RU2481855C2 - Enzymatic anticancer therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions refers to new methods of treating and suppressing the tumour growth, specifically malignant solid tumours by administering an effective amount of adenosine aminase conjugated with a non-antigen polymer for the purpose of the tissue adenosine and deoxyadenosine level recovery.

EFFECT: post-therapeutic tumour shows a reduced volume or a reduced growth rate.

19 cl, 5 tbl, 10 ex

Description

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of provisional patent application US 60 / 913,039, filed April 20, 2007, the contents of which are incorporated into this application by reference.

FIELD OF THE INVENTION

The invention provides new methods for treating and slowing the growth of tumors, and in particular malignant solid tumors, by administering adenosine deaminase to restore levels of adenosine and deoxyadenosine in tissues.

BACKGROUND

A tumor is a benign or malignant formation of cells or tissue that results from unregulated cell proliferation. A malignant tumor is a tumor that spreads from a site of its formation, and is also known in the art as cancer. Thus, tumors and cancerous tumors are a group of diseases that have the common property of unregulated or inappropriate cell growth. In a broad sense, malignant tumors are either tumors originating from hematopoietic cells, such as leukemia, or solid tumors. Malignant tumors originating from hematopoietic cells usually circulate in the blood, and solid malignant tumors spread from the primary tumor throughout the body. Then, common tumor cells can develop into multiple secondary tumors during metastasis. In order for a solid tumor to undergo such a metastatic spread, the cells of the solid tumor must leave the primary or original tumor, enter the bloodstream or lymphatic system, and then from there enter the tissue of other organs, where they divide and form new tumors. Metastasis is a complex multistage process, which includes changes in the adhesion and motility of tumor cells, secretion of proteolytic enzymes, chemoattractants and proteoglycans, as well as other factors. In addition, angiogenesis or the formation of new blood vessels is also an essential step in the metastatic process (Folkman, 1995, Nature Medicine 1: 27-31).

The immune system has also been shown to inhibit the metastasis of such malignant tumor cells, while it has been reported that adenosine, in turn, can inhibit such immune defense reactions. For example, Loshkin et al. (2006, Cancer Res. 66: 7758-7765) reported that adenosine inhibits cytokine activation and production in killer T cells. Adenosine adversely affects other free functions, including cellular elements and inflammatory functions (see, for example, reviews Spychala, 2000, Pharmacology & Therapeutics, 87: 161-173 and Sitkovesky et al., 2005, Nature Reviews Immunology, 5: 713- 721). Sitkovesky et al. In WO 03/050241, published June 19, 2003, also described methods of enhancing an immune response to an antigen and treating tumors by administering an adenosine receptor antagonist, which may include adenosine deaminase.

Adenosine has also been shown to promote tumor cell migration and angiogenesis (Barcz et al., 2000, Oncol. Rep., 7 (6): 1285-91; Adair, 2005, Am. J. Physiol. Regul. Integr. Comp. Physiol., 289: R283-R296) and that adenosine stimulates the proliferation of colorectal cancer cells (Mujoomdar et al., 2003, Biochemical Pharmacology 66: 1737-1747). Asmar et al. (1966, Proc. Am. Assoc. Cancer Res., Abstract No. 73) also reported that in a murine ascites model, the growth of certain tumor cells, by more than 50%, was inhibited by the injection of ADA. These were L1210 and L4946 lymphatic leukemia cells, 6C3HED lymphosarcomas, TA3 breast adenocarcinomas, and Ehrlich E2 carcinomas. In the same abstract, adenocarcinoma 755 was reported to be two times more stable, and sarcoma 180 was completely resistant to this effect. WO03050241 A2 describes the effects of an adenosine receptor inhibitor on B 16 melanoma cells. Although WO03050241 A2 refers to ADA as an adenosine inhibitor, there is no specific description of the use of ADA in the treatment of specific cancers, in particular ovarian and prostate cancers.

Thus, for some tumors, the presence of adenosine appears to provide a “trigger” signal for tumor proliferation and tumor angiogenesis, as well as a “stop” signal for killer T cells that would normally destroy these tumors.

In contrast to the results described above, Lind et al. (US Pat. No. 6,579,857) reported that adenosine in combination with an adenosine deaminase enzyme inhibitor and / or in combination with an antitumor agent such as coformicin can be used in a method for potentiating death of neoplastic cells of epithelial origin. Thus, this reference suggests that the role of adenosine in cancer is more complex and unknown.

As indicated above, the adenosine deaminase enzyme is a means to lower endogenous adenosine levels. Adenosine deaminase (ADA), designated as EC 3.5.4.4, is an important enzyme in purine metabolism. ADA converts adenosine or deoxyadenosine, in the presence of water, to inosine or deoxyinosine and ammonia. It is known that in people whose ADA gene contains unfavorable mutations, immunodeficiency states of varying degrees, from moderate to severe, that is, severe combined immunodeficiency (TKID), can develop. TCID has been confirmed to result from toxic accumulation of enzyme, adenosine and deoxyadenosine substrates in immature lymphocytes. The onset of the disease can vary from early childhood to adulthood, depending on the inherited mutations. ADA deficiency is one of the leading causes of TCID in children and serves as one of the main goals for gene therapy methods (R. Parkman et al., 2000, "Gene therapy for adenosine deaminase deficiency", Aim. Rev, Med., 51: 33- 47).

Earlier, ADA was isolated on an industrial scale from sources obtained from cattle, and was used in the treatment of a number of diseases, including TCID, in the form of bovine ADA conjugated to a polymer - polyethylene glycol (PEG). PEGylated ADA for medical use is commercially available from Enzon Pharmaceuticals under the brand name ADAGEN ^® (PEGylated ADA). Conjugation of the PEG molecule with ADA allows the enzyme to produce its full therapeutic effect by increasing the duration of circulation while preserving the essentially non-antigenicity of ADA in order to minimize the possibility of the development of immunogenic reactions. You can also obtain recombinant human or bovine ADA enzymes for use in the form of conjugates, as described in US patent application 11 / 738,012 (the present applicant), entitled "Stabilized Proteins" (Stabilized Proteins), and US patent application ______ (the present applicant), entitled "Stable Recombinant Adenosine Deaminase", filed on the same date as this application and claiming the priority of US patent application 60 / 913,009, which are incorporated by reference in their entirety.

Thus, in the prior art there is a long-term need for new and improved methods of treating or inhibiting the growth, spread and development of cancerous tumors.

SUMMARY OF THE INVENTION

In this regard, the invention provides a method of treating a patient having a tumor, comprising administering an effective amount of ADA to a patient in need thereof. An effective amount is an amount that can easily be determined by one of ordinary skill in the art to reduce levels of adenosine or deoxyadenosine in a patient’s tissues, and where tumor growth or spread is inhibited as a result of a significant decrease in adenosine levels in a patient’s tissues. The route of administration is such a route as subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal, inhalation and transurethral.

The tumor may be malignant or benign, and is preferably a solid tumor, for example, a tumor such as a prostate tumor, ovarian cancer and / or colorectal cancer.

Adenosine deaminase is preferably conjugated to a substantially non-antigenic polymer such as polyalkylene oxide (PAO). Preferably, the PAO size ranges from about 4,000 to about 45,000 daltons. PAO is preferably polyethylene glycol (PEG). The molar ratio of ADA to polymer can be 1: 1, or two or more ADA molecules can be present per polymer molecule, or, more preferably, from about 1 to about 20 polymer molecules (i.e. 11-18 PEG chains) per ADA molecule.

