RU2486246C2 - Stable recombinant adenosine deaminase - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: isolated recombinant adenosine deaminase is described, which comprises polypeptide SEQ ID NO: 1 or a version polypeptide SEQ ID NO: 1 of isolated recombinant adenosine deaminase, where the version polypeptide SEQ ID NO: 1 comprises one or more amino acid substitutions selected from the group consisting of: Gin instead Lysl98; Ala instead Thr245; and Arg instead of Gly351, and DNA encoding it. The conjugate of polyalkylene oxide with the said adenosine deaminase for treatment of adenosine deaminase-mediated diseases is presented where adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa for ADA protein. The methods of purification of the recombinant adenosine deaminase are proposed, including protein purification using ion exchange chromatography, or protein purification using hydrophobic interaction chromatography. Also the preparations of recombinant adenosine deaminase produced by these methods are provided.

EFFECT: invention enables to obtain recombinant adenosine deaminase, having increased stability.

14 cl, 1 tbl, 10 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of provisional patent application US 60/913,009, filed April 20, 2007, the contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention provides a recombinant adenosine deaminase mutated to increase stability.

BACKGROUND

Adenosine deaminase (ADA) has been used in the treatment of a disease caused by enzyme deficiency called severe combined immunodeficiency (TKID) or “bubble boy” (a boy in a bladder) for some time. For more than 15 years, Enzon Pharmaceuticals has made therapeutic ADA available to patients in the form of PEGylated ADA, obtained using the bovine ADA enzyme.

Recently, attempts have been made to replace the bovine enzyme with a recombinant enzyme (hereinafter “RADA”). As a substitute for purified natural bovine ADA, both recombinant human ("rhADA") and recombinant bovine ("rBADA") enzymes were considered. The rBADA and rhADA enzymes are somewhat less stable compared to the currently purified bovine enzyme, which is currently used. Both rhADA and rBADA are believed to decompose according to a mechanism consistent with the decomposition of cysteine: the addition of oxygen; the formation of dithiols; accelerated decomposition with increasing pH; precipitation, especially at elevated pH and sample concentration. In the reduced state, cysteine contains a reactive SH-group (sulfhydryl), and in this form is responsible for decomposition.

Experimental evidence suggests that a single, external cysteine may be responsible for the degradation observed for both rBADA and rhADA. The structure of bovine ADA (that is, native bovine ADA purified from a bovine source) is substantially similar to the structure of rhADA: both bovine ADA and rhADA have the same number of cysteines in the same positions of the primary sequence. Currently obtained recombinant human and recombinant bovine ADA contain decomposition products / impurities (dithiols), which are consistent with the reactivity of cysteine. The native bovine ADA differs in structure from the recombinant bovine ADA in that in the native bovine ADA, one mole of cysteine is associated with each mole of ADA. In addition, native bovine ADA is stable at high pH, suggesting that the cysteine associated with ADA functions as a blocking group.

One way to stabilize recombinant human and / or recombinant bovine ADA is to map the active Cys residue (Cys 74 in mature rBADA and mature rhADA) to any of oxidized glutathione, iodoacetamide, iodoacetic acid, cystine, other dithiols, and mixtures thereof. This method is described in US patent application 11 / 738,012 (of the present applicant), entitled "Stabilized Proteins", the contents of which are hereby incorporated by reference in their entirety.

Notwithstanding the foregoing, eliminating the need for an additional capping step by altering the protein structure in order to ensure intrinsic stability directly upon expression is preferred. US 5346823 describes the stabilization of prokaryotic proteases, such as subtilisin, as well as neutral proteases, by replacing destabilizing Cys residues with Ser and other amino acid residues through mutation. However, a mutation analysis of active sites in ADA showed that replacement of the Cys residue (Cys 262) led to a significant decrease in enzyme activity, Bhaumik et al 1993, The J. of Biol. Chem., 268 (8): 5464-5470. Thus, prior to the present invention, stabilization of adenosine deaminases by replacing the active and external Cys residue with another amino acid residue while maintaining the optimal beneficial activity of the enzyme was not known.

Thus, it is advantageous to provide stable rBADA and rhADA, that is, without significant decomposition during storage and processing, at pH levels used for optimal PEGylation of the enzyme.

SUMMARY OF THE INVENTION

Thus, the invention provides a recombinant ADA in which any oxidizable cysteine residue is replaced by an unoxidizable amino acid residue compared to the wild-type form of the ADA enzyme. ADA mutein includes a non-oxidizable amino acid residue, which is one of the natural L-amino acids, for example, alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine and / or known variants and derivatives of natural L-amino acids, for example, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic ki slot, 2-aminobutyric acid, 4-aminobutyric acid, piperidic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, , 2'-diaminopimelic acid, 2,3-diaminopropionic acid, n-ethyl glycine, n-ethyl asparagine, hydroxylisine, allo-hydroxylisine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycinine, n-methylglycine, n methylisoleucine, 6-n-methyllysine, -metilvalin, norvaline, norleucine, ornithine, etc. Optionally, methionine or tryptophan are excluded because they are potentially oxidizable.

More preferably, the non-oxidizable amino acid residue is one of serine, alanine, asparagine, glutamine, glycine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine. Serine is most preferred. In some preferred embodiments, oxidizable cysteine is located at approximately 74 positions of the mature ADA protein. Recombinant ADA is preferably recombinant bovine ADA or recombinant human ADA, that is, for example, translated from a DNA molecule corresponding to SEQ ID NO: 2 or SEQ ID NO: 4, and which preferably includes SEQ ID NO: 1 or SEQ ID NO: 3 When the recombinant ADA is a recombinant bovine ADA corresponding to SEQ ID NO: 1, the ADA is optionally expressed with a polymorphism selected from one or more Gln instead of Lys ₁₉₈ ; Ala instead of Thr ₂₄₅ ; and Arg instead of Gly ₃₅₁ .

The invention also provides a polyalkylene oxide-ADA conjugate, where the polyalkylene oxide is preferably polyethylene glycol. Optionally, polyethylene glycol is conjugated to recombinant adenosine deaminase via a chemical linker selected from the group consisting of succinimidyl carbonate, thiazolidinedione, urethane, succinimidyl succinate and amide linkers. Succinimidyl carbonate is preferred. The polyethylene glycol is preferably covalently attached to the epsilon amino group Lys of the recombinant adenosine deaminase.

The polyethylene glycol-ADA conjugate includes at least 1 (i.e., one or more) polyethylene glycol chains attached to the epsilon amino groups, preferably at least 5 (i.e., five or more) polyethylene glycol chains attached to the epsilon- amino groups, or, more preferably, from about 11 to about 18 polyethylene glycol chains attached to the epsilon amino groups of the Lys residues of the recombinant ADA.

The polyethylene glycol conjugates of the invention have a molecular weight of from about 2000 to about 100000 kDa, or, more preferably, from about 4000 to about 45000 kDa.

The invention further provides a method for purifying the recombinant adenosine deaminases of the invention. For example, recombinant adenosine deaminase is preferably purified by ion exchange chromatography (e.g., Capto Q, DEAE and SP chromatography), and recombinant adenosine deaminase SEQ ID NO: 1 is preferably purified by hydrophobic interaction chromatography.

In addition, the present invention further provides a method of treating an ADA-mediated disease in mammals, comprising administering an effective amount of a recombinant ADA according to the present invention. ADA-mediated disease includes, for example, TCID, cancer, and the like.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides stable recombinant adenosine deaminase enzymes. The adenosine deaminases of the invention are obtained by replacing the cysteine residue, which undergoes oxidation processes when the enzyme is in solution, with an acceptable alternative amino acid residue that retains the activity, charge and tertiary structure of the enzyme while eliminating the source of destructive instability.

A. Definitions

In order to provide clarity in the description of the invention, the following are definitions of several terms.

The term "recombinant" refers to a protein obtained using cells that in their native state do not contain an endogenous copy of DNA capable of expressing the protein. Cells produce a recombinant protein because they have been genetically altered by introducing an appropriate isolated nucleic acid sequence. The term also includes a reference to a cell, or nucleic acid, or a vector that has been modified by introducing a heterologous (exogenous or foreign) nucleic acid or by altering a native nucleic acid to obtain a form that is not native to the cell, or the cell is derived from cells modified in this way. Thus, for example, recombinant cells express genes that are not present in the native (non-recombinant) form of the cell, express mutant genes that are present in the native form, or express native genes that are otherwise incorrectly expressed, are insufficiently expressed or not expressed at all.

As used herein, a “nucleic acid” or “nucleic acid sequence” includes a reference to a deoxyribonucleotide or ribonucleotide polymer in either single or double stranded form and, unless otherwise limited, encompasses known analogs of natural nucleotides that hybridize with nucleic acids in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence includes an appropriate complementary sequence.

The term "coding" as applied to said nucleic acid includes reference to nucleic acids that include information for translation into a specific protein. Information is determined by codons.

A "host cell" is a cell that can support the replication or expression of an expression vector. The host cells may be prokaryotic cells, such as E. coli cells, or eukaryotic cells, such as yeast, insect, amphibian or mammalian cells.

As used herein, “polypeptide”, “peptide” and “protein” are used interchangeably and include reference to a polymer consisting of amino acid residues.

The term “residue” or “amino acid residue” or “amino acid” includes a reference to an amino acid that is part of a protein, polypeptide or peptide (collectively “peptide”).

An amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of naturally occurring amino acids that can function similarly to naturally occurring amino acids.

“Transfection” refers to the uptake of an expression vector by a host cell, regardless of whether any coding sequences are actually expressed or not. Numerous transfection methods are known to one of ordinary skill in the art. For example, transfection is carried out in the presence of an expression vector and high concentrations of CaPO ₄ , using electroporation, using phage or a viral expression vector to integrate into the host cell, by mechanically inserting the nucleic acid, and even by culturing the host cell in the presence of unpacked nucleic acid fragments acids. Transfection is usually considered successful when any indication of the function of the target vector is observed in the host cell.

