Use methods of compositions for amino acid depletion therapy

Abstract

To provide use methods of compositions in drug production for inhibiting cancer cells having low asparagine synthetase (ASNS) expression.SOLUTION: The disclosure relates to a use method of a composition comprising an arginine reducing compound and an asparagine reducing compound for preparing a medicament to inhibit a cancer cell, where an arginase being the arginine reducing compound is specifically effective in combination use with an asparaginase being the arginine reducing compound for inhibiting proliferation of cancer cells, in particular, cells having low ASNS expression. Further, the medicament comprises a compound that reduces glutamine concentration such as an aminotransferase inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 546,489, filed August 16, 2017, and U.S. Provisional Application No. 62 / 591,102, filed November 27, 2017. These and all referenced exogenous materials are incorporated herein by reference in their entirety. In the event that the definition or use of a term in a reference incorporated by reference is inconsistent or incompatible with the definition of that term provided herein, the definition of that term provided herein shall control.

[0002] The field of the invention is arginase purification, arginase modification, and medical uses of arginase and asparaginase. [Background technology]

[0003] The following description contains information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] Increasing evidence suggests that amino acid deprivation may be an effective candidate for treating cancer. Deprivation of certain amino acids (e.g., arginine, asparagine, or glutamine) has been found to be useful for treating different types of cancer (Feun, You et al., 2008 (Non-Patent Document 1); Hensley, Wasti et al., 2013 (Non-Patent Document 2); Krall, Xu et al., 2016 (Non-Patent Document 3)). All publications herein are incorporated by reference to the same extent as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. If the definition or use of a term in an incorporated reference contradicts or is incompatible with the definition of that term provided herein, the definition of that term provided herein applies, and the definition of that term in the reference does not apply. The effectiveness of amino acid deprivation is thought to be due to downstream effects such as inactivation of mTORCl and disruption of protein synthesis.

[0005] The application of recombinant human arginase (rhArg) to deplete arginine has been shown to be effective in inhibiting cancer cell proliferation in vitro (Lam, Wong et al., 2009; Tsui, Lam et al., 2009). Arginine was selected as a target amino acid not only because of its semi-essential role in protein synthesis but also because of its role in activating mTORCl (Carroll, Maetzel et al., 2016; Chantranupong, Scaria et al., 2016; Krall, Xu et al., 2016; Saxton, Chantranupong et al., 2016; Zheng, Zhang et al., 2016). It was found that arginine depletion could effectively inhibit various cancer cell lines, including cell lines derived from breast, colon, lung, and cervical cancers.

[0006] The enzyme arginase acts on arginine to produce ornithine and urea and is part of the urea cycle. Arginase is increasingly used as a chemotherapeutic agent and is utilized to reduce serum arginine levels. These depleted serum arginine levels can effectively "starve" cancer cells, many of which are auxotrophic for arginine.

[0007] The use of arginase as a therapeutic agent requires the availability of both large amounts and highly purified human arginase. Attempts have been made to provide highly purified recombinant human arginase. For example, U.S. Patent No. 8,507,245 (to Leung and Lo) describes a pseudo-affinity chromatography method for purifying recombinant human Arginase 1 modified to provide a single site for PEGylation. However, the described method is limited to forms containing a polyhistidine sequence that allows complexation with a metal pseudo-affinity medium. This pseudo-affinity medium is used in an affinity purification step that is necessitated by the use of a large excess of reactive PEG analogs in the conjugation reaction. Therefore, Arginase 1 purified by such a method cannot be considered fully human without additional processing to remove the polyhistidine sequence.

[0008] Unmodified arginase is unstable in plasma, severely limiting its therapeutic application. Numerous attempts to extend plasma half-life have been explored, including protein conjugation with the polymer polyethylene glycol (PEGylation). A widely used conjugation strategy is nonselective PEGylation of arginase amino groups (e.g., the ε-amine of lysine) as described in U.S. Patent No. 9,050,340 (to Georgiou and Stone). Such treatment requires the use of a significant molar excess of costly amine-reactive PEG reagents, due in part to the relatively rapid hydrolysis of such reagents. Such random conjugation also complicates qualitative and quantitative characterization of the modified arginase, thereby limiting its pharmaceutical use. Consistent product properties are unlikely to be achieved using such an approach unless the coupling reaction is performed under very tightly controlled conditions, which are often not amenable to scale-up. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 8,507,245 [Patent Document 2] U.S. Patent No. 9,050,340 [Non-patent literature]

[0010] [Non-Patent Document 1] Feun, L., M. You, et al. (2008). "Arginine deprivation as a targeted therapy for cancer." Curr Pharm Des 14(11): 1049-1057. [Non-patent document 2] Hensley, C. T., A. T. Wasti, et al. (2013). "Glutamine and cancer: cell biology, physiology, and clinical opportunities." J Clin Invest 123(9): 3678-3684. [Non-Patent Document 3] Krall, A. S., S. Xu, et al. (2016). "Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor." Nat Commun 7: 11457. [Non-Patent Document 4] Lam, T. L., G. K. Wong, et al. (2009). "Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest." Cancer Lett 277(1): 91-100. [Non-Patent Document 5] Tsui, S. M., W. M. Lam, et al. (2009). "Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)." Cancer Cell Int 9: 9. [Non-Patent Document 6] Carroll, B., D. Maetzel, et al. (2016). "Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity." Elife 5. [Non-Patent Document 7] Chantranupong, L., SM Scaria, et al. (2016). "The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway." Cell 165(1): 153-164. [Non-patent document 8] Saxton, RA, L. Chantranupong, et al. (2016). "Mechanism of arginine sensing by CASTOR1 upstream of mTORC1." Nature 536(7615): 229-233. [Non-Patent Document 9] Zheng, L., W. Zhang, et al. (2016). "Recent Advances in Understanding Amino Acid Sensing Mechanisms that Regulate mTORC1." Int J Mol Sci 17(10). Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, there remains a need for methods that can provide active, effective, and consistent PEGylated arginase in high purity. [Means for solving the problem]

[0012] (Summary of the Invention) The subject of the present invention is to provide compositions and methods for preparing and derivatizing highly pure arginase and utilizing the so-prepared arginase in combination with asparaginase in the treatment of cancer.