The ADA polymer conjugate is preferably administered at a dose ranging from about 10 U to about 30 U per kg or more, and for a sufficient amount of time to support tumor inhibition, for example, from about 1 to about 20 days or longer.

The amount of adenosine deaminase that is administered is effective to substantially lower levels of adenosine or deoxyadenosine in the patient’s tissues, and where tumor growth or spread is inhibited as a result of a significant decrease in adenosine levels in the tissues of the indicated patient. For example, this is a dose of adenosine deaminase ranging from about 10 U to about 30 U per kg. The dose is re-administered for a sufficient period of time to maintain tumor inhibition, for example, from about 1 to about 20 days or longer. The dose is administered in any suitable way, for example subcutaneously, intravenously, intramuscularly, intrathecally, intraperitoneally, inhalation and transurethrally.

Adenosine deaminase is optionally isolated from a source that refers to cattle or is a recombinant adenosine deaminase. Recombinant adenosine deaminase is, for example, recombinant bovine adenosine deaminase (Ser ₇₄ rhBADA) including SEQ ID NO: 1, recombinant human adenosine deaminase (Ser ₇₄ rhADA) including SEQ ID NO: 3, and recombinant bovine adenosine deaminase 5: , and / or their variants or polymorphisms. Recombinantly obtained bovine adenosine deaminase, for example SEQ ID NO: 5, is optionally capped at Cys ₇₄ to increase stability in the aquatic environment.

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 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 -alkylaminoalkyl .

As used herein, the term “alkenyl” 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 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.

As used herein, the term “alkynyl” 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 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” group 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, for example methylthiomethyl 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 by acyl 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, the terms "adenosine-mediated tumor" or "adenosine deaminase-reactive tumor", as it is understood, broadly include any types of tumors in which administration of ADA or its active fraction, etc., is successfully applied, regardless of the route of administration.

In the framework of the present invention, the terms “treatment of an adenosine-mediated tumor”, or “treatment of an adenosine deaminase-reactive tumor”, or “inhibition of the growth of an adenosine-mediated tumor”, or “inhibition of the growth of an adenosine-mediated tumor”, as understood, mean minimization or reduction manifestations of symptoms or conditions compared with those observed in the absence of treatment with ADA. The degree of convalescence can be confirmed, for example, by reducing the level of adenosine.

In a broad sense, treatment should be considered successful when the desired clinical response is obtained. For example, treatment success can be confirmed by obtaining, for example, 10% or higher (i.e., 20%, 30%, 40%) inhibition of tumor growth. 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 cancerous adenosine levels cells or tissues, including other clinical indicators considered by a specialist in this field, compared with that observed in the absence of treatment using the ADA described in this application.

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, process steps and materials described in this application, since such configurations, process steps 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.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention provides new methods for treating tumors, including cancer, by administering an ADA enzyme to a patient in need thereof, in an amount and over a period of time sufficient to reduce the amount of adenosine present in tissues and / or fluids in the body the patient. Preferably, the ADA enzyme is conjugated to a polymer. For tumors that depend on the presence of adenosine necessary for their growth, metastatic spread and / or to protect against the patient’s immune system, a sufficient decrease in the level of endogenous adenosine limits or prevents the development of cancer, the spread of metastases and / or ensures normal antitumor activity of the patient’s immune system .

It is also contemplated that the treatment of tumors using ADA in accordance with the invention is optionally carried out in combination or in conjunction with one or more other suitable, known from the prior art methods of treating cancer and antitumor agents, as described in more detail below. Combination treatment of the present invention includes administering an effective amount of the compounds described herein, alone or in combination, simultaneously or sequentially, with a second chemotherapeutic agent.

Examples of known antitumor agents for such combination treatment methods include, for example, Taxol ™, bevacizumab (Avastin 11), vincristine, vinblastine, neomycin, combretastatin (s), podophyllotoxin (s), TNF-alpha, angiostatin, endostatin, vasculostatin, integrin alpha _v beta ₃ antagonists, calcium ionophores, agents that induce the flow of calcium ions, as well as any derivatives or prodrugs thereof.

Additional antitumor agents also include chemotherapeutic agents, radiotherapeutic agents, cytokines, antiangiogenic agents, apoptosis-inducing agents or antitumor immunotoxins or coagulating ligands. One of these immunotoxins is, for example, Erbitux ^® (cetuximab). "Chemotherapeutic agents" used in this application, refer to the classical chemotherapeutic agents or drugs used in the treatment of malignant neoplasms. This term is used for simplicity, despite the fact that other compounds that have an antitumor effect may technically be indicated as chemotherapeutic agents. However, “chemotherapeutic” has a clear meaning in the prior art and is used according to this standard value.

Thus, the method of the present invention can be used in combination with one or more chemotherapeutic agents that are known to be effective in treating cancer, including, but not limited to, 5-azacytidine, 5-fluorouracil, optionally in combination with leucovorin, 5-fluorodeoxyuridine , 6-mercaptopurine, 6-thioguanine, mitoxantrone, aziridinylbenzoquinone (AZQ), carmustine (BCNU or BiCNU ^® , Bristol-Myers-Squibb), bleomycin, carboplatin (CBDCA), lomustine (CCNU), methyl-CCNU, MeN-chloro, CNNU, MeNuram, MeNuram chlordeoxyoxyadenosine, cisplatin, cyclophosphamide, qi tarabin, dactinomycin, daunorubicin, deoxycoformicin, doxorubicin, doxycoformicin, DTIC (dacarbazine), epirubicin, etoposide (VP-16), fludarabine, hexamethylmelamine, hydroxyurea, N-phosphamide amide, acetamide, acetaminophenide, -phenylalanine mustard, 4 '- (9-acridinylamino) -methanesulfon- m- anisidide (mAMSA), known in the prior art multidrug resistance inhibitors (i.e. MDR inhibitors), melphalan, methotrexate, optionally in combination with leucovorin, mitramycin, mitomycin-C, protein inhibitors, associated with multidrug resistance to drugs (MRP inhibitors), paclitaxel, procarbazine, streptozotocin, N, N'N'-triethylene thiophosphoramide (tioTEPA), topoisomerase I and / or topoisomerase II inhibitors, taxol, vinblastine, vincine vincinum, vincin VM-26 ^® ), as well as others too numerous to list. Other methods of treating cancer contemplated for use in combination with the methods of the present invention include irradiation with x-ray, gamma radiation, directly or by tomography, treatment of cancerous tissue with implanted radioactive granules or "grains", irradiation with a neutron beam of tissue that has undergone initial processing boron compounds, and / or other types of therapy using a particle beam known in the art.

Additional antitumor or anticancer agents (anticancer agents) that are optionally administered in combination or in conjunction with the methods of the present invention include those described in GOODMAN AND OILMAN'S, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, TENTH EDITION, Eds. Hardman and Limbird, incorporated herein by reference in its entirety.

In order to better understand the invention, the following terms are defined.

The reference to "adenosine" in the present description also covers deoxyadenosine and their known prior art variants and derivatives present in vivo , unless otherwise indicated.

The reference to adenosine deaminase or ADA includes any form of the enzyme known from the prior art, including purified natural enzyme, for example, purified natural ADA, recombinant human or bovine ADA, as well as their variants, polymorphisms and derivatives. The ADA enzyme isolated from cattle sources has the sequence according to SEQ ID NO: 5 with a naturally capped or cysteine protected Cys ₇₄ residue, with six C-terminal residues predicted based on the ADA encoding gene SEQ ID NO : 5, not present. However, it is contemplated that the invention may be practiced with alternatives to natural bovine ADA, including alternative alleles and polymorphisms, both natural and genetically engineered, with and without the predicted six C-terminal residues. Bovine ADA polymorphisms include, for example, glutamine at position 198 instead of lysine; alanine at position 245 instead of threonine; arginine at position 351 instead of glycine.