"Transformation" describes the introduction of a nucleic acid into the body so that the nucleic acid is capable of replication either as an extrachromosomal element or by integration into the host chromosome. Depending on the host cell used, the transformation is carried out using methods known in the art that are appropriate for the particular host cell. Calcium treatment using calcium chloride as described by Cohen, S. N. Proc. Natl. Acad. Sci. (USA), 69: 2110 (1972); and Mandel et al, J. Mol. Biol. 53: 154 (1970), usually used for prokaryotic or other cells that have cell walls (e.g., many bacterial and / or plant cells). For mammalian cells without such cell walls, the preferred method is precipitation of calcium phosphate Graham, F. and van der Eb, A., Virology, 52: 456-457 (1978). General aspects of transforming a mammalian host cell system have been described in US Pat. No. 4,399,216, published August 16, 1983. Yeast transformation is usually performed according to the method of Van Solingen, P., et a., J. Bad., 130: 946 (1977) and Hsiao, C. L., et al., Proc. Natl. Acad. Sci, (USA) 76: 3829 (1979). However, any other methods of introducing a nucleic acid, for example, DNA, into cells, such as, for example, microinjection, lipofection or fusion of protoplasts, can also be used.

As used herein, the term “complementary” as applied to a nucleic acid refers to a pair of opposite chains synthesized (using the Watson-Crick principle of complementarity) by replicating the first nucleic acid molecule using the specified molecule as a matrix to form a new, second nucleic acid chain. In one aspect of the invention, two nucleic acid molecules are considered complementary to each other when they hybridize or bind under stringent conditions.

The term “functionally connected” refers to such an adjacent arrangement of elements, for example, a regulatory region and an open reading frame, in which the normal functioning of the elements can be realized. Thus, an open reading frame that is “operably linked” to regulatory sequences refers to a configuration in which a coding sequence can be expressed under the control of said sequences.

"Regulatory sequences" refer to nucleic acid sequences necessary for the expression of a functionally linked coding sequence in a specific host organism. Regulatory sequences suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly other sequences that are still poorly understood. Eukaryotic cells are known to use, for example, regulatory sequences such as promoters, polyadenylation signals and enhancers, as well as many others.

An “expression system” or “expression vector” refers to nucleic acid sequences containing a desired coding sequence and regulatory sequences in functional communication, through which host organisms transformed with these sequences are capable of producing encoded proteins. For transformation, the expression system can be included in the vector, however, then the corresponding nucleic acid molecule can also be integrated into the chromosome of the host organism.

Used in the present description, "cell", "cell line" "cell culture" are used interchangeably, with all such designations include offspring. Thus, "transformants" or "transformed cells" include the primary target cell and cultures obtained from it without regard to the number of passages. It should also be understood that all offspring may not be strictly identical in genomic content due to intentional or random mutations. Also included is a mutant progeny having the same functional properties that were identified in the originally transformed cell. In cases where other definitions are needed, this will be clear from the context.

In the framework of the present invention, the term "residue", as it is understood, means that part of the compound to which it refers, for example, PEG, ADA, amino acid, etc., which remains after it has undergone a substitution reaction with another compound.

In the framework of the present invention, the term "polymer residue" for example, "PEG residue", as it is understood, means that part of the polymer or PEG that remains after it has undergone reaction with other compounds, groups, etc.

As used herein, the term “alkyl” as used herein refers to a saturated aliphatic hydrocarbon, including linear, branched and cyclic alkyl groups. The term “alkyl” also includes alkyl thioalkyl, alkoxyalkyl, cycloalkylalkyl, heterocycloalkyl and C _1-6 alkylcarbonylalkyl groups. Preferably, the alkyl group includes 1-12 carbon atoms. More preferably, the alkyl group is lower alkyl comprising from about 1-7 carbon atoms, most preferably about 1-4 carbon atoms. The alkyl group may be substituted or unsubstituted. When the alkyl group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C _1-6 hydrocarbonyl, aryl and amino.

As used herein, the term “substituted”, as used herein, refers to the addition or replacement of one or more atoms contained within a functional group or compound to one of the groups selected from halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkylthio, alkylthioalkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C _1-6 -carbonyl, aryl .

The term “alkenyl,” as used herein, refers to groups containing at least one carbon-carbon double bond, including linear, branched and cyclic groups. Preferably, the alkenyl group includes about 2-12 carbon atoms. More preferably, the alkenyl group is lower alkenyl comprising from about 2-7 carbon atoms, most preferably about 2-4 carbon atoms. The alkenyl group may be substituted or unsubstituted. When the alkenyl group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, cyano, nitro, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C _1-6 alkylcarbonylalkyl, aryl and amino.

The term “alkynyl,” as used herein, refers to groups containing at least one carbon-carbon triple bond, including linear, branched and cyclic groups. Preferably, the alkynyl group includes about 2-12 carbon atoms. More preferably, the alkynyl group is lower alkynyl comprising from about 2-7 carbon atoms, most preferably about 2-4 carbon atoms. The alkynyl group may be substituted or unsubstituted. When the alkynyl group is substituted, the substituted group (s) preferably includes halo, hydroxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C _1-6 hydrocarbonyl, aryl and amino. Examples of “alkynyl” include propargyl, propyne and 3-hexine.

As used herein, the term “aryl” refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring may optionally be condensed or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.

As used herein, the term “cycloalkyl” refers to a cyclic C _3-8 hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. cycloheptyl and cyclooctyl.

As used herein, the term “cycloalkenyl” refers to a cyclic C _3-8 hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl include cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,3-cyclohexadienyl, cycloheptenyl, cycloheptatrienyl and cyclooctenyl.

As used herein, the term “cycloalkylalkyl” refers to an alkyl group substituted with a C _3-8 cycloalkyl group. Examples of a cycloalkylalkyl group include cyclopropylmethyl and cyclopentylethyl.

In the framework of the present invention, the term "alkoxy" refers to an alkyl group with the specified number of carbon atoms attached to the main part of the molecule through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.

As used herein, an “alkylaryl” group refers to an aryl group substituted with an alkyl group.

As used herein, an “aralkyl” group refers to an alkyl group substituted with an aryl group.

As used herein, the term “alkoxyalkyl” refers to an alkyl group substituted with an alkoxy group.

As used herein, the term “alkyl-thio-alkyl” refers to an alkyl-S-alkyl-thioether group, for example methylthio methyl or methylthioethyl.

In the framework of the present invention, the term “amino” refers to a nitrogen-containing group, which, as is known in the art, is derived from ammonia by replacing one or more hydrogen radicals with organic radicals. For example, the terms “acylamino” and “alkylamino” refer to certain organic radicals N-substituted with aryl and alkyl substituent groups, respectively.

As used herein, the term “alkylcarbonyl” refers to a carbonyl group substituted with an alkyl group.

As used herein, the terms “halogen” or “halo” refer to fluoro, chloro, bromo and iodo.

As used herein, the term “heterocycloalkyl” refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen and sulfur. The heterocycloalkyl ring may optionally be fused or otherwise attached to other heterocycloalkyl rings and / or non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups include from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine and pyrazole. Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl and pyrrolidinyl.

As used herein, the term “heteroaryl” refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen and sulfur. The heteroaryl ring may be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings, or heterocycloalkyl rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isothiazolyl, benzisothiolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolaziolazolaziolazolazolazolazolazolazolyazolazol

As used herein, the term “heteroatom” refers to nitrogen, oxygen, and sulfur.

In some embodiments, substituted alkyls include carboxyalkyls, aminoalkyls, dialkylamino, hydroxyalkyls and mercaptoalkyls; substituted alkenyls include carboxyalkenyls, aminoalkenyls, dialkenylamino, hydroxyalkenyls and mercaptoalkenyls; substituted alkynyls include carboxyalkynyls, aminoalkynyls, dialkynylamino, hydroxyalkynyls and mercaptoalkynyls; substituted cycloalkyls include groups such as 4-chlorocyclohexyl; aryls include groups such as naphthyl; substituted aryls include groups such as 3-bromophenyl; aralkyls include groups such as tolyl; heteroalkyls include groups such as ethylthiophene; substituted heteroalkyls include groups such as 3-methoxy-thiophene; alkoxy includes groups such as methoxy; and phenoxy includes groups such as 3-nitrophenoxy. “Halo,” as understood, includes fluorine, chlorine, iodine, and bromine.

In the framework of the present invention, a "positive integer", as it should be understood, includes an integer greater than or equal to 1 and, as will be understood by those of ordinary skill in the art, is within the scope of rationality.

In the framework of the present invention, the term “bound”, as understood, includes covalent (preferably) or non-covalent attachment of one group to another, that is, as a result of a chemical reaction.

The terms "effective amounts" and "sufficient amounts" in the framework of the present invention should mean an amount that ensures the achievement of the desired effect or therapeutic effect, and the effect as such is understood by those of ordinary skill in the art.

In the framework of the present invention, the term "adenosine", as is understood, includes nucleosides adenosine and deoxyadenosine. Adenosine also includes adenosine and deoxyadenosine present in the form of AMP, ADP, ATP, dAMP, dADP or dATP.

In the framework of the present invention, "adenosine-mediated disease" or "adenosine deaminase-reactive disease", as it is understood, broadly includes any diseases, pathological conditions or disorders in which the administration of ADA or its active fraction, etc., is successfully applied, independently from the route of administration.

In the framework of the present invention, “treatment of an adenosine-mediated disease” or “treatment of an adenosine deaminase-reactive disease”, such as TCID, as understood herein, means eliminating, minimizing or reducing the onset of symptoms or conditions compared to those observed in the absence of treatment using ADA. The degree of convalescence can be confirmed, for example, by reducing the level of adenosine.