[0013] One embodiment of the inventive concept is a method of purifying arginase by obtaining cells (e.g., bacterial cells) that express arginase, disrupting the cells to produce a lysate containing arginase, and raising the temperature of the lysate to a precipitation temperature (e.g., at least 50°C or about 65°C) in the presence of CoCl (e.g., at a concentration of at least 20 mM) for a sufficient time (e.g., about 5-30 minutes) to precipitate contaminants from the lysate and produce a supernatant containing a first partially purified arginase. The supernatant is then contacted with an anion exchanger, thereby binding additional contaminants and forming a partially purified CoCl. 2+ Arginase (i.e., Mn 2+ Co 2+ The flow-through fraction containing the partially purified CoA (substituted arginase) is obtained. 2+ The arginase can have a purity of about 80% or greater.

[0014] In some embodiments, the method includes the further step of contacting the flow-through fraction with a cation exchanger to produce a bound fraction containing the arginase and a second flow-through fraction. An elution buffer is applied to the cation exchanger to form a bound fraction containing the arginase. 2+ The arginase is eluted. 2+ The arginase can have a purity of about 90% or more. The arginase can be human arginase 1. The anion exchanger and cation exchanger used can be strong ion exchangers.

[0015] Another embodiment of the inventive concept is a method for selectively derivatizing a protein (such as human Arginase 1 or a variant thereof) by obtaining a protein containing at least one cysteine, contacting the protein with PEG-maleimide in a buffer having a pH between 6.5 and 7.0 at a temperature between 2°C and 15°C, and incubating the protein with the PEG-maleimide for 24 to 72 hours at 2°C to 15°C to produce a PEG-derivatized protein. In such methods, the PEG-maleimide is present in less than a 4-fold molar excess over the protein. The PEG-maleimide can be derived from branched or linear PEG. In some embodiments, the protein is an Arginase mutated to remove all but one cysteine. In some embodiments, the protein does not contain a polyhistidine sequence. In some embodiments, the method includes the additional step of separating the PEG-derivatized protein from unreacted or hydrolyzed PEG-maleimide, e.g., by dialysis, size exclusion chromatography, and / or ion exchange chromatography.

[0016] Another embodiment of the inventive concept is a preparation of PEG-modified human arginase 1 comprising a peptide sequence corresponding to SEQ ID NO: 1 covalently attached to a single PEG moiety having a molecular weight of at least 20 kDa. The PEG-modified human arginase 1 represents at least 90% of the human arginase in such a preparation, and the PEG-modified human arginase 1 does not contain a polyhistidine sequence. The PEG moiety of the PEG-modified human arginase can be linear or branched (e.g., having a "Y" configuration or a "V" configuration). In some such embodiments, the PEG-modified human Arginase 1 may include a metal cofactor, such as manganese, nickel, and / or cobalt.

[0017] Another embodiment of the concept of the present invention is a method for inhibiting cancer cells by reducing the arginine concentration in the culture medium used to culture the cancer cells and reducing the asparagine concentration in the culture medium.The arginine concentration can be reduced using arginase (e.g., recombinant human arginase).Similarly, the asparagine concentration can be reduced using asparaginase (ASNase). In some such embodiments, the cancer cells have low asparagine synthetase (ASNS) expression. In some such embodiments, the method includes the further step of reducing glutamine levels, for example, with an aminotransferase inhibitor (such as aminooxyacetic acid).

[0018] Another embodiment of the present invention is a composition for inhibiting cancer cells, comprising an arginine reductase and an asparagine reductase. The arginine reductase can be an arginase, such as recombinant human arginase. Similarly, the asparagine reductase can be asparaginase (ASNase). In some such embodiments, the cancer cells have low expression of asparagine synthetase (ASNS). The composition can also include a compound that reduces glutamine levels, such as an aminotransferase inhibitor (e.g., aminooxyacetic acid).

[0019] That is, the present invention includes the following.

[0020] 1. A method for purifying arginase, comprising obtaining cells that express arginase, disrupting the cells to produce a lysate containing arginase, raising the temperature of the lysate to a precipitation temperature in the presence of CoCl for a time suitable to precipitate a first contaminant from the lysate and produce a supernatant containing a first partially purified arginase, and contacting the supernatant with an anion exchanger to produce a first bound fraction and a first flow-through fraction, wherein the flow-through fraction is enriched in Mn 2+ Co 2+Partially purified Co substituted with 2+ arginase, Preferably, the method further comprises contacting the first flow-through fraction with a cation exchanger to produce a second bound fraction and a second flow-through fraction; and applying an elution buffer to the cation exchanger to produce an eluted fraction; The eluted fraction contains Mn 2+ Co 2+ Purified Co substituted with 2+ Contains arginase, the cells express human arginase 1. the cell is a bacterial cell; the precipitation temperature is greater than 50°C or 65°C; The CoCl2 is provided at a concentration of at least 20 mM. The time is between 5 minutes and 30 minutes, or about 15 minutes. The anion exchanger is a strong anion exchanger. the cation exchanger is a strong cation exchanger; The partially purified Co 2+ The arginase has a purity of at least 80% or 90%.