Preferred derivatives of the ADA enzyme include a genetically engineered ADA enzyme that has been mutated to increase stability compared to the un mutated ADA recombinant enzyme. These include, for example, recombinant ADA enzymes modified based on SEQ ID NO: 5 and / or SEQ ID NO: 5 with one or more of the above polymorphisms, to replace the oxidizable hydrophilic Cys residue with a suitable non-oxidizable amino acid residue. Such an non-oxidizable residue includes any known amino acid residue and / or any derivatives thereof known from the prior art. For example, a mature recombinant ADA expressed from a gene originally isolated from a cattle or human source has an unstable Cys ₇₄ residue, which is preferably replaced by an non-oxidizable amino acid residue, such as Ser ₇₄ . Such recombinant ADA enzymes are shown in SEQ ID NO: 1 (bovine ADA structure) and SEQ ID NO: 3 (human ADA structure). SEQ ID NO: 2 and SEQ ID NO: 4 show useful DNA molecules designed for expression of the same that have been optimized for codons for expression in E. coli . Further details regarding such recombinant ADA muteins, as well as the preparation and purification of these proteins, are provided in US patent application _______, which claims the priority of US patent application 60 / 913,009, entitled "Stable Recombinant Adenosine Deaminase" filed on the same date as this application and included in this application by sending in full. The exact data regarding the vectors and methods can be found in the specified application, in particular in the Examples section, and in particular in Examples 1-4.

Alternatively, ADA enzymes can be stabilized, if necessary, by capping the hydrophilic oxidizable Cys residue by treating the ADA enzyme with a sufficient amount of capping agent, under reaction conditions that are sufficient to cope the reactive cysteine, without significant inactivation of the ADA protein. In this process, it is preferable to use ADA obtained by genetic engineering methods in the form of a mutein or a wild-type enzyme.

For example, capping agents include, but are not limited to, oxidized (preferably) glutathione, iodoacetamide, iodoacetic acid, cystine, and other dithiols known to those of ordinary skill in the art, as well as mixtures thereof. The amount and concentration of capping agent introduced during the reaction phase of the methods described in this application, to some extent depends on the specific capping agent used and the needs of a person skilled in the art, but will not be subjected to undue experimental research. When using oxidized glutathione as a prototype, its concentration used in the course of interaction with a recombinant protein, such as rhADA, can vary from about 25 μm to about 100 mm. Preferably, the oxidized glutathione interacts with the recombinant protein at a concentration of from about 5 nM to about 25 mm.

The reaction conditions used during the reaction of the capping agent and the recombinant protein further include the use of an aqueous solution having a pH of from about 6.5 to about 8.4, preferably from about 7.2 to about 7.8. In addition, the aqueous solution preferably includes a suitable buffer solution, such as sodium phosphate, potassium phosphate, Tris and HEPES, as well as mixtures thereof in concentrations from 10 to 150 mm (optional capping can be performed at buffer concentrations outside the specified range, for example below 10 or higher 150 mM). The reaction conditions further include carrying out the reaction at temperatures that will not contribute to protein degradation, i.e., at temperatures of about 4-37 ° C. Optionally, capping may be performed outside a specified temperature range, for example, in a temperature range below 0-4 ° C or above 31 ° C. The reaction is carried out for a time that is sufficient to achieve the desired stabilization of the reactive cysteine. Just as an example, the reaction is carried out for from about 5 seconds to about 8 hours (for example, overnight).

Further details regarding the capped, stabilized recombinant ADA are provided in US Patent Application 11 / 738,012, entitled "Stabilized Proteins", especially in the Examples section, which is incorporated herein by reference.

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.

A. ADA-polymer conjugates

A preferred form of ADA is a polymer conjugated enzyme. The ADA-polymer conjugate of the present invention generally correspond to formula (I):

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

wherein

(ADA) is an adenosine deaminase enzyme or, optionally, a derivative or fragment thereof;

NH- is the amino group of an amino acid present in ADA, a derivative or fragment thereof, which serves to attach to the 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 is a substantially non-antigenic polymer residue that is attached to the ADA in a reversible or irreversible manner.

The non-antigenic polymer portion (R) of the conjugate can be selected from a non-limiting list of polymer based systems, such as:

in which:

R _10-11 and R _22-23 may be the same or different and are independently selected non-antigenic polymer residues;

R _3-9 , R _12-21 and R ₂₄ (see below) are the same or different, each of which is independently selected from hydrogen, C _1-6 alkyls, branched C _3-12 alkyls, C _{3- 8} -cycloalkyl, substituted C _1-6 -alkyl, substituted C _3-8 -cycloalkyl, aryl, substituted aryl, aralkyl, C _1-6- heteroalkyl, C _1-6 -alkoxy, phenoxy and C _1-6 -heteroalkoxy;

Ar is a group that forms a multisubstituted aromatic hydrocarbon or a multisubstituted heterocyclic group;

Y _1-11 and Y ₁₃ may be the same or different and independently selected from O, S and NR ₂₄ ;

A is selected from alkyl groups, targeting groups, diagnostic agents and biologically active groups;

X is O, NQ, S, SO or SO ₂ , where Q is H, C _1-8 alkyl, branched C _1-8 alkyl, substituted C _1-8 alkyl, aryl or aralkyl;

Z and Z 'are independently selected from groups actively transported to the target cell, hydrophobic groups, bifunctional crosslinking groups, and combinations thereof;

L _1-6 and L ₈ may be the same or different and are independently selected bifunctional linker groups;

(a), (c), (d), (f), (g), (i), (j), (j '), (k), (l), (n), (o), ( p), (q) and (t) may be the same or different and preferably independently are 0 or a positive integer, in most aspects; (b), (e), (r), (r '), (s), (h), (h') and (m) may be the same or different and independently are 0 or 1;

MPEG is methoxy-PEG, and

(u) is a positive integer, providing polymers having a total molecular weight of from about 2000 to about 100000 Da, preferably from about 4000 to about 45000 Da.

In the framework of the foregoing, it is preferable that Y _1-11 and Y ₁₃ are O; all R _3-8 , R _12-21 and R ₂₄ are independently either hydrogen or C _1-6 alkyl, with methyl and ethyl being the most preferred alkyls, and R _{7-9 is} preferably CH ₃ .

In another aspect of the invention, the polymer portion of the conjugate may be one that provides multiple points for the attachment of an ADA. A non-limiting list of such systems includes:

and

,

,

where all variables are defined above.

Alternatively, and / or preferably, multiple PEG chains are attached to the ADA. In these aspects, the ADA polymer conjugate may include at least 5 chains to 80 polyethylene glycol chains attached to the Lys epsilon amino groups in the enzyme, and preferably 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.

Activated polymers that can be used to produce ADA conjugates usually directly correspond to the polymer moieties described above. The main difference is the presence of a leaving or activating group, sometimes referred to in the present description as B ₁ , which facilitates the attachment of the polymer system to an amino group (for example, the epsilon-amino group of lysine) present in ADA. Thus, compounds (i) to (xiii) include a leaving or activating group, such as:

or another suitable leaving or activating group, such as N-hydroxybenzotriazolyl, halogen, N-hydroxyphthalimidyl, imidazolyl, O-acylureas, pentafluorophenol or 2,4,6-trichlorophenol, or other suitable leaving groups that are obvious to those of ordinary skill in the art that are present in the art the place where the ADA joins after the conjugation reaction.