In a broad sense, treatment of an adenosine-mediated disease should be considered successful when the desired clinical response is obtained. Alternatively, treatment success can be confirmed by obtaining at least 20% or preferably 30%, more preferably 40% or higher (i.e., 50% or 80%) reduction in adenosine levels , including other clinical indicators considered by a specialist in this field, compared with that observed in the absence of treatment with ADA.

In addition, the use of the terms in the singular for the convenience of description should in no way be considered limiting in any way. Thus, for example, reference to a composition comprising an enzyme refers to one or more molecules of a given enzyme. You must also understand that the present invention is not limited to the specific configurations, stages of the methods and materials described in this application, since such configurations, stages of the methods and materials may vary slightly.

You must also understand that the terminology used in this application is used only to describe specific embodiments and should not be limiting, since the scope of the present invention will be determined by the attached formula and its equivalents.

B. ADA Enzymes Obtained by Genetic Engineering

Initial attempts to obtain a recombinant ADA enzyme, including enzymes expressed from human or bovine genes, revealed storage instability not previously observed for natural ADA obtained from the intestinal tract of cattle. Studies of the degradation products of rhADA and rBADA confirmed that both ADA enzymes decompose according to a mechanism consistent with the decomposition of cysteine. For example, the addition of oxygen to rhADA leads to the formation of more hydrophilic compounds than rhADA, having a mass of 16 and 32 Da higher than rhADA. In addition, this leads to the formation of dithiols (as indicated by the reversal of the formation of degradation products as a result of the addition of dithiothreitol [DTT]); increased decomposition rate with increasing pH; precipitation, especially with increasing pH and at a high concentration of samples, which suggests the possibility of the formation of intermolecular disulfide bonds, leading to the formation of insoluble aggregates.

The inventors of the present invention have determined that a single, external cysteine is responsible for the degradation observed for rhADA. Bovine (non-degradable) ADA has a structure similar to that of rhADA: both bovine ADA and rhADA contain the same amount of cysteines in the same positions of the primary sequence. In addition, rBADA also contains degradation products / impurities (dithiols), which are consistent with the reactivity of cysteine. Native bovine ADA differs in structure from rBADA in that one mole of cysteine is associated with each mole of ADA, and the native bovine ADA is stable at high pH, which suggests that cysteine associated with ADA functions as a blocking group. Cysteine associated with native bovine ADA can be removed by treatment with a reducing agent, such as monothioglycol or DTT. Not wanting to associate this with any theory or hypothesis, it can be assumed that the cysteine group is conjugated to ADA via a disulfide bond as follows:

ADA-S-S-cysteine,

where one cysteine in the primary ADA sequence is associated with a cysteine molecule. The cysteines present in such disulfide bonds are resistant to the oxidative degradation processes mentioned in the first section. Cysteine residues are located at positions 74, 152, 153, 168 and 261 of the human and bovine mature ADA. A study of the three-dimensional structure of bovine ADA obtained using x-ray diffraction analysis (Kinoshita et al., 2005, Biochemistry, 44: 10562-10569) showed that cysteines at positions 74, 152, 153, 168 and 261 cannot participate in the formation of intramolecular disulfide bonds . Structural geometric constraints are known to generally prevent the formation of disulfide bonds by vicinal cysteine residues, such as those located at positions 152 and 153 of the ADA. Thus, all cysteine residues are potentially in a reduced state and, therefore, are potential candidate sites for oxidative destruction reactions. However, the aforementioned visual analysis of the three-dimensional structure of bovine ADA showed that cysteine 74 quite accurately is exposed to the solvent to a greater extent than the other four cysteines and, in addition, that the other four cysteines seem to be deepened into the structure of the enzyme to such an extent that significant interaction with solvated reagents is probably prevented (if the protein is not denatured). The existence of a single reactive cysteine residue could explain the mono-derivatization of native bovine adenosine deaminase, which apparently occurs as a result of post-translational modification.

The facts set forth above indicate that reactive cysteine at position 74 may be responsible for the degradation observed for rhADA and rBADA, and that capping of the reactive -SH cysteine group protects rhADA or rBADA from the obvious oxidative degradation processes observed for these recombinant enzymes. In order to confirm this, the following experiment was carried out. Recombinant chADA at a concentration of approximately 0.6 mg / ml was treated with 125 mm iodoacetamide (IAA) in sodium phosphate buffer with a pH of 7.4 for 16 hours at 37 ° C. Within a few minutes after the start of the reaction, analysis of the sample by RP-HPLC with UV and mass spectrometric detection showed that approximately 70.9% of rhADA underwent IAA mono-derivatization and 17.2% underwent derivatization at two sites. After incubation for 2 and 16 hours, the chromatographic profile was not significantly changed, indicating that the derivative was resistant to the oxidative degradation typical of rhADA. A similar rhADA sample was then prepared, but without IAA, which was analyzed in a similar manner. After incubation for 16 hours at 37 ° C and pH 7.4, the rhADA protein degraded by 30% (the degree of decomposition of the sample compared to the initial degradation). The results correspond to a single, predominant external cysteine that can be protected by capping with iodoacetamide. These experiments are described in more detail in US patent (the present applicant) 11 / 738,012, entitled "Stabilized proteins", which is incorporated into this description by reference, as described above.

Although capping effectively prevents the oxidative degradation of reactive cysteine in ADA, the use of such a blocked enzyme requires an additional process step. Thus, the direct elimination of the unstable Cys residue from the coding gene by substituting another amino acid was investigated. An amino acid suitable for substitution is one that does not undergo the same type of oxidation, does not interfere with the formation of the tertiary structure of the folded ADA protein, and in a typical embodiment of the invention is selected so as not to undergo accidental binding to activated polyalkylene oxide during the formation of the conjugate. Suitable for replacing the oxidizable cysteine according to the invention is any of the prior art natural amino acids and / or unnatural amino acids and / or their derivatives that meet the specified criteria. An exemplary list of such amino acids includes natural L-amino acids, such as: alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan and tyrosine. Tryptophan and methionine can be oxidized relatively readily and are less preferred in some optional embodiments.

Methods for producing recombinant proteins using site-specific incorporation of unnatural amino acids into host cells are described in the literature, for example, Liu et al., 2007, Nat. Methods 4 (3): 239-44, Xie et al., 2006 Nat. Rev. Mol. Cell. Biol. 7 (10): 775-82, Ryu et al., 2006, Nat. Methods 3 (4): 263-65, Deiters et al, 2004, Bioorg. Med. Chem Lett. 14 (23): 5743-5, Bogosian et al., 1989, J. Biol. Chem. 264 (1): 531-9, Tang et al., 2002, Biochemistry 41 (34): 10635-45, Budisa et al., 1995. Eur. J. Biochem. 230 (2): 788-96; and Randhawa et al., 1994, Biochemistry, 33 (14): 4352-62. Thus, a substitute amino acid may also include a modified or less typical amino acid, such as: 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidic acid, 6 aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, n- ethyl glycine, n- ethylasparagin, hydroxylisine, allo-hydroxylisine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, n-methylglycine, sarcosine, n-methylisoleucine, 6-n-methyllysine, n-methylvaline, norvaline, norleucine and ornithine.

More preferred natural amino acids to which cysteine is optionally substituted in recombinant ADA include, for example, alanine, serine, aspargin, glutamine, glycine, isoleucine, leucine, phenylalanine, threonine, tyrosine and valine. Serine is most preferred and is given as an example hereinafter.

Thus, DNA molecules expressing human and bovine wild-type adenosine deaminase were obtained that were optimized for codons for expression in E. coli and mutations to express the rbADA mutein and rhADA mutein, including the Ser residue at position 74 of the corresponding mature proteins (position 75 of the translated protein) instead of the natural residue Cys. These are Ser ₇₄ rhBADA (SEQ ID NO: 1) and Ser ₇₄ rhADA (SEQ ID NO: 3), respectively. In addition, it should be noted that natural bovine ADA isolated from the intestinal tract of cattle also contains 6 residues post-translationally removed from the C-terminal region. An optional feature of the present invention is that the Ser ₇₄ -RBADA according to the invention is either expressed without 6 C-terminal residues (as mutein) or post-translationally modified to remove said 6 C-terminal residues not found in purified natural bovine ADA.

It should also be noted that natural bovine ADA isolated from the intestinal tract of cattle has polymorphisms: in relation to SEQ ID NO: 5, polymorphisms of bovine ADA include, for example, glutamine at position 198 instead of lysine, alanine at position 245 instead of threonine; arginine at position 351 instead of glycine. Thus, it is contemplated that the recombinant mutein (at position 74) of a bovine ADA according to the invention may also include additional substitutions at one or more of the indicated positions or analogues of these positions: Gln instead of Lys ₁₉₈ ; Ala instead of Thr ₂₄₅ ; Arg instead of Gly ₃₅₁ .

In a further aspect of the invention, the present invention provides isolated DNAs that encode an ADA mutein having the amino acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 3 described herein. Other DNAs encoding an ADA mutein with one or more substitutions: Gln instead of Lys ₁₉₈ ; Ala instead of Thr ₂₄₅ ; Arg instead of Gly ₃₅₁ , are also included in the scope of the present invention.

A suitable expression vector can be obtained from genomic or cDNA encoding rhADA or rBADA, respectively, which are optionally under the control of a suitable functionally linked inducible promoter. The DNA is preferably codon-optimized for the appropriate host cell and mutated using any suitable method known in the art, for example, highly efficient site-directed mutagenesis using oligonucleotides (Olsen DB and Eckstein F, Proc Natl Acad Sci USA 87: 1451-5; 1990), synthesis of full-sized genes using overlapping long oligonucleotides (Vasantha N and Filpula D., Gene 76: 53-60; 3989), PCR-mediated gene synthesis (Jayaraman K et al., Proc Natl Acad Sci USA 88: 4084- 88: 1991) or PCR with elongation of overlapping primers (Pogulis RJ et al., Methods Mol Biol 57: 167-76; 1996).

