained.

In a preferred embodiment, the biologically active molecule is a polypeptide. A particularly preferred polypeptide is uPA_1-48. uPA_1-48 is herein demonstrated to have a short serum half-life. Increasing the serum half-life of rapidly cleared compounds is desirable, particularly where the compounds are recombinant molecules which are difficult and costly to produce. Such an increase in half-life can reduce treatment costs, decrease the amount of agent administered, decrease the duration of administration, and lessen the exposure of patients to large volumes of pharmaceutical preparations. Conjugation of PEG to uPA_1-48, is shown herein to dramatically increase its serum half-life by as much as 28.8 fold.

The polypeptide can be produced by any suitable means, such as expression in a recombinant host cell or by chemical synthesis. The polypeptide is then purified through standard methods. Where the polypeptide is uPA_1-48, production in a yeast host cell, as described in published PCT patent application WO 94/28145, is suitable. For example, DNA encoding residues 1-48 of mature human uPA are cloned into a yeast expression vector as a fusion with the yeast alpha-factor leader (.alpha.F1), under transcriptional control of a hybrid ADH2-GAP promoter. The PCR fragment of the gene encoding huPA primer and a template plasmid, and the alkaline phosphatase treated pCBR subcloning vector containing the yeast expression cassette are digested with BgIII, followed by ligation. The subclone thus obtairned (pCBRuPA.alpha.13) is subjected to. BamHI digestion and the isolated expression cassette is ligated into the yeastshuttle vector. The expression plasmid is then transformed into the yeast host under conditions to promote the expression of the polypeptide. uPA_1-48 can then be purified as described therein, or by suitable techniques known in the art, such as centrifugation, column chromatography, anion exchange chromatography, cation exchange chromatography, or combinations thereof. Diafiltration can be used to bring the polypeptide solution to a desired concentration and/or to change the composition of the solution.

The biologically active molecule can be linked to a polymer through any available functionality using standard methods well known in the art. It is preferred that the biologically active molecule be linked at only one position in order to minimize any disruption of its activity and to produce a pharmacologically uniform product. Nonlimiting examples of functional groups on either the polymer or biologically active molecule which can be used to form such linkages include amine and carboxy groups, thiol groups such as in cysteine resides, aldehydes and ketones, and hydroxy groups as can be found in serine, threonine, tyrosine, hydroxyproline and hydroxylysine residues.

The polymer can be activated by coupling a reactive group such as trichloro-s-triazine (Abuchowski et al., (1977), J. Biol. Chem. 252:3582-3586), carbonylimidazole (Beauchamp et al., (1983), Anal. Biochem. 131:25-33), or succinimidyl succinate (Abuchowski et al., (1984), Cancer Biochem. Biophys. 7:175-186) in order to react with an amine functionality on the biologically active molecule. Another coupling method involves formation of a glyoxylyl group on one molecule and an arninooxy, hydrazide or semicarbazide group on the other molecule to be conjugated (Fields and Dixon, (1968), Biochem. J. 108:883-887; Gaertner et al., (1992), Bioconjugate Chem. 3:262-268; Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146; Gaertner et al., (1994), J. Biol. Chem. 269:7224-7230). Other methods involve formation of an active ester at a free alcohol group of the first molecule to: be conjugated using chloroformate or disuccinimidylcarbonate, which can then be conjugated to an amine group on the other molecule to be coupled (Veronese et al., (1985), Biochem. and Biotech. 11:141-152; Nitecki et al., U.S. Pat. No. 5,089,261; Nitecki, U.S. Pat. No. 5,281,698). Other reactive groups which may be attached via free alcohol groups are set forth in Wright, published European patent application 0 539 167 A₂, which also describes the use of imidates for coupling via free amine groups.