Some preferred activated PEGs include activated PEGs as described in U.S. Patent Nos. 5,226,214, 5,324,444, 5,612,460 and 5,808,096 (succinimidyl carbonate-activated polyethylene glycol (SC-PEG) and similar activated PEGs) and US Pat. No. 534,9001 (cyclic imidion activated PEG) the contents of which are incorporated into this application by reference. As will be appreciated by those of ordinary skill in the art, 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 PEGs, such as the above SC-PEG, may contain, on average, from about 1 to about 80 PEG chains per enzyme molecule. Thus, in this case, molar excesses of several hundred times, for example 200-1000x, can be used. The molar excess used for branched polymers and polymers 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 the framework of the present invention, activated groups, as understood, are those groups that are capable of reacting with an amino group (nucleophile) present in an ADA, for example, on Lys.

In the framework of the present invention, the above is also referred to as activated polymer linkers. The polymer residues are preferably polyalkylene oxide based polymers, and more preferably polyethylene glycol (PEG) based polymers, where the PEG is linear, branched or multipath.

Further, referring to the polymers described above, it can be noted that Ar is a group that forms a multisubstituted aromatic hydrocarbon or a multisubstituted heterocyclic group. The main feature is that the Ar group is aromatic in nature. In general, in order to be an aromatic compound, π-electrons must be distributed in a “cloud” above and below the plane of the cyclic molecule. In addition, the number of π electrons must satisfy the Hückel rule (4n + 2). The average experts in this field are aware that a huge number of groups meets the requirement of aromaticity of the group and, thus, is suitable for use in the present invention.

In some preferred aspects of the invention, activated polymer linkers of polymer systems based on benzyl cleavage or lactonization of a trimethyl lock are prepared in accordance with U.S. Patent Nos. 6180095, 6720306, 5965119, 6624142 and 6303569, the contents of which are incorporated herein by reference. Within this context, the following activated polymer linkers are preferred:

In one alternative aspect of the invention, an ADA-polymer conjugate is prepared from 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) . A description of each such patent application is hereby incorporated by reference. Some of the preferred activated polymers include:

You must also understand that the leaving or activating group shown above is only one of the suitable groups, and other groups mentioned in this application can also be used without undue experimental work.

In alternative aspects, activated polymer linkers are prepared using branched polymer residues, such as those described in US Patents 5,651,567, 5756593, 5643575, 5919455, 6113906, 6153655, 6395266 and 6638499, 6251382, and 6824766, the disclosures of which are incorporated herein application by sending. Such activated polymers correspond to polymer systems (iv) to (ix), an example of which is the following compound:

,

,

in which B ₁ is an activating group, and all variables are defined above.

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 from about 10 to about 2300, preferably providing polymers having a total molecular weight of from about 4000 to about 45000.

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

Methods for producing 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 process for producing PAO carboxylic acids involves the formation of an intermediate, such as t- butyl ether of a polyalkylene oxide-carboxylic acid. This intermediate is formed in 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 potassium salt of methyl tert-butylimidodicarbonate (KNMeBoc) or 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%.

1. Essentially non-antigenic polymers

As indicated above, R _10-11 and R _22-23 are preferably water soluble polymer residues, which are preferably substantially non-antigenic, such as polyalkylene oxides (PAO), and most preferably polyethylene glycols, such as mPEG. For purposes of non-limiting illustration, a portion of the polyethylene glycol (PEG) residue R _10-11 and R _22-23 can be selected from the following:

JO- (CH ₂ CH ₂ O) _u -,

JO- (CH ₂ CH ₂ O) _u -CH ₂ C (O) -O-,

JO- (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ NR ₂₅ - and

JO- (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ SH-,

Where:

(u) is the degree of polymerization, that is, equal to from about 10 to about 2300;

R _{25 is} selected from hydrogen, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, branched C _3-12 alkyl, C _3-8 cycloalkyl, substituted C _1-6 alkyl, substituted C _2-6 alkenyl, substituted C _2-6 alkynyl, substituted C _3-8 cycloalkyl, aryl, substituted aryl, aralkyl, C _1-6 heteroalkyl, substituted C _1-6 heteroalkyl, C _1-6 alkoxy, phenoxy and C _1-6 heteroalkoxy, and

J is a capping group, that is, a group that is present at the end of the polymer and, in some aspects, may be selected from any of NH ₂ (or CH ₂ CH ₂ NH ₂ ), H, SH (or CH ₂ CH ₂ SH), CO ₂ H (or CH ₂ CO ₂ H), C _1-6 -alkyls, preferably methyl, or other PEG-terminal activating groups, these groups being known to those of ordinary skill in the art.

In one most preferred embodiment, R _10-11 and R _22-23 are selected from:

CH ₃ -O- (CH ₂ CH ₂ O) _u -, CH ₃ -O- (CH ₂ CH ₂ O) _u -CH ₂ C (O) -O-,

CH ₃ -O- (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ NH- and CH ₃ -O- (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ SH-,

where (u) is a positive integer, preferably selected so that the average total molecular weight of the polymer part ranges from about 2000 to about 100000 Da. More preferably, R _10-11 and R _22-23 independently have an average total molecular weight of from about 4000 Da to about 45000 Da, with the most preferred weight average molecular weight being from about 5000 Da. Other molecular weights are also considered suitable in accordance with the needs of a person skilled in the art.

PEG has the general formula:

-O- (CH ₂ CH ₂ O) _u -,

and R _10-11 and R _22-23 preferably include residues of this formula. The degree of polymerisation of the polymer is the number of repeating units in the polymer chain and depends on the molecular weight of the polymer.

In the alternative, a portion of the polyethylene glycol (PEG) residue according to the invention can be represented by the structure:

-Y ₃₁ - (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ Y ₃₁ -,

-Y ₃₁ - (CH ₂ CH ₂ O) _u -CH ₂ C (= Y ₃₂ ) -Y ₃₁ -,

-Y ₃₁ - (= Y ₃₂ ) - (CH ₂ ) _a11 -Y ₃₃ - (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ -Y ₃₃ - (CH ₂ ) _a11 -C (= Y ₃₂ ) -Y ₃₁ -,

-Y ₃₁ - (CR ₃₁ R ₃₂ ) _a12 -Y ₃₃ - (CH ₂ ) _b11 -O- (CH ₂ CH ₂ O) _u - (CH ₂ ) _b11 -Y ₃₃ - (CR ₃₁ R ₃₂ ) _a12 -Y ₃₁ -,

-Y ₃₁ - (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ -,

-Y ₃₁ - (CH ₂ CH ₂ O) _u -CH ₂ C (= Y ₃₂ ) -,

C (= Y ₃₂ ) - (CH ₂ ) _a11 -Y ₃₃ - (CH ₂ CH ₂ O) _u -CH ₂ CH ₂ -Y ₃₃ - (CH ₂ ) _a11 -C (= Y ₃₂ ) - and

- (CR ₃₁ R ₃₂ ) _a12 -Y ₃₃ - (CH ₂ ) _b11 -O- (CH ₂ CH ₂ O) _u - (CH ₂ ) _b11 -Y ₃₃ - (CR ₃₁ R ₃₂ ) _a12 -,

Where:

Y ₃₁ and Y ₃₃ are independently O, S, SO, SO ₂ , NR _33, or a bond;

Y ₃₂ is O, S or NR ₃₄ ;

R _{31-34 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 - 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 arylcarbonyl deputy substituted C _2-6 alkanoyloxy, substituted aryloxycarbonyl, substituted C _2-6 alkanoyloxy and substituted arylcarbonyloxy;

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

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

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

-C (= Y ₃₄ ) - (CH ₂ ) _m11 - (CH ₂ CH ₂ O) _u -,

-C (= Y ₃₄ ) -OY) - (CH ₂ ) _m11 - (CH ₂ CH ₂ O) _u -,

-C (= Y ₃₄ ) -NR ₃₁ - (CH ₂ ) _m11 - (CH ₂ CH ₂ O) _u -,

-CR ₃₅ R ₃₆ - (CH ₂ ) _m11 - (CH ₂ CH ₂ O) _u -,

Where

R ₃₁ , R ₃₅ and R _{36 are} independently selected from H, C _1-6 alkyl, aryl, substituted aryl, aralkyl, heteroalkyl, substituted heteroalkyl and substituted C _1-6 alkyl;

(m11) is zero or a positive integer, and is preferably 1 or 2;

Y ₃₄ is O or S; and

(u) represents the degree of polymerization.