Typically, prokaryotes are preferred for the initial cloning of DNA sequences and vector constructs used in the invention. For example, E. coli strain K12 MM 294 (ATCC No. 31,446) is most preferred. Other microbial strains that can be used, simply by way of example, include strains of E. coli such as E. coli B and E. coli X1776 (ATCC No. 31,537). The above strains can be used, as well as, for example, strains of E. coli W3110 (F-, lambda, prototrophic, ATCC No.27,325), K5772 (ATCC No.53,635) and SR101, bacilli such as Bacillus subtilis, and other enterobacteria such as Salmonella typhimurium or Serratia marcescens, and various types of pseudomonads.

Typically, plasmid vectors containing replicon and regulatory sequences derived from species compatible with the host cell are used in conjunction with these host organisms. Conventional plasmid vectors are double-stranded DNA molecules, preferably containing enzyme recognition sites suitable for embedding exogenous DNA sequences, a selective antibiotic resistance gene, a replication initiation site for autonomous reproduction in a host cell, and a gene for selecting or selecting clones that contain a recombinant insert DNA Available plasmid vectors suitable for use in E. coli cells include, for example, pET3, pET9, pET11 and other pET series (included in the Novagen Corporation catalog), pBAD, trc, phoA, trp and plasmids O _{L / R} / P _{L / R.}

Just as an example, E. coli is usually transformed using the plasmid pBR322 obtained from E. coli (see, for example, Bolivar et al., 1977, Gene. 2:95). pBR322 contains ampicillin and tetracycline resistance genes, and thus provides an easy way to identify transformed cells. Similarly, pUC plasmids are convenient vectors for the selection and replication of DNA molecules (Yanisch-Perron, et al., 1985, Gene 33: 103-119, the contents of which are fully incorporated into this application by reference). Plasmid pBR322 or another microbial plasmid, or phage, must also contain, or are modified to include promoters that can be used by the microbial organism to express its own encoded proteins.

Promoters commonly used in recombinant DNA constructs include systems based on the beta-lactamase promoter (penicillinase) and the lactose promoter (Chang et al., 1978, Nature, 375: 615; Itakura ef al., 1977, Science, 198: 1056; Goeddel et al., 1979, Nature, 281: 544), as well as the tryptophan (trp) promoter (Goeddel et al., 1980, Nucleic Acids Res., 8: 4057; EPO Appl. Publ. No.0036,776). They are used most often, although other microbial promoters have been discovered and used, their nucleotide sequences have been published, which allows a qualified specialist to functionally integrate them into known vectors, for example, plasmid vectors.

Just as an example, the regulation of transcription in E. coli can be provided by any of the following inducible promoters: lac, trp, phoA, araBAD, T7, trc and derivatives of lambda promoters P _L and P _R , as well as others known in the art (for example, Makrides, 1996, Microbiol. Rev. 60: 512-538, the entire contents of which are incorporated herein by reference).

Suitable induction conditions optionally compatible with the vector include, for example, the addition of arabinose, lactose or thermal induction, a lack of phosphates, a lack of tryptophan and the like. Preferably, the inducing element is the Lac operon, which is induced by isopropylthiogalactoside (IPTG).

A suitable signal sequence (signal peptide) can be obtained from pelB, fd pIII or ompA.

Suitable selective antibiotic resistance markers are known in the art and include, for example, those that confer resistance to ampicillin, kanamycin, chloramphenicol, rifampicin or tetracycline, among others.

Suitable replication initiation sequences include the corresponding sequences from the following plasmids: pUC19, pACYC117, pUB110, pE194, pAMB1, pIJ702, pBR322, pBR327 and pSC101.

Suitable termination sequences include, for example, the main phage terminator fd, TΦ, and rrnB.

In addition to prokaryotes, eukaryotic microorganisms such as yeast cultures may also be used. Of the eukaryotic microorganisms, Saccharomyces cerevisiae or conventional baker's yeast is most commonly used, although there are also many other strains available. For expression in Saccharomyces, the YRp7 plasmid is usually used, for example (Stinchcomb et al., 1979, Nature, 282: 39; Kingsman et al., 1979, Gene, 7: 141; Tschemper et al., 1980, Gene, 10: 157) . This plasmid already contains the trp1 gene, which provides a selective marker of choice for a mutant strain of yeast lacking the ability to grow in the absence of tryptophan, for example, ATCC No.44,076 or PEP4-1 (Jones, 1977, Genetics, 85:12). The presence of an abnormality in trp1, which is a feature of the genome of the yeast host cell, provides an effective medium for detecting growth transformants in the absence of tryptophan.

An expression system based on Pichia pastoris has been shown to provide a high level of production of several proteins (Cregg, JM et al., 1993, Bio / Technology 11: 905-910, the contents of which are fully incorporated herein by reference) and can be used to expression of ADA in the form of soluble protein in the cytoplasm of Pichia pastoris.

Suitable promoter sequences in yeast vectors include promoters of 3-phosphoglycerate kinase (Hitzeman et al., 1980, J. Biol. Chem., 255: 2073) or other glycolytic enzymes (Hess et al., 1968, J. Adv. Enzyme Reg., 7: 149; Holland et al., 1978, Biochemistry, 17: 4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphatisurase aminose, 3-phosphoglycerate phosphatasease, glucokinase. When constructing suitable expression plasmids, the termination sequences associated with the indicated genes are also inserted into the expression vector, ligating them to the 3'-end of the expressed sequence to ensure mRNA polyadenylation and transcription termination. Other promoters that have the added benefit of regulated transcription growth conditions include the promoter region of alcohol dehydrogenase 2, isocytotochrome C, acid phosphatase, degradation enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase and enzymes responsible for the use of maltose and maltose. Suitable is any plasmid vector containing a yeast compatible promoter, a replication initiation site, and termination sequences.

The replication initiation site can be provided either by the construction of a vector including an exogenous initiation site, for example, which can be derived from SV40 or another viral source (e.g., polyoma virus, adenovirus, VSV, BPV), or can be provided by the mechanism of chromosome replication of the host cell. If the vector is integrated into the chromosome of the host cell, the latter is often sufficient. Other useful plasmid elements may include expressed genes encoding chaperone, proline isomerase or protein disulfide isomerase proteins.

C. Polymer conjugates

In another aspect of the invention, an ADA mutein, such as Ser ₇₄ β-rBADA (SEQ ID NO: 1) and Ser ₇₄ β-rhADA (SEQ ID NO: 3) proteins, is conjugated to a suitable polymer to form polymer conjugates.

In preferred aspects, the ADA mutein polypeptide is conjugated to a substantially non-antigenic polymer, preferably polyalkylene oxide (PAO).

ADA-polymer conjugate generally correspond to the formula (I):

(I) [R-NH] _z - (ADA)

wherein

(ADA) is a recombinant adenosine deaminase mutein or active fragment thereof;

NH- is an amino group of an amino acid present in an ADA mutein that serves to attach to a polymer;

(z) is a positive integer, preferably from about 1 to about 80, more preferably from about 5 to about 80, most preferably from about 11 to about 18; and

R includes a substantially non-antigenic polymer residue that is attached to the ADA in a reversible or irreversible manner.

In more preferred aspects, the polymers include polyethylene glycol (PEG), where the PEG may be linear, branched or multipath. Polyethylene glycol has the general formula:

-O- (CH ₂ CH ₂ O) _n -,

in which (n) is a positive integer, preferably from about 10 to about 2300, more preferably from about 40 to about 2300. The average molecular weight of the polymers is in the range from about 2000 to about 300,000 Da. More preferably, the polymers have an average molecular weight of from about 4,000 Da to about 45,000 Da, even more preferably from 4,000 Da to about 20,000 Da. Most preferably, the PEG has a molecular weight of approximately 5000 daltons. Other molecular weights are also considered in order to meet the needs of a person skilled in the art.

In the alternative, part of the remainder of the polyethylene glycol (PEG) of the invention can be represented by the structure:

-Y ₁₁ - (CH ₂ CH ₂ O) _n -CH ₂ CH ₂ Y ₁₁ -,

-Y ₁₁ - (CH ₂ CH ₂ O) _n -CH ₂ C (= Y ₁₂ ) -Y ₁₁ -,

-Y ₁₁ - (= Y ₁₂ ) - (CH ₂ ) _a11 -Y ₁₃ - (CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y ₁₃ - (CH ₂ ) _a11 -C (= Y ₁₂ ) -Y ₁₁ -,

-Y ₁₁ - (CR ₁₁ R ₁₂ ) _a12 -Y ₁₃ - (CH ₂ ) _b11 -O- (CH ₂ CH ₂ O) _n - (CH ₂ ) _b11 -Y ₁₃ - (CR ₁₁ R ₁₂ ) _a12 -Y ₁₁ -,

-Y ₁₁ - (CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -,

-Y ₁₁ - (CH ₂ CH ₂ O) _n -CH ₂ C (= Y ₁₂ ) -,

C (= Y ₁₂ ) - (CH ₂ ) _a11 -Y ₁₃ - (CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -Y ₁₃ - (CH ₂ ) _a11 -C (= Y ₁₂ ) -, and

- (CR ₁₁ R ₁₂ ) _a12 -Y ₁₃ - (CH ₂ ) _b11 -O- (CH ₂ CH ₂ O) _n - (CH ₂ ) _b11 -Y ₁₃ - (CR ₁₁ R ₁₂ ) _a12 -,

Where:

Y ₁₁ and Y ₁₃ independently represent O, S, SO, SO ₂ , NR ₁₃ or a bond;

Y ₁₂ are O, S, or NR ₁₄

R _{11-14 are} independently selected from hydrogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, branched C _3-19 alkyl, C _3-8 cycloalkyl substituted with C _1-6 -alkyl, substituted C _2-6 alkenyl, substituted C _2-6 alkynyl, substituted C _3-8 cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, C _1-6 heteroalkyl, substituted C _1-6 - heteroalkyl, C _1-6 alkoxy, aryloxy, C _1-6 heteroalkoxy, heteroaryloxy, C _2-6 alkanoyl, arylcarbonyl, C _2-6 alkoxycarbonyl, aryloxycarbonyl, C _2-6 alkanoyloxy, arylcarbonyloxy, substituted C _{2 -6} alkanoyl, substituted rilkarbonila substituted C _2-6 alkanoyloxy, substituted aryloxycarbonyl, substituted C _2-6 alkanoyloxy and substituted arylcarbonyloxy;

(a11), (a12) and (b11) are independently zero or a positive integer, preferably 0-6, and more preferably 0, 1 or 2; and

(n) is an integer from about 10 to about 2300.