[0021] The present invention further provides a method for selectively derivatizing a protein, comprising the steps of obtaining a protein of interest comprising at least one cysteine; contacting the protein of interest with PEG-maleimide in a buffer solution having a pH between 6.5 and 7.0 at a temperature between 2°C and 15°C; and incubating the protein of interest with the PEG-maleimide at 2°C to 15°C for 24 to 72 hours to produce a PEG-derivatized protein, wherein the PEG-maleimide is present in less than a 6-fold molar excess compared to the protein of interest; Preferably, the PEG-maleimide comprises branched PEG or linear PEG. The target protein is human arginase 1 or a variant thereof. the variant comprises the removal of all but one cysteine, the target protein or the variant thereof does not contain a polyhistidine sequence; further comprising the additional step of separating the PEG-derivatized protein from unreacted or hydrolyzed PEG-maleimide. the additional step of separation is carried out by one of the group consisting of dialysis, size exclusion chromatography, and ion exchange chromatography; The PEG-maleimide is present in less than a four-fold molar excess compared to the protein of interest.

[0022] The present invention also provides a preparation of PEG-modified human Arginase 1, comprising a peptide sequence corresponding to SEQ ID NO: 1 covalently attached to a single polyethylene glycol moiety having a molecular weight of at least 20 kDa, wherein the PEG-modified human Arginase 1 represents at least 90% of the human arginase in the preparation, and the PEG-modified human Arginase 1 does not contain a polyhistidine sequence; Preferably, the polyethylene glycol moiety is linear or branched. the polyethylene glycol moiety is branched; the polyethylene glycol moiety comprises a "Y" branched structure or a "V" branched structure The PEG-modified human Arginase 1 comprises a metal cofactor selected from the group consisting of manganese, nickel, and cobalt.

[0023] The present invention also provides a method for inhibiting cancer cells, comprising the steps of: reducing the arginine concentration in a medium contacting the cancer cells; and reducing the asparagine concentration in the medium; Preferably, arginase is used to reduce the arginine concentration. The arginase is recombinant human arginase. reducing the asparagine concentration using asparaginase; the cancer cells have low asparaginase expression. Further comprising the step of reducing glutamine concentration. reducing glutamine levels with aminotransferase inhibitors; The aminotransferase inhibitor is aminooxyacetic acid.

[0024] The present invention also provides a composition for inhibiting cancer cells, comprising an arginine reductase and an asparagine reductase, Preferably, the arginine reductase is arginase. The arginase is recombinant human arginase. the asparagine-reducing enzyme is asparaginase; the cancer cells have low asparaginase expression. further comprising a compound that reduces glutamine levels, the compound that reduces glutamine levels is an aminotransferase inhibitor; The aminotransferase inhibitor is aminooxyacetic acid.

[0025] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements. [Brief explanation of the drawings]

[0026] [Figure 1] 1 is a flowchart of an exemplary process of the inventive concept. [Figure 2] Electrophoresis gel photograph showing results obtained from reducing SDS-PAGE on an 8-16% gradient gel for homogenates and products of thermal precipitation. [Figure 3] Graph of UV absorbance during chromatography on Capto Q™. [Figure 4] Electrophoresis gel photograph showing results obtained from reducing SDS-PAGE on an 8-16% gradient gel for a typical Capto Q™ chromatography step. [Figure 5]Graph of UV absorbance during chromatography on Capto S™. [Figure 6] Electrophoresis gel photograph showing results obtained from reducing SDS-PAGE on an 8-16% gradient gel for a typical Capto Q™ chromatography step. [Figure 7] Graph of PEGylation kinetics for human kinase 1 at different molar excesses of PEG-maleimide at 2-8°C. [Figure 8] Graph of plasma arginine concentrations in healthy rats after a single intravenous administration of PEG-modified human arginase 1 on Day 0. [Figure 9] Graph of plasma arginine concentrations in healthy rats treated with a single intravenous dose of PEG-modified human arginase 1 on Day 0. [Figure 10] Graph of body weight of healthy rats treated with a single intravenous administration of PEG-modified human Arginase 1 on Day 0. [Figure 11] Graph of plasma arginine concentrations in healthy rats treated with a single intravenous dose of linear and branched PEG-modified human arginase 1 on Day 0. [Figure 12] Graph of body weight of healthy rats treated with a single intravenous dose of linear or branched PEG-modified human Arginase 1 on Day 0. [Figure 13] Molecular mass determination by LC / Q-TOF MS. The analyzed mass of PEGylated arginase is 34,572.3 Da. [Figure 14] Peptide map of mutant human arginase 1. [Figure 15] Graph of arginase enzyme activity in rat plasma over time after a single intravenous dose of 2 mg / kg. [Figure 16] Graph of plasma concentrations of immunoactive PEG-modified human arginase 1 in rat plasma over time after a single intravenous dose of 2 mg / kg. [Figure 17]Graph showing the efficacy of ASNase alone in (i) low-ASNS-expressing cell lines (MDA-MB-231, ZR-75-1, MCF7) and (ii) high-ASNS-expressing cell lines (HeLa, HepG2, and MIA-Paca2). Three independent experiments were performed for each experiment. Error bars represent one standard deviation (SD). [Figure 18] Graphs showing the efficacy of rhArg alone and rhArg-ASNase combination in (i) low-ASNS-expressing cell lines (MDA-MB-231, ZR-75-1, MCF7) and (ii) high-ASNS-expressing cell lines (HeLa, HepG2, and MIA-Paca2). Three independent experiments were performed per experiment. Error bars represent one standard deviation (SD). DETAILED DESCRIPTION OF THE INVENTION