In these aspects, a capping group (J), such as methyl, is attached at the end of the PEG.

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 the NOF Corp. catalog. Drug Delivery System catalog. Ver. 8, April 2006, the contents of which are incorporated herein by reference, into a suitable activated polymer using activation methods described in US Patents 5,226,214 or 5808096 described above. See also Shearwater's Polyethylene Glycol and Derivatives for Biomedical Application catalog, 2001, incorporated herein 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 particular, 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 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:

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.

In another embodiment, and as an alternative to PAO-based polymers, each R _10-11 and R _{22-23 is} optionally selected from one or more non-allergenic materials, such as dextran, polyvinyl alcohols, carbohydrate-based polymers, polyvinylpyrrolidones, polyacrylamides, for example hydroxypropylmethacrylamide (HPMA), polyalkylene oxides and / or copolymers of the above polymers. See also U.S. Patent 6153655, the contents of which are incorporated herein by reference. The average specialists in this field will be able to understand that the same type of activation is used as described in this application with respect to PAO, such as PEG. Those of ordinary skill in the art will also understand that the above list is merely illustrative, and that all polymeric materials having the qualities described herein are considered, and that other derivatives of polyalkylene oxides, such as polypropylene glycols, etc., are also contemplated.

2. Bifunctional linker groups

In many aspects of the invention, L _1-6 and L ₈ are linker groups that facilitate the attachment of a polymer chain, for example R _10-11 and / or R _22-23 . The bond formed may be direct or include additional crosslinking groups known to those of ordinary skill in the art. In this aspect of the invention, L _1-6 and L ₈ may be the same or different and may be selected from a wide variety of groups known to those of ordinary skill in the art, such as bifunctional and heterobifunctional aliphatic and aromatic-aliphatic groups, amino acids, etc. Thus, L _1-6 and L ₈ may be the same or different and include groups such as:

Preferably, L _1-6 and L ₈ are selected from:

Alternatively, suitable amino acid residues may be selected from any known natural L-amino acids, for example alanine, valine, leucine, etc., and / or combinations thereof, as well as many others. L _1-6 and L ₈ may also include a peptide, the size of which varies, for example, from about 2 to about 10 amino acid residues.

Derivatives and analogues of natural amino acids, as well as various non-natural amino acids (D or L) known in the art, hydrophobic or non-hydrophobic, are also included in the scope of the invention.

3. Z-groups and their function

In one aspect of the invention, Z and Z 'are L ₇ -C (= Y ₁₂ ), where L ₇ is a bifunctional linker selected from the group that defines L _1-6 , and Y _{12 is} selected from the same groups that define Y ₁ . In this aspect of the invention, the Z group serves as a bond between the ADA and the rest of the polymer delivery system. In other aspects of the invention, Z is a group that is actively transported to the target cell, a hydrophobic group, and combinations of the above. If Z 'is present, it can serve as a bifunctional linker, a group that is actively transported to the target cell, a hydrophobic group, including combinations of the above.

In this aspect of the invention, reversible-coupled polymer systems are obtained which undergo in vivo hydrolysis with cleavage of the polymer from ADA, which leads to the release of the enzyme, still bound to the Z group, into the intercellular fluid. For example, some possible ZB combinations include leucine-ADA and Gly-Phe-Leu-Gly-ADA.

B. Preparation of ADA Conjugates

For purposes of illustration, the present application describes suitable conjugation reactions involving the reaction of ADA with a polymer system activated as desired. The reaction is preferably carried out using protein modification conditions known to those skilled in the art, including the use of a PBS buffer system, etc., with a pH in the range of about 6.5-8.5. It is believed that in most cases an excess of activated polymer reacts with ADA.

Reactions of this type often lead to the formation of conjugates in which one or more polymers are attached to the ADA. It should be understood that it is often necessary to isolate various fractions and obtain a more homogeneous product. In most aspects of the invention, the reaction mixture is collected, applied to a column loaded with a suitable resin, and the desired fractions are eluted sequentially with increasing buffer levels. Fractions are analyzed by suitable analytical methods to determine the purity of the conjugated protein before further processing. Regardless of the route of synthesis and the selected activated polymer, the conjugates are in accordance with Formula (I) as defined herein. Some of the preferred conjugates obtained using the synthesis methods described in this application include:

where B is an ADA.

Still other conjugates obtained in accordance with the present invention include:

where all variables are defined above, and B is an ADA.

Other conjugates include:

where B is an ADA.

A particularly preferred conjugate is:

where the molecular weight of MPEG is from about 4000 to about 45000.

When bicin-based polymer systems are used, two preferred conjugates are:

where the molecular weights of MPEG are the same as described above. More preferred conjugates include:

It should be noted that the PEGylation of ADA is empirically optimized with respect to the total PEG addition per protein molecule, the size of the PEG polymer, as well as the structure of the PEG linker. Key characteristics of PEGylated ADA to evaluate the optimization of PEGylation include both in vitro assays (e.g., enzyme activity and stability analyzes) and in vivo assays (e.g. pharmacokinetics and pharmacodynamics).

C. Tumors to be treated

The methods of the invention can be used to treat all types of tumors, including cancers that are sensitive to lower levels of adenosine or deoxyadenosine in the blood and / or tissue of a human or animal being treated. In a broader sense, they include tumors originating from hematopoietic cells, as well as solid tumors. Solid tumors include those that are suppressed when a decrease in adenosine levels allows the patient’s immune system to more effectively suppress the tumor, and / or tumors that are suppressed when a decrease in adenosine levels inhibits blood supply, for example, in already hypoxic tumors.

More preferably, tumors that are sensitive to treatment using the methods of the invention are solid tumors that are successfully suppressed as a result of the additional effect of reducing angiogenic stimulation, concomitant with a decrease in tissue adenosine levels.

Tumors to be treated include, by way of example only, those tumors that initially form in the immune system, skeletal system, muscles and heart, mammary gland, gastrointestinal tract, central and peripheral nervous system, renal system, reproductive system, respiratory system system, skin, systems of connective tissues, including joints, adipose tissues, cardiovascular system, including the walls of blood vessels, etc.

Skeletal system tumors include, for example, sarcomas and blastomas, such as osteosarcoma, chondrosarcoma, chondroblastoma, etc. Muscle and cardiac tumors include skeletal and smooth muscle tumors, such as leiomyomas (benign smooth muscle tumors), leiomyosarcomas, rhabdomyomas (benign skeletal muscle tumors), rhabdomyosarcomas, heart sarcoma, etc. Tumors of the gastrointestinal tract include, for example, tumors of the mouth, esophagus, stomach, small intestine, colon and colorectal tumors, as well as tumors of the secretory organs of the gastrointestinal tract, such as salivary glands, liver, pancreas, bile ducts, etc. P.