For example, PEG may be functionalized in the following non-limiting manner:

-C (= Y ₁₄ ) - (CH ₂ ) _m - (CH ₂ CH ₂ O) _n -,

-C (= Y ₁₄ ) -OY) - (CH ₂ ) _m - (CH ₂ CH ₂ O) _n -,

-C (= Y ₁₄ ) -NR ₁₁ - (CH ₂ ) _m - (CH ₂ CH ₂ O) _n -,

-CR ₁₅ R ₁₆ - (CH ₂ ) _m - (CH ₂ CH ₂ O) _n -

Where

R ₁₁ , R ₁₅ and R _{16 are} independently selected from H, C _1-6 alkyl, aryl, substituted aryl, aralkyl, heteroalkyl, substituted heteroalkyl and substituted C _1-6 alkyl;

(m) is zero or a positive integer, and is preferably 1 or 2;

Y ₁₄ is O or S; and

(n) represents the degree of polymerization.

In these aspects, the polymer (R-group) includes a blocking group, that is, a group located at the end of the polymer. The blocking group may be selected from any of NH ₂ , OH, SH, CO ₂ H, C _1-6 alkyl, preferably methyl, with these groups being known to those of ordinary skill in the art.

In another aspect, the polymer portion of the conjugate may be one that contains many points for the attachment of ADA. In the alternative, many PEG molecules can be attached to the ADA.

The pharmacokinetic and other properties of PEGylated ADA can be adjusted, if necessary, depending on the desired clinical application by changing the molecular weight of PEG, chemical linkers, as well as the ratio of PEG and enzyme chains.

In these aspects, the ADA can be attached to the non-antigenic polymer in a reversible or irreversible manner using various linkers known in the art.

Reversible-coupled polymer systems can be based on the removal of benzyl or the lactonization of a trimethyl lock. Activated polymeric linkers of reversibly coupled polymeric systems can be obtained in accordance with US patents 6180095, 6720306, 5965119, 6624142 and 6303569, the contents of which are incorporated herein by reference. Alternatively, ADA polymer conjugates are prepared using certain bicin polymer residues, such as those described in US Pat. Nos. 7,121,289 and 7,087,229, as well as in US patent applications 10 / 557,522, 11 / 502,108 and 11 / 011,818 (all of this applicant), included in the present description by reference. Other contemplated reversible bound polymer systems are also described in PCT / US07 / 78600, the contents of which are incorporated herein by reference.

Illustrative examples of reversibly-linked or irreversibly-linked ADA-polymer conjugates discussed in this application are described in US patent application 60 / 913,039, the contents of which are incorporated herein by reference.

Conjugation with the polymer is preferably a PEGylation reaction, wherein such reactions are known to those of ordinary skill in the art. Briefly, the rBADA or rhADA mutein reacts with the activated polymer to form an ADA-polymer conjugate. In this regard, a wide variety of activated or functionalized polyethylene glycols can be used, including, for example, 5122614, 5324844, 5612460 and 5808096 (succinimidyl carbonate-activated polyethylene glycol (SC-PEG) and similar activated PEGs), US Pat. No. 5349001, described for example (PEG activated by cyclic imidthione), US Pat. No. 5,650,234, as well as others known to those of ordinary skill in the art. A description of each of the above documents is included in this application by reference. See also activated polymers supplied by Nektar / Shearwater Polymers. Specialists in this field can use various activated forms of polymers to join without unnecessary experimental work.

Those of ordinary skill in the art will appreciate that such conjugation reactions are usually carried out in a suitable buffer using a molar excess (several times) of activated PEG. Some preferred conjugates obtained with linear PEG molecules, such as the above SC-PEG, may contain, on average, from about 10 to about 80 PEG chains per ADA enzyme molecule. Thus, in this case, molar excesses of several hundred times, for example, 200-1000x, can be used. The molar excess used for the branched PEG and PEG attached to the enzyme is usually lower and can be determined using the procedures described in patents and patent applications, describing the same, which are listed below in this application.

In these aspects, the polyalkylene oxide is conjugated to a protein using a chemical linker, including, for example, succinimidyl carbonate, thiazolidinedione, urethane and amide linkers. The polyalkylene oxide is preferably covalently attached to the epsilon amino group Lys in ADA, although other covalent attachment sites are known in the art. The ADA-polymer conjugate may include at least 5 polyethylene glycol chains attached to the Lys epsilon amino groups in the enzyme, and in the alternative, may include about 11-18 PEG chains attached to the Lys epsilon amino groups in the enzyme.

Although ADA is conjugated to from about 11 to about 18 PEG molecules per enzyme molecule via lysine bonds, the ratio of PEG to ADA can be different in order to change the physical and kinetic properties of the combined conjugate to suit any particular clinical situation.

From the above it is obvious that additional aspects of the invention include the use of any commercially available or known activated PEG, or a similar polymer, for conjugation with the ADA enzyme, or a fragment thereof, in order to provide the conjugates used in the treatment methods described in this application. See, for example, the Nektar Advanced Pegylation 2004 catalog (Nektar, San Carlos, California), all incorporated herein by reference.

Activated PEGs may include linear, branched, or U-PEG derivatives, such as those described in US Pat. Nos. 5,681,567, 5,756,593, 5,643,575, 5,919,455, 6,113,906, 6,566,506, 6,135,555, 6,395,266 and 6,638,499, 6,251,382 and 6,824,766 (also incorporated herein by reference) . An unlimited list of such polymers corresponds to polymer systems (i) - (vii) with the following structures:

in which:

Y _61-62 independently represent O, S or NR ₆₁ ;

Y ₆₃ represents O, NR ₆₂ , S, SO or SO ₂ ;

(w ₆₂ ), (w ₆₃ ) and (w ₆₄ ) independently represents 0 or a positive integer, preferably from about 0 to about 10, more preferably from about 1 to about 6;

(w ₆₁ ) is 0 or 1;

MPEG is methoxy PEG,

where PEG is defined above, and the total molecular weight of the polymer part is from about 2000 to about 100000 daltons; and

R ₆₁ and R ₆₂ independently represent the same groups that can be used as R ₁₁ .

In addition, it should be understood that in addition to PEG-based polymers, many other polyalkylene oxides can also be used. For example, the conjugates of the present invention can be prepared by methods that include the conversion of multipath PEG-OH and star-shaped PEG products, such as those described in Shearwater's catalog of Polyethylene Glycol and Derivatives for Biomedical Application, 2001. See also NOF Corp. catalog. Drug Delivery System catalog. Ver. 8, April 2006. The contents of each of these directories are hereby incorporated by reference. Multipath polymers contain four or more polymer chains, and preferably four or eight polymer chains. For purposes of non-limiting illustration, the remainder of multipath polyethylene glycol (PEG) may have the formula:

wherein:

(x) is 0 and a positive integer, that is, from about 0 to about 28; and

(n) is the degree of polymerization.

In one specific embodiment of the present invention, a multipath PEG has the following structure:

in which (n) is a positive integer. In one preferred embodiment, the polymers have a total molecular weight of from about 2000 Da to about 100000 Da, and preferably from 4000 Da to 45000 Da.

In another specific embodiment, the multipath PEG has the following structure:

in which n is a positive integer. In one preferred embodiment, the polymers have a total molecular weight of from about 2000 Da to about 100000 Da, and preferably from 4000 Da to 45000 Da.

The polymers can be converted into a suitably activated polymer using the activation techniques described in US Pat. Nos. 5,226,214 or 5,808,096. In particular, such a PEG may have the formula:

wherein:

(u ') is an integer from about 10 to about 570, to preferably provide polymers having a total molecular weight of from about 2000 Da to about 100000 Da, and preferably from about 4000 Da to about 45000 Da; and up to 3 ends of the residue are capped by methyl or other lower alkyl.

In some preferred embodiments, all 4 PEG chains are converted to suitable functional groups, i.e., SC, etc., in order to facilitate attachment to the recombinant protein. Such compounds before conversion include:

In the most preferred aspects of the invention, the activated polyethylene glycol is polyethylene glycol, which provides the formation of a urethane bond or an amide bond with a protein.

In other alternative aspects, sterically hindered ester linker may be used in activated polymers. See PCT / US07 / 78593 entitled "Polyalkylene Oxides Having Hindered Ester-Based Biodegradable Linkers", the contents of which are incorporated herein by reference. For example, a non-limiting list of such compounds includes:

where (u) is an integer, preferably providing polymers having a total molecular weight of from about 2000 Da to about 100000 Da.

In one preferred embodiment, the PEG conjugate comprises:

,

,

where (u) is an integer providing a polymer part having a molecular weight of from about 2000 Da to about 100000 Da, and preferably from about 4000 Da to about 45000 Da, more preferably about 5000 Da.

Suitable polymers vary widely in weight. Polymers having a number average molecular weight in the range of from about 2000 to about 100000 are typically included within the scope of the present invention. Molecular weights of from about 4,000 to about 45,000 are preferred, and from 5,000 to about 12,000 are most preferred. The incorporated polymeric substances are also preferably soluble in water at room temperature. A non-limiting list of such polymers includes homopolymers of polyalkylene oxides such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylene polyols, their copolymers and block copolymers, provided that the water solubility of the block copolymers is maintained. In addition to MPEG, polymers with terminal C _1-4 alkyls may also be used.