[0027] The following description contains information that may be useful in understanding the present invention. No admission is made that any of the information provided herein is prior art or relevant to the claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0028] The present invention provides devices, compositions, and methods for scalable purification of human arginase. In the compositions and methods of the present invention, a preparation containing recombinant human arginase is incubated at high temperatures, thereby forming a precipitate. The precipitate is removed, and the supernatant is collected and subjected to ion exchange on an anion exchanger. The flow-through fraction from this anion exchange treatment is collected and subjected to a further polishing step on a cation exchanger, where the human arginase is eluted using a salt gradient. The resulting human arginase can be modified, for example, by PEGylation. Such PEGylation can be performed at low temperatures using a relatively small molar excess of a reactive PEG analog to produce highly pure PEGylated human arginase. The present invention also provides devices, systems, and methods for using arginine- and asparagine-reducing compounds to inhibit cancer cell growth. Such compounds can be enzymes, such as arginase and / or asparaginase, that catalytically reduce the concentrations of arginine and asparagine. In a preferred embodiment, such enzymes are human enzymes, such as recombinant human arginase (rhArg).

[0029] Such amino acid-depleted enzymes may be provided as purified enzymes from natural sources or as the product of recombinant bacteria, fungi, plant cells, or animal cells. Enzymes utilized in such treatments can have a purity of greater than 80%, 85%, 90%, 95%, 98%, 99% or more, and may be modified after conversion. Such modifications may include modifications that improve absorption and / or half-life (e.g., PEGylation). The enzyme-containing pharmaceutical formulation may be administered intravenously, for example, by injection or infusion. Such preparations may include or be co-administered with chemotherapeutic agents, immunotherapeutic agents, and / or radiation therapy utilized for cancer treatment.

[0030] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, taken in conjunction with the accompanying drawings in which like numerals represent like elements.

[0031] It should be appreciated that the disclosed approach provides many advantageous technical effects, including the scalable production of highly purified human arginase having a native sequence from recombinant sources.

[0032] In some embodiments, numbers expressing properties such as amounts and concentrations of ingredients, reaction conditions, and the like, used to describe and claim certain embodiments of the present invention are understood to be modified in some instances by the term "about." Accordingly, in some embodiments, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the reported significant digits and by applying ordinary rounding techniques. Nevertheless, the numerical ranges and parameters setting forth the broad scope of some embodiments of the present invention are approximations, and the numerical values ​​set forth in the specific examples are reported to the extent practicable. The numerical values ​​set forth in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0033] As used herein and throughout the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used herein, the meaning of "in" includes "in" and "on," unless the context clearly dictates otherwise.

[0034] Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") provided with respect to certain embodiments herein is intended merely to better clarify the invention and does not otherwise limit the scope of the claimed invention. No language herein should be construed as indicating any non-claimed element essential to the practice of the invention.

[0035] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limiting. Members of each group may be referenced and claimed individually or in combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When such inclusions or deletions are made, the specification is considered herein to include groups modified in accordance with the specification of all Markush groups used in the appended claims.

[0036] The following discussion provides many exemplary embodiments of the inventive subject matter. Although each embodiment represents a single combination of the inventive elements, it is contemplated that the inventive subject matter includes all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, a second embodiment includes elements B and D, and it is contemplated that the inventive subject matter also includes any other remaining combination of A, B, C, or D, even if not explicitly disclosed.

[0037] In embodiments of the inventive concept, one of the amino acid-depleting enzymes is an arginase, such as a mammalian, avian, reptilian, plant, fungal, and / or bacterial arginase. In some such embodiments, the arginase can be a human arginase provided as a product of genetic engineering of, for example, bacterial, yeast, fungal, insect, plant, or mammalian cells in culture. Such arginase can include one or more sequence modifications that serve to improve the specificity of subsequent coupling reactions, e.g., removal or substitution of one or more amino acids containing potentially modifiable side chains. In a preferred embodiment, recombinant human arginase can be produced in an E. coli clone, e.g., strain BL21 (T7 Express, New England Biolabs), containing the kanamycin-resistant expression vector pET-30a into which a cDNA encoding human arginase 1 has been inserted. Cultivation of such transformed E. coli can be carried out in an appropriate medium at any suitable scale, e.g., 0.1 L, 1 L, 5 L, 10 L, or more. The optical density of such a bacterial culture can be monitored to determine when it has reached an optimal density for recovery of recombinant human Arginase 1. Alternatively, cultivation can be continued for a predetermined period of time before harvesting and further processing the bacteria.

[0038] Figure 1 provides a flow chart outlining an exemplary process of the inventive concept. As shown, bacteria or other cells expressing human Arginase 1 are harvested, disrupted, and extracted to liberate the desired enzyme. Bacteria may be harvested by any suitable means, including filtration, sedimentation, and / or centrifugation. If desired, bacteria harvested in this manner may be rinsed or washed prior to further processing.