Tumors of the central nervous system include tumors of the brain, retina, and spinal cord, and can also form in connected connective tissue, bone, blood vessels, or nerve tissue. Tumors of the peripheral nervous system can also be treated. In addition, peripheral nervous system tumors include peripheral nerve sheath malignancies.

Tumors of the renal system include tumors of the kidney, for example, renal cell carcinoma, as well as tumors of the ureter and bladder. Tumors of the reproductive system include tumors of the cervix, uterus, ovary, prostate gland, male genital glands and associated secretory glands. Tumors of the immune system include both hematopoietic-based tumors and solid tumors, including lymphomas, such as Hodgkin and non-Hodgkin.

Tumors of the respiratory system include tumors of the nasal passages, bronchi and lungs. Breast tumors include, for example, both lobular and ductal carcinoma.

In particular, the common types of cancer to be treated include, by way of example only: prostate cancer, lung cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic cancer, endometrial cancer, ovarian cancer, cutaneous melanoma, lymphoma , non-Hodgkin’s lymphoma, pancreatic cancer, neuroblastoma, Wilms tumor, rhabdomyosarcoma (occurring in the muscles), retinoblastoma, osteosarcoma and Ewing's sarcoma, as well as many others.

D. Dose selection

The specialist will be able to evaluate that in a clinical situation, the dosage of Adagen ^{® is} individualized depending on the clinical response of the tumor and the profile of side effects in an individual patient - animal or human. In the examples below, the highest dose is the maximum dose that can be tolerated. Adagen ^{® is} supplied commercially at a dosage of 250 U / ml. This corresponds to 2000 U / kg for mice weighing approximately 25 g after administration of 0.2 ml Adagen ^®. The minimum dose used in the examples below is approximately the same as the clinical dose for humans. The recommended dosage regimen for the treatment of patients with SCID is 10 U / kg for the first dose, 15 U / kg for the second dose and 20 U / kg for the third dose. Subsequent dose increases of 5 U / kg / week are allowed, up to a maximum single dose of 30 U / kg. The dose in the following exemplary protocol (100 U / kg) is a mouse dose equivalent to a clinical child dose of approximately 12 U / kg.

The dose based on the amount of the enzyme varies, for example, from about 0.10 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.

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 + 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.

Detailed information regarding the dosage of ADA is provided on the medical instructions leaflet for ADAGEN ^® (Enzon, Inc.), the contents of which are included in 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

Antitumor efficacy of Adagen ^®  in a xenograft model of a human prostate tumor DU145.

A) Test system

View: House mouse, Mus musculus Line: hairless, hairless Provider: Harlan-sprague dawley Floor: female Average starting weight: 27.2 g Test Number: 40 Adaptation Period: 7 days after arrival Identification: cell number and ear stamp

B) Methods

Experiment Design:

Human prostate cancer cells DU 145 were obtained from the American Tissue Culture Collection (ATTC), Manassas, VA. Tumors were engrafted into nude mice by subcutaneous injection of 2.0 × 10 ⁶ DU 145 cells / mouse into the right axillary region. Tumor growth was checked twice a week and measurements were taken immediately after the tumor began to be felt. When the tumors reached an average volume of 78 mm ³ , the mice were divided into experimental groups (8 per group). Mice were Adagen ^® 2,000, 500 or 100 IU / kg twice a week for 5 weeks. As a positive control, mice were injected with Avastin ^® (bevacizumab, an anti-VEGF monoclonal antibody) at a dose of 5 mg / kg with the same frequency as Adagen ^® . The experimental groups were processed according to the scheme shown in Table 1. The first dosing day was designated as day 1. The tumor volume in each mouse was determined by measuring in two dimensions using a caliper and then calculating using the formula: tumor volume = (length × width ² ) / 2. Mouse weight and tumor size were determined at the beginning of the study and twice a week for an 8-week period.

Table 1 Group number Group Number of animals (n) Dose (IU / kg) Route of administration Dosing schedule one The control 8 saline in / b twice a week × 5 2 Adagen ^® 8 2000 in / b twice a week × 5 3 Adagen ^® 8 500 in / b twice a week × 5 four Adagen ^® 8 one hundred in / b twice a week × 5 5 Avastin ^® 8 5 mg / kg in / b twice a week × 5

Dose selection:

Adagen ^® or Avastin ^® was administered intraperitoneally (ip) twice a week for five weeks (10 doses in total).

Adagen ^® : Batch number: NV0604, concentration: 229 IU / ml.

Avastin ^® : Batch number: M66781, concentration: 25 mg / ml.

Dose calculations were based on body weight on day 1.

Clinical Trials:

Mice were examined visually after arrival. Then the mice were examined individually, twice a week after the start of palpation of the tumor, for clinical symptoms, general changes in behavior, and body weight was also monitored. All deaths and clinical symptoms were recorded. Food and water consumption were not monitored. Tumor mice that showed open necrotic lesions were euthanized. Mice losing more than 20% of body weight were also humanely killed.

Statistical analysis:

Differences in% change in tumor volume between different treatments were compared using one-way analysis of variance. All paired multiple comparisons were performed using the Hill-Sidak method.

C) Results

Definitions of terms used:

(a)% of the initial tumor volume: (tumor volume on any day / tumor volume on day 1) × 100.

(b)% change in tumor volume: [(tumor volume on any day — tumor volume on day 1) / tumor volume on day 1] × 100.

(c)% tumor growth inhibition (TGI): [(average tumor volume in the control group — average tumor volume in the treated group) / average tumor volume in the control group] × 100.

(d) Tumor regression is defined as a decrease in tumor volume compared to day 1.

(e) Recovery is defined as the complete absence of a tumor when observed with the naked eye.

The average tumor size at the beginning of the study was 78 mm ³ . The average body weight at the beginning of the study was 27.20 g. Mice in all groups gained weight, and by the end of the study, the weight of mice in different groups increased by 20-25% of the weight before treatment. The study was completed on day 57, when most of the animals had either ulcerated tumors or had a tumor volume of more than 1500 mm ³ . Tables 2 and 3 below show the final results recorded on the 49th day of the study (the last day, on which 100% survival of the control animals was still preserved), as well as a comparison of survival between groups of animals treated with Adagen ^® and groups animals treated with Avastin ^® . Tumors in the control group grew stably during the study. On the 49th day, the average tumor volume was 783.1 (± 556.2) mm ³ . The percentage change in tumor growth was 833.3% (± 622.7). Treatment with Adagen ^® at all three dose levels provide effective inhibition of tumor growth. It should be noted that in all treated groups, tumors grew at a slow rate until the last day of dosing (day 33), after which the tumors grew at a higher rate (data not shown). This observation is consistent with the fact that the% change in tumor volume at all three dose levels of Adagen ^{® was} significantly different from the change in the control group up to 36 days (P <0.05) (data not shown).

This suggests that Adagen ^® has a cytostatic effect on tumor growth. Although the% change in tumor volume did not statistically differ from the corresponding indicator in the control group after day 36 and until the end of the study, treatment with Adagen ^® led to detectable inhibition of tumor growth. In particular, tumors treated with 2,000 IU / kg Adagen ^®, had an average volume 343,0 (± 249,8) mm ³ in 49 days. The mean change in tumor volume was 424.2% (± 360.6). Adagen ^® in a dose of 2000 IU / kg showed 56% inhibition of tumor growth.