Methods for the preparation of polymers having terminal carboxyl groups with high purity are described in US Patent Application 11 / 328,662, the contents of which are incorporated herein by reference. The methods include first preparing a tertiary alkyl ester of a polyalkylene oxide followed by conversion to the corresponding carboxylic acid derivative. The first step in the preparation of the PAO carboxylic acids of the process involves the formation of an intermediate, such as polyalkylene oxide-carboxylic acid t-butyl ester. This intermediate is formed by the reaction of PAO with t-butyl haloacetate in the presence of a base such as potassium t-butylate. After the formation of the t-butyl ether intermediate, the polyalkylene oxide-carboxylic acid derivative can be easily obtained with a purity in excess of 92%, preferably in excess of 97%, more preferably in excess of 99% and most preferably in a purity in excess of 99.5%.

In other alternative aspects, polymers having terminal amino groups can be used to produce ADA conjugates. Methods for producing high purity terminal amine containing polymers are described in US patent applications 11 / 508,507 and 11 / 537,172, the contents of which are incorporated by reference. For example, polymers having azides react with a phosphine-based reducing agent, such as triphenylphosphine, or with an alkali metal borohydride, such as NaBH ₄ . Alternatively, polymers comprising leaving groups are reacted with salts of protected amines, such as the potassium salt of methyl tert-butylimidodicarbonate (KNMeBoc) or the potassium salt of di-tert-butylimidodicarbonate (KNBoc ₂ ), followed by deprotection of the protected amino group. The purity of the terminal amine-containing polymers obtained by these methods is greater than about 95% and preferably greater than 99%.

The branching provided by the polymers of Patent 6,153,655 mentioned above allows secondary or tertiary branching as a way to increase the polymer load on a biologically active molecule from a single attachment point. It should be understood that a water-soluble polymer can be functionalized to attach bifunctional crosslinking groups, if necessary, without unnecessary experimental work.

The polymeric substances included in the present invention are preferably soluble in water at room temperature. A non-limiting list of such polymers includes homopolymers of polyalkylene oxides such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylene polyols, their copolymers and block copolymers, provided that the water solubility of the block copolymers is maintained.

As an alternative to PAO-based polymers, virtually non-allergenic materials such as dextran, polyvinylpyrrolidones, polyacrylamides such as HPMA (hydroxypropylmethacrylamides), polyvinyl alcohols, carbohydrate-based polymers, copolymers of the above polymers, etc. can be used. Those of ordinary skill in the art are aware that the above list is merely illustrative, and that all polymeric materials having the qualities described herein are considered. In the framework of the present invention, the term “substantially or substantially non-allergenic” means all materials that are known to be non-toxic and do not cause a noticeable immune response in mammals.

D. Application

One skilled in the art will appreciate that the ADA mutein of the present invention will readily find use in a clinical situation to treat any disease or disorder that is susceptible to the effects of the ADA enzyme. Such a disease or disorder is a disease or disorder that responds to decreased levels of adenosine or deoxyadenosine in tissues or blood. Such a disease or disorder may include, for example, TCID, lung diseases, such as asthma, as well as cancers that respond to decreased local or systemic levels of adenosine or deoxyadenosine. More detailed information on the use of ADA in the treatment of tumors or cancer is given in the application filed on the same date with this application (of the present applicant) for US patent ________ (which claims the priority of provisional patent application US 60 / 913,039), entitled "Enzymatic antitumor therapy "and is fully incorporated by reference in this application. A therapeutic agent may be, for example, rhADA mutein or rBADA mutein. Preferably, the therapeutic RADA mutein is conjugated to a polymer as described above, for example, PEGylated. The dose of ADA or ADA-polymer conjugate is individualized depending on the clinical response of the tumor and the side effect profile in an individual patient - animal or human. In the examples below, the highest dose is the maximum dose that can be tolerated.

For example, Adagen ^® available commercially in the form 250E bovine ADA / mL. This corresponds to 2000 U / kg for mice weighing approximately 25 g after administration of 0.2 ml Adagen ^®. Of course, one skilled in the art will appreciate that the dose of the ADA-polymer conjugate can also be adjusted depending on the size of the particular polymer, chemical linker, and valency. For example, the dosage regimen for a polymer conjugate comprising two or more ADA enzymes per polymer is controlled according to the ADA units in ml of a solution of any particular ADA polymer conjugate.

When providing an ADA or conjugate of ADA with PEG by injection, the optimal dose range is preferably set by plasma control. In general, it is desirable to provide the recipient with a dosage that supports plasma ADA activity (minimum levels) in the range of from about 10 to 100 μmol / h / ml, preferably from about 15 to about 35 μmol / h / ml (assayed at 37 ° C ), and shows a decrease in erythrocyte adenosine, i.e., DATP, to ≤ approximately 0.001-0.057 μmol / ml, preferably to approximately 0.005 to approximately 0.015 μmol / ml in packed red blood cells, or ≤ approximately 1% of the total erythrocyte adenosine (i.e., ATP content + DATP), relative to the normal level of adenosine, when measured in a sample taken before injection. The normal value of DATP is less than approximately 0.001 μmol / ml.

The dose based on the amount of enzyme varies, for example, from about 0.1 U / kg to about 30 U / kg inclusive, or higher, preferably from about 0.5 U / kg to about 20 U / kg, and more preferably from from about 0.5 U / kg to about 12 U / kg (i.e., per kg of patient weight), such as from about 0.5 U / kg to about 5 U / kg. The total weekly dose may be up to 40 U / kg or more if tolerated by the recipient. Subsequent dose increases of 5 U / kg / week are allowed, up to a maximum single dose of 30 U / kg or more if the indicated dose is tolerated by the recipient. Typically, after weekly injections of ADAGEN ^® at a dosage of 15 U / kg, the average minimum level of ADA activity in plasma is 20 to 25 µmol / h / ml.

It should be noted that a dose of 100 U / kg is a mouse dose equivalent to a clinical children's dose of approximately 12 U / kg.

Detailed information regarding the dosage of ADA is known in the art and is provided on the medical information leaflet for ADAGEN ^® (Enzon, Inc.), the contents of which are incorporated into this application.

EXAMPLES

The following examples serve to provide a more complete understanding of the invention, but in no way limit the actual scope of the invention.

EXAMPLE 1

Construction of an expression strain of E. Coli expressing recombinant human ADA with Cys / Ser substitution at position 74 of the mature protein

Published amino acid sequence 363 a.k. in length human adenosine deaminases (GenBank NP_000013, incorporated herein by reference) were analyzed for the presence of cysteine codons. Five positions in the mature (with cleaved N-terminal Met) polypeptide encode cysteine (C74, C 12, C153, C168, C261). In the constructed and modified gene expressing human ADA, only one of the five cysteine codons (Cysteine 74, TGC) was replaced by a serine codon (TCC) (this is the 75 position in the translated protein). A specific polypeptide sequence (see SEQ ID NO: 3) was provided by Blue Heron Corporation (Bothell, Washington, USA) for the synthesis of a new full-sized gene, including codons optimized for expression in E. coli, using standard chemical synthesis with overlapping oligonucleotide fragments. Briefly, the sequence was optimized for bacterial expression in accordance with the standard bacterial codons used for Escherichia coli K12 using the codon data described by Grantham R. et al .; 1981, "Codon catalog usage in genome strategy modulated for gene expressivity," Nucleic Acid Res. 9: r43-r47, and Lathe, R. 1985, "Synthetic oligonucleotide probes deduced from amino acid sequence data, Theoretical and practical considerations," J. Mol. Biol .; 183: 1-12.

Then the corresponding RNA sequence was analyzed for the formation of hairpin structures or loops and subjected to calculations of the minimum free energy. Flanking restriction sites NdeI and BamHI were introduced into the terminal regions of the gene. After cleavage of synthetic DNA with restriction enzymes NdeI and BamHI 1.1 kb the gene was ligated with T4 DNA ligase into the plasmid vector pET-28a (Novagen), which was also digested with these two enzymes. The recombinant plasmid was introduced into the E. coli BLR strain (DE3) or HMS 174 (DE3) by electroporation using the BTX Electro Cell Manipulator 600 according to the manufacturer's instructions. The transformed culture was plated on LB agar plates containing kanamycin (15 μg / ml) to select colonies containing the plasmid pET-28a / ADAcysSer (designated ADAc75s / pET28a: BLR (DE3) or ADAc75s / pET28a: HMS174 (DE3)). The nucleotide sequence of an ADA gene variant was confirmed by DNA sequence analysis using an ABI Prism 310 Genetic Analyzer using Big Dye Terminators. The DNA sequence encoding the open reading frame of the Ser ₇₄ rfADA was consistent with SEQ ID NO: 4.

The isolated colonies were then purified by transferring to plates and analyzed for expression induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) in LB medium using standard methods such as those described in the ninth issue of Novagen pET System Manual, incorporated by reference in this application.

Several induction parameters were investigated, including time, temperature, and inductor concentration. Preferred induction conditions, including the induction of 50 μM IPTG for 12 hours at 25 ° C, ensured the production of ADA with a high level in the cytoplasm of the host bacteria in an amount of approximately 20% of the total protein of the cell. Expressed ADA protein pronounced by SDS-PAGE electrophoresis had a desired molecular weight of approximately 40 kDa (data not shown).

EXAMPLE 2

Construction of an expression strain of E. Coli expressing recombinant bovine ADA with Cys / Ser substitution at position 74 of the mature protein

The purified mature ADA protein obtained from preparations of the intestinal tract of cattle is a protein of 356 amino acids in length, without an N-terminal methionine, and also without six C-terminal residues predicted from the cDNA sequence (GenBank NP_776312, incorporated by reference in this application) . The bovine ADA amino acid sequence was analyzed for the presence of cysteine codons. Five positions in the mature polypeptide encode cysteine (C74, C152, C153, C168, C261). In the constructed and modified synthetic bovine ADA gene, only one of the five positions of cysteine (cysteine 74) was replaced by a serine residue. This was accomplished by introducing a serine codon (TCC) instead of the normal cysteine codon at position 74 of the mature polypeptide (or translation product position 75). The gene was also optimized for codons for expression in E. coli.