[0039] The disruption of the bacteria or other cells can be carried out by any suitable process, including, but not limited to, enzymatic digestion, osmotic shock, and sudden pressure changes (e.g., by compressive squeezing and / or sonication). In some embodiments, these processes can be carried out under temperature-controlled conditions. For example, the temperature of the bacteria or other cells undergoing sonication can be controlled to ensure that it does not exceed a temperature compatible with the subsequent activity of human arginase. In other embodiments, one or more protease inhibitors can be added before, during, or after the disruption of the bacteria or other cells. In yet other embodiments, one or more stabilizers (e.g., antioxidants) can be added before, during, or after the disruption of the bacteria or other cells.

[0040] After disruption of the bacteria or other cells, residual debris can be removed (e.g., by sedimentation, filtration, and / or centrifugation) and the solution containing the human arginase can be left standing. The inventors have found that arginase is surprisingly stable at elevated temperatures (i.e., above 37°C, 40°C, 45°C, 50°C, 60°C, and / or 70°C) that can result in the denaturation and subsequent precipitation of undesired contaminating proteins. For example, the inventors have found that human arginase I is stable at 74°C, a temperature that results in the precipitation of many contaminating proteins. Without being limited by theory, it is believed that this stability is provided by complexation with divalent ions. This allows the use of Mn to generate a precipitate (containing the contaminating proteins) and a supernatant (containing the human arginase). 2+ or Co 2+ In the presence of divalent ions such as cobalt, it becomes possible to extract arginase at high temperatures (i.e., temperatures above 37°C). Furthermore, cobalt-chelated arginase exhibits greatly enhanced catalytic activity (k cat / K M ), which not only provides a high catalytic potential but also enhances the Mn 2+ Co 2+CoCl can also be utilized during extraction to replace the arginase. The temperature and incubation time can be selected to provide adequate to optimal recovery, purity, and activity of the human arginase.

[0041] Incubation temperatures can range from about 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C or greater than about 90°C. Incubation times can range from 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, or more than 12 hours. In some embodiments, the temperature can be changed during the incubation period. In a preferred embodiment, the incubation temperature is 65°C and the incubation period is 15 minutes. The inventors have found that such methods can simultaneously induce Mn in the arginase. 2+ Co 2+ It has been found that the majority of undesired heat-sensitive proteins can be advantageously removed while replacing the hydroxyl group with hydroxyl group.

[0042] After this precipitation step, the supernatant containing the arginase is recovered for further processing. The supernatant can be separated from the precipitate by any suitable method, including sedimentation, filtration, and / or centrifugation. The recovered supernatant is then transferred (e.g., by dialysis, gel filtration, and / or diafiltration) to an aqueous buffer compatible with anion exchange. The composition of such a buffer depends at least in part on the nature of the anion exchange medium used, but generally can have a relatively low molarity (e.g., less than 100 mM) and a high pH (e.g., greater than 7). Such an anion exchanger can be a weak anion exchanger with a relatively low affinity for anions, such as an anion exchanger containing ammonium groups. Alternatively, such an anion exchanger can be a strong anion exchanger with a relatively high affinity for anions, such as an anion exchanger containing quaternary amine groups. In a preferred embodiment, a strong anion exchanger is used. For example, when a strong anion exchanger such as Capto Q™ is used, a suitable buffer can be 20 mM Tris at pH 8.05. The final protein concentration of the supernatant may be adjusted to provide an appropriate concentration for optimal separation of the arginase from at least a portion of any contaminating materials present.

[0043] After transfer to an appropriate anion exchange buffer, the supernatant is treated with an anion exchange medium. As described above, such an anion exchange medium can be a strong anion exchange medium, such as an anion exchange medium containing immobilized quaternary amines. The anion exchange medium can be provided in any suitable form, such as a filter, an immiscible liquid, porous particles, and / or non-porous particles. An example of such a strong anion exchange medium is Capto Q™ medium (GE Healthcare), which is provided as porous particles containing pendant quaternary amine groups. Such an anion exchange medium can be applied as a bulk solid phase mixed and / or suspended in the supernatant and then removed (e.g., by sedimentation, centrifugation, and / or filtration). In a preferred embodiment, the anion exchange medium is provided as a chromatographic bed in a chromatography column.

[0044] The supernatant can be applied to a bed of an anion exchange medium, allowing a flow-through fraction containing the partially purified arginase to pass through while at least some contaminants bind to the medium. The volume and configuration of such a column, along with the flow rate during application, can be optimized to provide an appropriate one for optimal capture of contaminants from the supernatant. After passage and recovery of the partially purified human arginase, the anion exchange medium can be rinsed (e.g., with a buffer containing a high salt concentration) and regenerated for reuse. In such embodiments, the anion exchange medium can be selected to withstand sterilization and / or pyrogen reduction procedures.

[0045] The recovered, partially purified arginase can be used as is (depending on the application) or can be subjected to a further polishing step. Presumably, pharmaceutical uses will require such further processing. If a polishing step is desired, the partially purified arginase can be transferred to an aqueous buffer suitable for cation exchange chromatography. This transfer can be achieved by any suitable means, including dialysis, gel filtration, and / or diafiltration. In some embodiments, this transfer can be effectively achieved by diluting the partially purified human arginase in a dilution buffer that provides an appropriate cation exchange buffer composition. In yet other embodiments, the buffer used in the previous anion exchange step can be adjusted (e.g., by manipulating pH and / or ionic strength) to make it suitable for cation exchange.