An aldehyde functionality useful for conjugating the biologically active molecule can be generated from a functionality having adjacent amino and alcohol groups. Where the biologically active molecule is a polypeptide, for example, an N-terminal serine, threonine or hydroxylysine can be used to generate an aldehyde functionality via oxidative cleavage under mild conditions using periodate. These residues, or their equivalents, can be normally present, for example at the N-terminus of a polypeptide, may be exposed via chemical or enzymatic digestion, or may be introduced via recombinant or chemical methods. The reaction conditions for generating the aldehyde typically involve addition of a molar excess of sodium meta periodate and under mild conditions to avoid oxidation at other positions in the protein. The pH is preferably about 7.0. A typical reaction involves the addition of a 1.5 fold molar excess of sodium meta periodate, followed by incubation for 10 minutes at room temperature in the dark.

The aldehyde functionality can then be coupled to an activated polymer containing a hydrazide or semicarbazide functionality to form.a hydrazone or sernicarbazone linkage. Hydrazide-containing polymers are commercially available, and can be synthesized, if necessary, using standard techniques. PEG hydrazides preferred for the invention can be obtained from Shearwater Polymers, Inc., 2307 Spring Branch Road, Huntsville, Ala. 35801. The aldehydeis then coupled to the polymer by mixing the solution of the two components together and heating to about 37^oC. until the reaction is substantially complete,. An excess of the polymer hydrazide is typically used to increase the amount of conjugate obtained. A typical reaction time is 26 hours. Depending on the thermal stability of the reactants, the reaction temperature and time can be altered to provide suitable results. Detailed determination of reaction conditions for both oxidation and coupling is set forth in Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146, and in Geoghegan, U.S. Pat. No. 5,362,852.

Such a conjugate formed between uPA_1-48 and a polymer can be used therapeutically to treat uPA- and uPA receptor-mediated disorders. A pharmaceutically acceptable solution containing the conjugate is prepared, and a therapeutically effective dose of the conjugate is administered to an individual having a uPA-mediated or a uPA receptor-mediated disorder. The conjugate is preferably administered via injection either intravenously or, more preferably, subcutaneously. Administration is repeated as necessary in order to maintain therapeutically effective levels of the conjugate.

Pharmaceutical compositions comprising a conjugate of a biologically active molecule and a polymer can be prepared. by mixing the conjugate with any pharmaceutically acceptable component, such as, for example, a carrier, a medicinal agent, an adjuvant, a diluent, and the like, as well as combinations of any two or more thereof. Suitable pharmaceutical carriers, medicinal agents, adjuvants, and diluents: are described in "Remington's Pharmaceutical Sciences," 18^th edition, by E. W. Martin (Mack Publ. Co., Easton, Pa.).

The composition may be administered in a variety of ways, including, for example, orally, parenterally (e.g., intravenously), by intramuscular: injection, by intraperitoneal injection, as suppositories, etc. The specific amount of active conjugate administered will, of course, depend on the subject being treated, the subject's weight, the manner of administration and the judgment of the prescribing physician. Information concerning dosages of various pharmacological agents may be found in standard pharmaceutical reference books, e.g., "Remington's Pharmaceutical Sciences," supra. The pharmaceutical compositions may be in solid, semi-solid or liquid dosage forms, such as, for examnple, tablets, pills, capsules, powders liquids, suspensions, and the like.

It is to be understood that while the invention has been described in conjunction with the preferred specific embodiments thereof, the foregoing description as well as the examples which follow are intended to illustrate and not limit the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

Claim 1 of 13 Claims

What is claimed is:

1. A method for conjugating urokinase plasminogen activator_1-48 having adjacent amino and alcohol groups at the N-terminus thereof to polyethylene glycol in the form of polyethylene glycol hydrazide or semicarbazide, comprising:

(a) oxidatively cleaving between the adjacent amino and alcohol groups to yield an aldehyde functionality in place thereof, and

(b) reacting the aldehyde-containing urokinase plasminogen activator_1-48 provided in step (a) with the polyethylene glycol hydrazide or semicarbazide under reaction conditions effective to promote formation of PEGylated polypeptide, wherein the polypeptide is bound to polyethylene glycol through a hydrazone or semicarbazone linkage.
 

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