Briefly, a specific polypeptide sequence (see SEQ ID NO: 1) was provided to BioCatalytics for the synthesis of a new full-sized gene, including codons optimized for expression in E. coli, using BioCatalytics proprietary methods that involve chemical synthesis with overlapping oligonucleotide fragments. BioCatalytics methods are described in more detail in US patent 6366860, the contents of which are fully incorporated by reference in this application.

Bovine ADA expression was analyzed in several expression systems. For example, flanking restriction sites NdeI and BamHI were introduced into the terminal regions of the gene. After cleavage of synthetic DNA with restriction enzymes NdeI and BamHI, a 1.1 kb gene were ligated with T4 DNA ligase into plasmid vector pET-9d (Novagen), which was also digested with these two enzymes. The recombinant plasmid was introduced into the E. coli BLR strain (DE3) or HMS 174 (DE3) by electroporation using the BTX Electro Cell Manipulator 600 according to the manufacturer's instructions. The transformed culture was plated on LB agar plates containing kanamycin (15 μg / ml) to select colonies containing the plasmid pET-9d / bADA (designated bADA / pET9d: BLR (DE3) or bADA / pET9d: HMS 174 (DE3)) . The nucleotide sequence of an ADA gene variant was confirmed by DNA sequence analysis using an ABI Prism 310 Genetic Analyzer using Big Dye Terminators. The DNA sequence containing the open reading frame of the Ser ₇₄ rfADA is shown in SEQ ID NO: 2.

Then, the isolated colonies were purified by transferring to plates and analyzed for IPTG-induced gene expression in LB medium by standard methods, such as those described in the ninth edition of Novagen pET System Manual. Several induction parameters were analyzed, including time, temperature, and inductor concentration. Preferred induction conditions, including induction with 0.3% lactose for 12 hours at 37 ° C, ensured the production of ADA with a high level in the cytoplasm of the host bacteria in an amount of approximately 20% of the total protein of the cell. The ADA product pronounced by SDS-PAGE electrophoresis had a desired molecular weight of about 40 kDa.

EXAMPLE 3

Purification of mutein rHADA

Purification of the rhADA mutein was performed using a 3-step chromatographic purification developed by Enzon. Bacterial cells were cultured with an E. coli strain expressing rhADA protein from the synthetic gene in plasmid pET28a (Novagen) in the HMS 174 (DE3) host cell. Rifampicin (200 μg / ml) and kanamycin (μg / ml) with yeast extract (30 g / l) were added to the minimal medium with glycerol, and cells were grown at 28 ° C to OD ₆₀₀ 11, after which the IPTG inducer was added to the final concentration of 5 mm. After 40 hours (OD ₆₀₀ ~ 110), cells were harvested by centrifugation and frozen at -20 ° C. Briefly, thawed cell paste (50 g) was resuspended in 1800 ml of buffer containing 10 mM Tris [trishydroxymethylaminomethane], 1 mM DTT, pH 8.0, and homogenized at 1200 rpm for 10 seconds on Tempest Virtis ( ^SentryTM, Microprocessor) , Boston, MA). The resulting suspension was passed through a stainless steel sieve (with holes of 250 μm, No. 60, WS Tyler) to remove large particles. A homogeneous cell suspension was homogenized using a high pressure homogenizer in 3 cycles at 103.4 MPa (15000 psi) (unit was ice-cooled) (MicroFluidizer, Microfluidics Corp., Model # 110Y, Boston, MA). At the end of microhomogenization, 200 ml of the same buffer indicated above was used to wash the unit, and the resulting solution was combined with the above suspension. The soluble protein from cell lysates were pelleted by centrifugation at 16,000 rev / min for 40 minutes at 4 ^{° C (Sorvall ® RC 5C plus} , SLA- 1000 rotor). The supernatant was collected carefully to avoid undesired mixing. The pH was adjusted to 8.0, after which 1 mM MgCl ₂ and 20 mg / ml DNase were added and incubated at room temperature for 2 hours. Then the pH was adjusted to 6.5 1 N. HCl. A second centrifugation was carried out as described above, the supernatant was collected and EDTA was added to a concentration of 2 mM, followed by filtration using a 90 mm Nalgene ^® filter apparatus. The volume of the filtered supernatant was 500 ml, the concentration of total protein according to the BCA method (with bicinchoninic acid) was 8.5 mg / ml.

The cell extract (100 ml) was adjusted to a pH of 7.2, 4.5 mS / cm and applied to HiTrap ^® DEAE ff (ff means high flow rate) in 20 mM Bis-Tris, 20 mM NaCl, pH 6.5, and eluted in 20 mM Bis-Tris, 500 mM NaCl, pH 6.5. Peak fractions were analyzed by enzyme analysis and SDS-PAGE, then ammonium sulfate was added to a concentration of 1.5 M in 20 mM NaHPO ₄ , pH 6.5, and applied to a HiTrap Phenyl ff column. Protein was suirable in the gradient of the buffer for applying and 20 mm NaHPO ₄ , pH 6.5. The peak fraction (55 ml, 0.4 mg / ml) was diafiltered against a buffer containing 20 mM NaHPO ₄ , 1 mM EDTA, 1 mM DTT, pH 6.5 and applied to HiTrap SP-Sepharose ff and eluted in 20 mM NaHPO ₄ , 500 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 6.5. The collected fraction contained purified ADA protein (77 ml, 0.1 mg / ml).

EXAMPLE 4

Purification of Recombinant Bovine ADA Protein

Purification of the rBADA mutein expressed by the clone of Example 2 was performed using a 3-step chromatographic purification developed by Enzon. Briefly, 200 g of thawed cell paste (obtained from Blue Hereon or Biocatalytics, respectively) that was stored at −80 ° C. was resuspended in 1800 ml of buffer containing 20 mM Bis-Tris, 1 mM EDTA, pH 7.4, and homogenized at 1200 rev / min for 5 minutes ^{Tempest Virtis (Sentry TM, Microprocessor,} Boston, MA). The resulting suspension was passed through a stainless steel sieve (with holes of 250 μm, No. 60, WS Tyler) to remove large particles. A homogeneous cell suspension was homogenized using a high pressure homogenizer in 3 cycles at 103.4 MPa (15000 psi) (the unit was ice-cooled) (MicroFluidizer, Microfluidics Corp., Model # 110Y, Boston, MA). At the end of microhomogenization, 200 ml of the same buffer indicated above was used to wash the unit, and the resulting solution was combined with the above suspension. Soluble protein from cell lysates was isolated by centrifugation at 7100 rpm (12000 × g) for 60 minutes at 4 ° C (Avanti J-201, Beckman Coulter, JLA8.1000 rotor). The supernatant was collected carefully to avoid undesired mixing.

To remove the nucleotides in this cell extract, polyethyleneimine (PEI) was added to the above supernatant (to a final concentration of 0.15%, w / v) and mixed thoroughly for 10 minutes. Then the resulting cell extract was left overnight at 4 ° C. The precipitate from the indicated night sample was removed by centrifugation at 7100 rpm (12000 × g) for 60 minutes at 4 ° C (Avanti J-201, Beckman Coulter, JLA8.1000 rotor). Similarly, the supernatant was carefully collected to avoid undesired mixing. To facilitate ADA binding on the first column, 10% PEG4600 was slowly added to this cell extract, and the pH of the resulting cell extract was slowly adjusted to 6.5 1 N. NaOH and 1 N. HCl. The supernatant was then centrifuged again at 7100 rpm (12000 × g) for 60 minutes at 4 ° C (Avanti J-201, Beckman Coulter, JLA8.1000 rotor) before application to the next column.

Cell extract was applied to a pre-equilibrated Capto Q column (Cat. No. 17-5316-01, GE Healthcare, Piscataway, NJ). The volume of the 350 ml layer in an XK-50 column was pre-saturated with a buffer containing 20 mM Bis-Tris, 1 mM EDTA, pH 6.5. Before eluting ADA from the column with equilibration buffer with 80 mM NaCl, elution was first performed with 60 mM and 70 mM NaCl to remove impurities. The elution profile was analyzed for ADA activity by SDS-PAGE, Western blotting and RP-HPLC.

After the Capto Q column, two purifications were performed one after the other using hydrophobic interaction chromatography (HIC) to further increase the purity of the protein. The first HIC was performed on 4FF octyl sepharose (Cat. No. 17-0946-02, GE Healthcare, Piscataway, NJ). The combined ADA fractions after the Capto Q column were directly precipitated with ammonium sulfate powder at a concentration of 1.5 M (NH ₄ ) ₂ SO ₄ , and the pH was adjusted to 6.5. A filtered sample (Nalgene Nunc, Cat. 540887, MEMB 0.2 PES, Rochester, NY) was applied to a first HIC column, pre-equilibrated with 1.5 M (NH ₄ ) ₂ SO ₄ , 20 mM potassium phosphate, 1 mM EDTA, pH 6.5 (layer volume 150 ml, in XK-50, GE Healthcare, Piscataway, NJ). The ADA protein was eluted in a gradient of ammonium sulfate, and the purity profile of the obtained eluate was determined by SDS-PAGE and RP-HPLC. ADA protein in the fractions of the first HIC column was combined and the concentration of (NH ₄ ) ₂ SO _{4 was} adjusted to 1 M, after which it was directly applied to the second HIC column (layer volume 150 ml, XK-50, HIC Phenyl HP, Cat. 17- 1082-01, Piscataway, NJ), pre-equilibrated with 1 M (NH ₄ ) ₂ SO ₄ , 20 mM KH ₂ PO ₄ -K ₂ HPO ₄ , 1 mM EDTA, pH 6.5. ADA was suirable in a gradient of ammonium sulfate from 1 M to 300 mm in 20 mm KH ₂ PO ₄ -K ₂ HPO ₄ , 1 mm EDTA, pH 6.5. The purity of the ADA in the obtained fractions was analyzed by SDS-PAGE and RP-HPLC electrophoresis. The purified rBADA or rhADA was then desalted and concentrated using LabScale ™ TFF systems (Membrane BioMax 5, Bedford, MA) against storage buffer (e.g., 100 mM sodium phosphate, 1 mM EDTA, pH 6.5).