Tumors treated with 500 IU / kg Adagen ^®, had an average volume 696,3 (± 290,4) mm ³ in 49 days. The mean change in tumor volume was 797.6% (± 492.7), and the change from the initial tumor volume was 897.6% (± 492.7). Tumor growth inhibition was 11.1%. Tumors in this group showed similar growth rates, as in other groups treated with Adagen ^® , up to 40 days, after which they grew at a much higher rate. The reason for this discrepancy is unknown.

Tumors were treated with 100 IU / kg of Adagen ^® , had an average volume of 414.8 (± 219.0) mm ³ on the 49th day. The average change in tumor volume was 489.9% (± 307.0), and the average percentage change from the initial tumor volume was 589.9% (± 307.0). Inhibition of tumor growth or TGI was 47.0%.

The therapeutic effect of Adagen ^® reached its maximum level with a minimum dose of 100 IU / kg. It should be noted that the dose of 100 IU / kg is approaching the clinical dose of Adagen ^® for humans (the dose used to treat children with SCID). Avastin ^® showed the most effective tumor size reduction with inhibition of tumor growth or TGI of 92.5%.

In conclusion, it should be noted that treatment with Adagen ^® at all three dose levels provided effective inhibition of tumor growth of DU 145 in vivo .

table 2 Final Results (Day 49) Group number Compound The average final tumor volume The median value of the final tumor volume % change in tumor volume % of the initial tumor volume % of the initial tumor volume Cases of symptom relief Cases of Recovery
laziness % survivors one saline 783.1 (556.2) 680.0 833.3
(622.7) 933.3
(622.7) - 0 0 62.5 2 Adagen
(2000 IU / kg) 343.0
(249.8) 283.8 424.2
(360.6) 524.2
(360.6) 56.2 0 0 75.0 3 Adagen
(500 IU / kg) 696.3
(290.4) 766.2 797.6
(492.7) 897.6
(492.7) 11.1 0 0 75.0 four Adagen
(100 IU / kg) 414.8
(219.0) 349.8 489.9
(307.0) 589.9
(307.0) 47.0 0 0 75.0 5 Avastin
(5 mg / kg) 58.5
(36.1) 49.3 -15.7
(59.6) 84.3
(59.6) 92.5 7/8 0 87.5 The number of cases of symptom alleviation, cases of recovery, and% survivors on day 56 are given.
The mean tumor volumes, median values,% change in tumor volume,% of the initial tumor volume and% inhibition of tumor growth on day 49 are shown.

Table 3 Comparison of survival in groups (number of surviving animals) Group one 5 8 12 fifteen 19 22 26 29th 33 36 40 43 49 51 55 57 1: saline 8 8 8 8 8 8 8 8 8 8 8 8 8 5 ^a 5 5 5 2: Adagen
(2000 U / kg) 8 8 8 8 8 8 8 8 8 8 8 7 ^b 7 6 ^c 6 6 6 3: adagen
(500 U / kg) 8 8 8 8 8 8 8 8 8 8 8 7 ^b 7 6 ^c 6 5 ^d 5 4: adagen
(100 U / kg) 8 8 8 8 8 8 8 8 8 8 8 6 ^e 6 6 6 5 ^d 5 5: Avastin
(5 mg / kg) 8 8 8 8 8 8 8 8 8 8 8 8 8 7 ^c 7 7 7 a Two animals were killed because the tumor size exceeded 1500 mm ³ . One animal was killed due to ulceration of the tumor.
b The animal was killed due to ulceration of the tumor on day 49.
c The animal was killed due to ulceration of the tumor on day 49.
d The animal was killed due to ulceration of the tumor on day 55.
e Two animals were killed due to ulceration of the tumors on day 49.

EXAMPLE 2

Antitumor efficacy of Adagen ^®  in a xenograft model of a human ovarian tumor sk-ov-3

A) Test system

View: House mouse, Mus musculus Line: hairless, hairless Provider: Harlan-sprague dawley Floor: female Average starting weight: 22.18 g Test Number: 54 Adaptation Period: 7 days after arrival

B) Methods

Experiment Design:

Cells of SK-OV-3 human ovarian adenocarcinomas were implanted into nude mice by subcutaneous injection of 3 × 10 ⁶ cells / mouse into the right axillary region. Tumor growth was checked twice a week and measurements were taken immediately after the tumor began to be felt. Tumor volume in each mouse was determined by measuring in two dimensions using a caliper and subsequent calculation using the formula: tumor volume = (length × width ² ) / 2. When the tumors reached an average volume of 90 mm ³ , the mice were divided into experimental groups (9 per group). The experimental groups were processed according to the scheme shown in the table below. The first day of dosing was designated as day 1. The weight of the mouse and tumor size were determined at the beginning of the study and twice a week until the end of the study. The study was completed after approximately 7 weeks (52 days), when most animals developed large or ulcerated tumor masses.

Table 4 Group number Group Number of animals (n) Dose (E / kg) Route of administration Dosing schedule one the control 9 saline in / b twice a week × 5 2 Adagen ^® 9 2000 in / b twice a week × 5 3 Adagen ^® 9 500 in / b twice a week × 5 four Adagen ^® 9 one hundred in / b twice a week × 5 5 Avastin ^® 9 100 mcg / mouse in / b twice a week × 5 6 Native ADA 9 2000 in / b twice a week × 5

Dosing Scheme:

Adagen ^® or Avastin ^® was administered intravenously (iv) twice a week for five weeks (10 doses in total)

Test Products:

Adagen ^® : Lot number: NV0604, concentration: 229 IU / ml

Avastin ^® : Batch number: M66781, concentration: 25 mg / ml

Native ADA: Lot Number: 06-0315-111

Dose calculation:

Based on body weight per day 1.

Clinical Trials:

Mice were examined visually after arrival. Then the mice were examined individually, twice a week after the start of palpation of the tumor, for clinical symptoms, general changes in behavior, and body weight was also monitored. All deaths and clinical symptoms were recorded. Food and water consumption were not monitored. Tumor mice that showed open necrotic lesions were euthanized. Mice losing more than 20% of body weight were also humanely killed.

Statistical analysis:

Differences in% change in tumor volume between different treatments were compared using one-way analysis of variance. All paired multiple comparisons were performed using the Tukey-Cramer method.

C) Results

Definitions of terms used:

(a)% of the initial tumor volume: (tumor volume on any day / tumor volume on day 1) × 100.

(b)% change in tumor volume: [(tumor volume on any day — tumor volume on day 1) / tumor volume on day 1] × 100.

(c)% tumor growth inhibition (TGI): [(average tumor volume in the control group — average tumor volume in the treated group) / average tumor volume in the control group] × 100.

(d) Tumor regression is defined as a decrease in tumor volume compared to day 1.

(e) Recovery is defined as the complete absence of a tumor when observed with the naked eye.

The average tumor size at the beginning of the study was 90 mm ³ . The average body weight at the beginning of the study was 22.2 g. The average weight of mice in all groups did not significantly increase during the study. The study was completed on day 52. Four animals were killed because their tumor volume was more than 1500 mm ³ .

Table 5 below shows the final results on day 32 (on the last day of 66% survival of animals in the control group). Tumors in the control group grew stably during the study. On day 32, the average tumor volume was 754.9 (± 700.7) mm ³ . The percentage change in tumor growth was 693.2% (± 673.6) mm ³ .