[0046] The partially purified arginase can be applied to a cation exchange medium in a suitable cation exchange buffer. Suitable buffers typically have low to moderate ionic strength (e.g., less than 200 mM) and a neutral pH (e.g., pH 7). An example of a suitable cation exchange buffer is 50 mM Tris (pH 7). Such a cation exchanger can be a weak cation exchanger with a relatively low affinity for cations, such as a cation exchanger containing carboxyl groups. Alternatively, such a cation exchanger can be a strong cation exchanger with a relatively high affinity for cations, such as a cation exchanger containing sulfonic acid groups. In a preferred embodiment, the cation exchange medium can be a strong cation exchange medium, such as Capto S™ (GE Healthcare), containing pendant sulfonic acid groups. Because arginase from the partially purified arginase preparation binds to and selectively elutes from the cation exchange medium, the cation exchange medium is preferably provided as a solid (e.g., particle) that can be fixed in place to support gradient elution. In a preferred embodiment, the cation exchange medium is provided as a chromatographic bed in a chromatography column. The volume and configuration of such a column, along with the flow rate during application, can be optimized to provide optimal capture and subsequent release of human arginase from the cation exchange medium.

[0047] When using such a cation exchange column, the partially purified arginase transported in a cation exchange buffer is applied to the column at a flow rate that allows capture of the arginase on the cation exchange medium. After application of the partially purified arginase, the cation exchange column may be rinsed with an additional volume of cation exchange buffer (e.g., 1-10 column volumes) to remove unbound material. Optionally, UV absorbance can be monitored during this process to determine when the wash is complete. In some embodiments, an additional wash step can be used in which a more stringent cation exchange buffer (e.g., a wash buffer having a higher pH and / or ionic strength than the cation exchange buffer) is applied to the cation exchange column to displace loosely bound material. An example of such a wash buffer is 50 mM Tris + sodium chloride (NaCl) at less than 0.5 M (pH 7). In some embodiments, a series of such wash buffers of varying stringency can be used.

[0048] After application of the partially purified arginase and any subsequent washing steps, the purified arginase is eluted from the cation exchange medium by application of an elution buffer. This elution can also be achieved by applying a fixed composition elution buffer as a single bolus to provide a step elution. In other embodiments, the elution buffer can be applied as a mixture with the cation exchange buffer, where the ratio of elution buffer to cation exchange buffer increases over time. In such a gradient elution approach, the rate at which this ratio changes can be linear or nonlinear over time. In a preferred embodiment, elution is achieved using a linear gradient that transitions the column buffer composition from cation exchange buffer to elution buffer at a constant rate over time. The elution buffer can differ from the cation exchange buffer in pH, ionic strength, or both. In a preferred embodiment, the elution buffer substantially replicates the composition and pH of the cation exchange buffer, but contains a higher concentration of NaCl (e.g., greater than 0.2 M). An example of such an elution buffer is 50 mM Tris + 0.5 M NaCl (pH 7).

[0049] During such gradient elution, the UV absorbance of the material exiting the column can be monitored to determine which fractions are collected. Fractions can be selected based on arginase content and / or the presence of contaminants to provide the desired yield and purity. Such fractions can be collected, pooled, and transferred to an appropriate buffer for stability. In some embodiments, such purified arginase can be subsequently frozen and / or lyophilized. In yet other embodiments, the purified arginase thus obtained can be derivatized, e.g., by PEGylation, for pharmaceutical use. Typical results of the methods and compositions of the present concepts can provide highly pure (>90%) human arginase in approximately 30% yield. After passage and collection of the purified human arginase, the cation exchange medium can be rinsed (e.g., with a buffer containing a high salt concentration) and regenerated for reuse. In such embodiments, the cation exchange medium can be selected to withstand sterilization and / or pyrogen reduction procedures.

[0050] In some embodiments, the arginase purified as described above can then be chemically modified. Suitable chemical modifications include biotinylation, charge modification, crosslinking, and conjugation (e.g., grafting of a hydrophilic polymer (e.g., dextran), PEG, etc.). For example, the purified arginase can be grafted to a polymer bearing an amine-reactive group and / or a thiol-reactive group using a reactive form of the polymer by contacting the purified arginase with the polymer. Suitable reactive groups include N-hydroxysuccinimide (NHS) ester, sulfo-NHS ester, epoxide, halogenated triazine, aldehyde, hydrazine, iodoacetamide, maleimide, and other crosslinking groups known in the art. In some embodiments, the human arginase used in such treatment can be genetically modified, for example, to limit or reduce the number and / or locations of modifiable amino acid side chains present on the human arginase. For example, the number of reactive amines can be reduced by substituting one or more lysines in the sequence of the human arginase with another amino acid. Similarly, the number of reactive thiols can be reduced by substituting one or more cysteines in the sequence of human arginase with another amino acid.

[0051] It should be understood that the addition of polyhistidine sequences (e.g., at the amino and / or carboxyl termini), which are commonly used to generate fusion proteins with nickel affinity, is not required in the compositions or methods of the present invention. An example of a suitable recombinant arginase is provided in SEQ ID NO: 1. The inclusion of such polyhistidine sequences may render the human arginase antigenic and therefore unsuitable for repeated use as a therapeutic agent. Furthermore, the inclusion of polyhistidine sequences would interfere with the isolation of the arginase during initial purification and following the above-described modification reaction using cation exchange.