EXAMPLE 5

Stability Assays for rBADA and Ser ₇₄ -RBADA

The following analyzes were performed to demonstrate that the stability of rBADA during oxidative degradation was indeed improved due to a mutation with cys74 / ser74 replacement. For stability analysis, samples of recombinant bovine ADA (rBADA) and recombinant bovine ADA with the cys74 / ser74 mutation (Ser-74-rbADA) were used at concentrations of approximately 0.5 mg / ml in sodium phosphate buffer (pH 7.8). Stability was monitored by reverse phase HPLC (RP-HPLC) using UV detection at 220 nm and mass spectrometric detection (Micromass Q-TOF mass spectrometer with electrospray ionization). The HPLC conditions were as follows:

Speaker: Zorbax 300 SB-C8 (Agilent, 250 × 4.6 mm, pore size 300 angstroms, particle size 5 microns). Mobile phase A: 0.1% trifluoroacetic acid in water. Mobile phase B: 0.1% trifluoroacetic acid in a mixture of acetonitrile / water (80/20, v / v). Gradient: Time % mobile phase B 0 twenty 5 twenty 45 80 46 twenty 60 twenty Column Temperature: 40 ° C Flow rate: 1.0 ml / min Input Volume: 50 μl

The purity of the compounds was determined using RP-HPLC analysis at the initial moment of the beginning of the stability analysis and at various points in time, including 4, 8 and 17 days after the start of the analysis. It should be noted that samples of rBADA (not mutein) were stored for approximately two months before this study and had already undergone some decomposition. A Ser ₇₄ -RBADA sample was prepared recently and was relatively pure. However, in the framework of this study, the analyzed parameter was the difference in purity between the initial point in time and after 17 days of incubation at 25 ° C.

As shown in Table 1, the rbADA purity was 83.7% at the initial time point and decreased to 66.1% after 17 days, which indicates that 17.6% rbADA decomposed over the indicated time period. Mass spectrometric analysis of chromatographically separated peaks showed that the main degradation product, eluting at 31.851 minutes and occupying 30.5% of the chromatogram area, had a mass of 32 Da higher than rBADA. This mass change corresponds to the addition of 2 oxygen atoms to rBADA with the formation of a degradation product containing sulfinic acid instead of free cysteine at position 74 rbADA. The mass of the degradation product, which had a minor peak, eluting at 32.538 min, corresponded to the addition of 1 oxygen atom to rBADA with the formation of a degradation product containing sulfenic acid instead of free cysteine at position 74 rbADA. Ser ₇₄ -rbADA, containing a serine residue replacing the reactive cysteine residue 74, showed a slight decomposition for 17 days, while the purity after 17 days (97.9%) did not actually change compared to the purity at the initial time point (97 , 2%). This proves that cysteine 74 is indeed a source of oxidative degradation that occurs in rBADA, and a mutation with the substitution of this residue with serine, which is not prone to oxidation, prevents such decomposition.

Table 1 Stability of rBADA and Ser ₇₄ -RBADA in sodium phosphate buffer
(pH 7.8) at 25 ° C Moment of time % purity as determined by RP-HPLC rBADA Mut-rBADA Elementary 83.7 97.2 4 days 83.6 96.7 8 days 76.3 97.7 17 days 66.1 97.9

EXAMPLE 6

The use of ADA muteins in the treatment of ADA-deficient patients suffering from TCID

The described mutated ADA enzymes have been used in therapeutic regimens currently used for ADAGEN ^® . Ser ₇₄ -rb or rhADA can be modified by conjugation with polyethylene glycol (PEG), for example, with 11-17 PEG 5 kDa polymers per ADA protein. The pegylated ADA mutein preparations are introduced into a sterile solution of sodium chloride at a pH of 7.2-7.4 and at a concentration of approximately 250 units per milliliter. The pegylated mutein ADA is administered to patients transversely, for example, intramuscularly. Patients with indications for such therapy include patients with severe combined immunodeficiency caused by insufficient ADA activity. The introduction of PEG-ADA mutein is usually performed every seven days according to the dosing schedule of 10 U / kg for the first dose and 20 U / kg per week for subsequent doses. Ser ₇₄ rb or rh PEG-ADA is stored at 2-8 ° C in a single dose aqueous solution in a 1.5 milliliter vial. The dosage schedule is designed in such a way as to ensure plasma levels of ADA activity of the order of 15-35 μmol / h / ml (in the analysis at 37 ° C) and to reduce red blood cell DATP to ≤0.005-0.015 μmol / ml of packed red blood cells.

EXAMPLE 7

Preparation of Pegylated Ser ₇₄ -RBADA via Education

urethane bond

SK-PEG (N-hydroxysuccinimidyl carbonate activated polyethylene glycol, 0.084 mmol) was added to a solution of Ser ₇₄ -RBADA (0.00027 mmol) in 3 ml of sodium phosphate buffer (0.1 M, pH 7.8) with gentle stirring. The solution was stirred at 30 ° C for 30 minutes. To control PEG conjugation, a GPC column (Zorbax GF-450) was used. At the end of the reaction (as evidenced by the absence of a native enzyme), the mixture was diluted with 12 ml of preparation buffer (0.05 M sodium phosphate, 0.85% sodium chloride, pH 7.3), and then diafiltered using a Centriprep concentrator (Amicon ) to remove unreacted PEG. Diafiltration was continued at 4 ° C for as long as needed until free PEG was no longer detected by mixing equal amounts of filtrate and 0.1% PMA (polymethacrylic acid in 0.1 M HCl).

EXAMPLE 8

Preparation of Pegylated Ser ₇₄ rhADA via Education

urethane bond

The reaction of SK-PEG (0.084 mmol) and Ser _74- rchADA (0.00027 mmol) was carried out using the same conditions as described in Example 7.

EXAMPLE 9

Preparation of Pegylated Ser ₇₄ -RBADA via Education

amide bond

SS-PEG (N-hydroxysuccinimidyl succinate-activated polyethylene glycol, 0.084 mmol) was added to a solution of Ser ₇₄ -RBADA (0.00027 mmol) in 3 ml of sodium phosphate buffer (0.1 M, pH 7.8) with gentle stirring. The solution was stirred at 30 ° C for 30 minutes. To control PEG conjugation, a GPC column (Zorbax GF-450) was used. At the end of the reaction (as evidenced by the absence of a native enzyme), the mixture was diluted with 12 ml of preparation buffer (0.05 M sodium phosphate, 0.85% sodium chloride, pH 7.3), and then diafiltered using a Centriprep concentrator (Amicon ) to remove unreacted PEG. Diafiltration was continued at 4 ° C for as long as needed until free PEG was no longer detected by mixing equal amounts of filtrate and 0.1% PMA (polymethacrylic acid in 0.1 M HCl).

EXAMPLE 10

Obtaining pegylated rhADA mutein through the formation of an amide bond

The reaction of CC-PEG (0.084 mmol) and rhADA mutein (0.00027 mmol) was carried out using the same conditions as described in Example 9.

Claims (14)

1. An isolated recombinant adenosine deaminase comprising the SEQ ID NO: 1 polypeptide or a variant SEQ ID NO: 1 polypeptide of the isolated recombinant adenosine deaminase, where the SEQ ID NO: 1 variant polypeptide includes one or more amino acid substitutions selected from the group consisting of: Gln instead of Lys198 ; Ala instead of Thr245; and Arg instead of Gly351. 2. The recombinant adenosine deaminase according to claim 1, encoded by the DNA molecule of SEQ ID NO: 2. 3. The conjugate of polyalkylene oxide with adenosine deaminase for the treatment of adenosine deaminase-mediated diseases, where the adenosine deaminase is the recombinant adenosine deaminase according to claim 1, where the recombinant adenosine deaminase comprises from 11 to 17 chains of polyalkylene oxide with a molecular weight of 5 kDa. 4. The conjugate of polyalkylene oxide with adenosine deaminase according to claim 3, where the polyalkylene oxide is polyethylene glycol. 5. The polyalkylene oxide conjugate of adenosine deaminase according to claim 4, wherein the polyethylene glycol is conjugated to recombinant adenosine deaminase via a linker selected from the group consisting of succinimidyl carbonate, thiazolidinedione, urethane and amide linkers. 6. The polyalkylene oxide conjugate of adenosine deaminase according to claim 4, wherein the polyethylene glycol is covalently attached to the epsilon amino group Lys of the recombinant adenosine deaminase. 7. The polyalkylene oxide conjugate of adenosine deaminase according to claim 4, wherein the polyethylene glycol is conjugated to recombinant adenosine deaminase via a succinimidyl carbonate linker. 8. The method of purification of recombinant adenosine deaminase according to claim 1, including the purification of the protein using ion exchange chromatography. 9. The method for purification of the recombinant adenosine deaminase according to claim 1, comprising purifying the protein using hydrophobic interaction chromatography. 10. A recombinant adenosine deaminase preparation obtained by the method of claim 8. 11. The preparation of recombinant adenosine deaminase obtained by the method according to claim 9. 12. Isolated DNA that encodes a recombinant adenosine deaminase having an amino acid sequence comprising SEQ ID NO: 1. 13. The isolated DNA that encodes the recombinant adenosine deaminase according to claim 1. 14. The selected recombinant adenosine deaminase according to claim 1, comprising the polypeptide SEQ ID NO: 1.

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