Table 5 Final Results (Day 32) Group number Compound The average final tumor volume The median value of the final tumor volume % change in tumor volume % of the initial tumor volume % of the initial tumor volume Cases of symptom relief Cases of Recovery
laziness % survivors one saline 754.9
(700.7) 485.5 693.2
(673.6) 793.2
(673.6) - 0 0 66 2 Adagen
(2000 IU / kg) 437.7
(122.2) 446.4 402.1
(177.7) 502.1
(177.7) 42.0 0 0 one hundred 3 Adagen
(500 IU / kg) 471.1
(60.0) 465.8 421.3
(102.2) 521.3
(102.2) 37.6 0 0 89 four Adagen
(100 IU / kg) 497.7
(152.6) 478.9 450.7
(172.7) 550.7
(172.7) 34.1 0 0 one hundred 5 Avastin
(5 mg / kg) 210.2
(65.9) 198.6 161.6
(88.3) 261.6
(88.3) 72,2 0 0 one hundred 6 Native ADA (2000 IU / kg) 356.6
(213.0) 329.7 340.1
(239.1) 440.1
(239.1) 52.8 0 0 77

Tumors treated with 2,000 IU / kg Adagen ^®, had an average volume 437,7 (± 122,2) mm ³ in 32 days. The mean change in tumor volume was 402.1% (± 177.7). Adagen ^® in a dose of 2000 IU / kg showed 42% inhibition of tumor growth.

Tumors treated with 500 IU / kg Adagen ^®, had an average volume 471,1 (± 60,0) mm ³ in 32 days. The mean change in tumor volume was 421.3% (± 102.2), and the change from the initial tumor volume was 521.3% (± 102.2). Tumor growth inhibition was 37.6%.

Tumors treated with 100 IU / kg Adagen ^®, had an average volume 497,7 (± 152,6) mm ³ in 32 days. The average change in tumor volume was 450.7% (± 172.7), and the average percentage change from the initial tumor volume was 550.7% (± 172.7). Tumor growth inhibition was 34.1%.

Tumors treated with 2000 IU / kg of native ADA had an average volume of 356.6 (213.0) mm ³ on day 32 of the study. The average change in tumor volume was 340.1% (± 210.0), and the average percentage change from the initial tumor volume was 440.1% (239.1). Tumor growth inhibition was 52.8%.

Treatment with Adagen ^® at any dose level was comparable with the results in the control groups. However, Adagen ^® at each dose level caused TGI, as described above.

The therapeutic effect of Adagen ^® reached its maximum level with a minimum dose of 100 IU / kg. It should be noted that the dose of 100 IU / kg is approaching the clinical dose of Adagen ^® for humans (the dose used to treat children with SCID). Avastin ^® , which served as a positive control in this study, showed the most effective tumor size reduction with a TGI of 72%.

In conclusion, it should be noted that treatment with Adagen ^® at a dosage of 100, 500 or 2000 IU / kg led to inhibition of tumor growth in the range from 34-42%.

Alternative recombinant ADA enzymes for the methods of the invention are described below.

EXAMPLE 3

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 Nde I and Bam HI were introduced into the terminal regions of the gene. After cleavage of synthetic DNA with restriction enzymes Nde I and Bam HI 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.

Then, the isolated colonies were purified by transferring to plates and analyzed for expression induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG) in LB medium by 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 analyzed by SDS-PAGE electrophoresis had a desired molecular weight of approximately 40 kDa (data not shown).

EXAMPLE 4

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 Pat. No. 6,366,860, 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 Nde I and Bam HI were introduced into the terminal regions of the gene. After cleavage of synthetic DNA with restriction enzymes Nde I and Bam HI, 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 5

Purification of mutein by recombinant human ADA

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 glycerin and the cells were grown at 28 ° C to OD ₆₀₀ 11, after which the IPTG inducer was added to a final concentration 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 buffer containing 10 mM Tris, 1 mM DTT, pH 8,0, and homogenized at 1200 rev / min for 10 seconds ^{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) (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 16,000 rpm for 40 minutes at 4 ° C (Sorvall RC 5C plus, rotor SLA-1000). 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 with 1N 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 Nalgene filter (90 mm filter device). 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 onto HiTrap ^® DEAE ff 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 6

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 with 1N NaOH and 1N 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. Prior to eluting ADA from the column with equilibration buffer with 80 mM NaCl to remove impurities, elution was first performed at 60 mM and 70 mM NaCl. 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 7

Preparation of Pegylated Ser ₇₄ rbADA through 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 through 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 through 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 (19)

1. A method of treating a patient having a tumor, comprising administering an effective amount of adenosine deaminase to a patient in need thereof, where the tumor is a solid tumor and where adenosine deaminase is conjugated to a substantially non-antigenic polymer, where, after treatment, the tumor exhibits a reduced volume or reduced growth rate. 2. The method according to claim 1, where the tumor is malignant. 3. The method according to claim 1, where the amount of adenosine deaminase administered is effective to significantly reduce the levels of adenosine or deoxyadenosine in the tissue of the specified patient, and where the growth or spread of the tumor is inhibited by lowering the levels of adenosine in the tissue of the specified patient. 4. The method according to claim 1, where the tumor is selected from the group consisting of a prostate tumor, ovarian cancer and colorectal cancer. 5. The method according to claim 1, where the essentially non-antigenic polymer is polyalkylene oxide. 6. The method according to claim 5, where the polyalkylene oxide is polyethylene glycol. 7. The method according to claim 5, where the size of the essentially non-antigenic polymer varies from about 4000 to about 45000 Daltons. 8. The method according to claim 1, where the conjugated adenosine deaminase is administered at a dose of from about 10 E to about 30 E per kg. 9. The method according to claim 1, where the conjugated adenosine deaminase is administered for a period of time sufficient to maintain tumor inhibition. 10. The method according to claim 1, where adenosine deaminase is administered over a period of time from 1 to about 20 days. 11. The method according to claim 1, where the conjugate comprises two or more adenosine deaminase molecules on a substantially non-antigenic polymer. 12. The method according to claim 1, where the adenosine deaminase comprises from about 11 to about 18 chains of polyethylene glycol attached to the epsilon amino groups of one or more Lys residues in adenosine deaminase. 13. The method of claim 12, wherein the adenosine deaminase is conjugated to polyethylene glycol via a urethane bond. 14. The method according to claim 1, where adenosine deaminase is administered by a route selected from the group consisting of subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal, inhalation and transurethral routes of administration. 15. The method according to claim 1, where the adenosine deaminase is an adenosine deaminase isolated from a source related to cattle, or is a recombinant adenosine deaminase. 16. The method according to clause 15, where the recombinant adenosine deaminase selected from the group consisting of recombinant bovine adenosine deaminase, including SEQ ID NO: 1, recombinant human adenosine deaminase, including SEQ ID NO: 3, and recombinant bovine adenosine deaminase, including: . 17. The method according to clause 16, where the recombinant bovine adenosine deaminase, including SEQ ID NO: 5, includes Cys74, which is capped to prevent oxidation in the aquatic environment. 18. The method according to clause 16, where the recombinant adenosine deaminase corresponds to SEQ ID NO: 1 or SEQ ID NO: 5 with an amino acid substitution selected from the group consisting of Gln instead of Lys ₁₉₈ ; Ala instead of Thr245; Arg instead of Gly ₃₅₁ , and their combinations. 19. A method of inhibiting tumor growth in a mammal, comprising administering an effective amount of adenosine deaminase or its active fragment to a mammal in need thereof, where the tumor is a solid tumor and where adenosine deaminase or its active fragment is conjugated to a substantially non-antigenic polymer, where, after treatment, the tumor shows reduced volume or reduced growth rate.

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