[0052] PEGylation is often used to extend the serum half-life of therapeutically valuable proteins. Such treatments are generally performed using a large molar excess (e.g., a 10-fold or greater molar excess) of a reactive form of PEG to ensure PEGylation of a significant proportion of the target protein. This is particularly true for proteins, such as human arginase 1, that present stereochemical problems that reduce conjugation efficiency. Typical reactive forms of PEG include PEG-NHS or PEG-sulfoNHS (used when amine conjugation is desired) or PEG-maleimide (used when thiol conjugation is desired). The use of a large molar excess of such activated PEG is undesirable for a number of reasons, including the potential for low selectivity in conjugation at large molar excesses, the subsequent difficulty of separating the excess unreacted PEG from the reacted protein, and expense.

[0053] Surprisingly, the inventors have found that human arginase (such as human arginase 1 purified as described above) can be efficiently PEGylated using a very low (e.g., 3-4x molar excess relative to protein content) molar excess of PEG maleimide. Even more surprisingly, this PEGylation can be carried out with high efficiency under selected conditions (e.g., mildly acidic pH, which blocks potentially reactive amines by protonation), at low temperatures (both of which reduce reactivity), and for extended reaction times (i.e., greater than 1 hour and / or up to 72 hours). The inventors have found that such a method allows for the PEGylation of substantially all (i.e., greater than 90%) of the provided human Arginase 1, and separation from the relatively small amount of remaining unreacted or hydrolyzed PEG can be easily achieved by gel filtration and / or ion exchange. In some embodiments, the human arginase modified in this manner can be genetically modified to provide a reduced amount of potentially reactive cysteines compared to the native sequence. In a preferred embodiment, the human arginase can be a modified human Arginase 1 that provides a single cysteine ​​and does not contain a polyhistidine sequence. In some embodiments, the human arginase can include a non-natural (i.e., non-manganese) metal cofactor, such as cobalt or nickel. [Example]

[0054] Cell disruption, extraction and clarification An ampicillin-resistant E. coli clone (strain: BL21 Star™, Invitrogen) containing the transient and constitutive expression vector pET-3a was transformed with cDNA encoding human arginase-1 and established in culture. The cell paste produced from such a culture, with a wet weight of 42.2 g, was washed with 230 mL of lysis buffer (50 mM Tris pH 7.9, 1 mM MgSO4) and then centrifuged to recover the cell paste. After resuspension of the cell paste with 210 mL of lysis buffer, 35 mL was aspirated for further processing.

[0055] Human arginase was released from 35 mL of the cell resuspension by sonication using a Q700 Sonicator (Qsonica) and the following duty cycle: (10 seconds on, 30 seconds off, 3.5 minutes on) to produce a homogenate. After centrifugation (10,000 rpm, 15 minutes), the resulting supernatant was collected and subjected to thermal precipitation in the presence of CoCl2 in a water bath (65°C for 15 minutes). The precipitate was removed by centrifugation, and the supernatant was subjected to further purification steps. Figure 2 shows the results of SDS-PAGE for the homogenate and thermal precipitation in the presence of various amounts of CoCl2, indicating that optimal recovery of arginase was observed at a temperature of 65°C for approximately 15 minutes when at least 20 mM CoCl2 was present. This step of treatment not only removed heat-sensitive impurities but also replaced chelated ions with cobalt.

[0056] Partial purification by anion exchange chromatography The supernatant produced by thermal precipitation (26.5 mL) was filtered through a 0.45 μm filter (Agilent, Captiva™ Econofilter PES membrane, 25 mm, 0.45 μm) and diluted with 100 mL of MilliQ™ water and 110 mL of 20 mM Tris buffer (pH 8.1) to produce the loading sample (pH 7.95, conductivity: 1.213 mS / cm).

[0057] The purification was carried out by FPLC in an AKTA™ prime plus (GE Healthcare Life Sciences) equipped with a chromatography column packed with Capto Q™ (1 x 6.8 cm, GE Healthcare Life Sciences) previously equilibrated with 20 mM Tris buffer (pH 8.1). The flow-through was collected for further purification at a flow rate of 3.5 mL / min. UV absorbance was monitored during this process. The results are shown in Figure 3. SDS-PAGE analysis of the fractions collected during Capto Q™ chromatography is shown in Figure 4. It is clear that human Arginase 1 remains in the flow-through fractions, along with some minor contaminants.

[0058] Further purification by cation exchange chromatography Fifteen milliliters of 50 mM MES (MES monohydrate) buffer (pH 6.7) was added to the flow-through fraction from the Capto Q™ column, and the pH was adjusted to 6.2 by adding 6N hydrochloric acid before loading onto a Capto S™ column (1 x 10 cm, GE Healthcare Life Sciences) at a flow rate of 3.9 mL / min. The Capto S™ column was previously equilibrated with 50 mM MES buffer (pH 6.7). Human Arginase 1 was eluted with a linear gradient of 0 to 0.5 M NaCl in the equilibration buffer. UV absorbance was monitored. Typical results are shown in Figure 5. SDS-PAGE analysis of fractions collected during Capto S™ chromatography is shown in Figure 6. It is clear that human Arginase 1 elutes in high purity using a simple NaCl gradient, with only very small amounts of contaminants evident, as evidenced by overloading the SDS-PAGE gel.

[0059] The apparent absence of contaminants, even at protein concentrations that overload the SDS-PAGE gel, indicates a purity of at least 90%, 95%, 98%, 99%, or more for this preparation of human Arginase 1. It should be understood that this purity was achieved without the need for a polyhistidine or other affinity "tag" sequence and represents purification of the native protein sequence. The inventors contemplate that the methods and compositions of the present inventive concepts can be utilized to purify sequence-modified and / or derivatized (e.g., PEGylated) human Arginase, as well as to separate derivatized from unreacted human Arginase in the reaction product of the derivatization reaction.

[0060] Estimation of treatment yield and expression level Table 1 provides estimates of process yields at various points in the purification process. Protein concentrations were measured by densitometry using Image Studio Lite™ Version 5.2 (LI-COR Biosciences), except for the Capto S pool, which was measured by UV absorbance at 280 nm using a UV spectrophotometer (Multiskan™ GO, Thermo Scientific™). The extinction coefficient at 280 nm is 0.703 for 1 mg / mL.

[0061] [Table 1]

[0062] The overall yield in this example was estimated at 28.5%.

[0063] Preparation of conjugated human arginase 1 with various PEG moieties Sequence-modified cobalt-chelating human arginase-1 (SEQ ID NO: 1) with catalytic activity of 300-450 U / mg and purified as described above was obtained for subsequent PEGylation with different maleimide-derivatized PEGs. For selective conjugation, four PEG-maleimides were used: 20L (20-KD linear PEG-maleimide (purchased from Jenkem Technology, USA, Cat. #M-MAL-20K)), 20V Sinopeg #06020101912 (20-KD "V"-configuration PEG-maleimide (purchased from NOF Corp., Cat. #GL2-200MA)), 20Y (20-KD "Y"-configuration PEG-maleimide (purchased from Sinopeg, #06020501954)), and 40Y (40-KD "Y"-configuration PEG-maleimide (purchased from Jenkem Technology, USA, Cat. #Y-MAL-40K)). For conjugation, 5 mg / mL of the above human arginase 1 was reacted with different molar ratios of each of the PEG reagents at pH 6.7 and 4–10 °C. The reaction mixture was incubated at this low temperature for at least 48 hours. The monoPEGylated product was then separated by size exclusion chromatography or cation exchange chromatography (which may be more suitable for large-scale preparations). For cation exchange chromatography, the PEGylated product was purified using a MacroCap™ SP column with 20 mM MES pH 6.3 buffer containing 0.1 M NaCl as the elution buffer.

[0064] Fractions containing monoPEGylated product, as determined by SDS-PAGE, were pooled, concentrated, and dialyzed against PBS pH 7.4 buffer. The final protein concentration was determined directly by ultraviolet spectroscopy at 280 nm using an extinction coefficient of 0.703 for a 0.1% (1 mg / mL) solution.

[0065] Maleimide-derivatized PEG is effective for protein conjugation of sulfhydryl groups to form stable thioether bonds at pH 6.5-7.5. Reactivity toward primary amines may occur at pH above 7.5, and the stability of the maleimide group decreases as the pH increases. Human arginase is known to be relatively resistant to conjugation with such derivatized hydrophilic polymers. Human arginase is a homotrimer. The inventors (without wishing to be bound by theory) believe that even if the sulfhydryl conjugation reaction is selective (e.g., due to steric hindrance), conjugation of the first PEG molecule may prevent conjugation of the second on the homotrimer. As a result, a large molar excess of PEG reagent is typically used to ensure complete reaction. For example, under conventional reaction conditions, addition of a 6-fold molar excess of PEG yielded 71.1% monoPEGylated product after 35 hours of incubation at room temperature, and addition of an 8-fold molar excess yielded 86.3% monoPEGylated product after 46 hours of incubation at room temperature.

[0066] Surprisingly, the inventors have found that even when stringent conjugation conditions (such as a weakly acidic pH) are used, high conjugation yields can be achieved using minimal PEG reagents if the conjugation reaction occurs at low temperatures (e.g., below 10°C). Without being bound by theory, the inventors believe that low temperatures extend the half-life of the maleimide linking group, thereby increasing the probability of secondary and tertiary conjugation. The pH for the reaction can be about pH 6.7 to favor selective conjugation of sulfhydryl groups.

[0067] PEGylation kinetics was investigated using different molar ratios (e.g., 2x, 4x, 6x) of the PEG-maleimide reagent. After addition of PEG-maleimide and incubation at approximately 4°C, the crude reaction mixture was diluted with 4 volumes of 20 mM MES pH 6.3 buffer before purification on a cation exchanger (MacroCap™ SP). After loading the diluted reaction mixture onto the column, excess PEG was removed by washing with 20 mM MES pH 6.3 buffer. MonoPEGylated arginase was then eluted with the same buffer containing 0.1 M NaCl. After extensive dialysis and concentration against PBS buffer, the final product had a specific activity of 425 U / mg.

[0068] The PEGylation yield (monoPEGylated product) over time at low temperature was determined by reversed-phase ultra-performance liquid chromatography (RP-UPLC). The results, shown in Figure 7, indicate that a 4-fold molar excess of PEG-maleimide over human kinase 1 is sufficient to reach a 95% yield in 48 hours. We believe that a 3-fold molar excess is sufficient to reach similar yields with extended incubation (e.g., 48 hours or more) at low temperature.

[0069] Pharmacodynamic study of PEGylated human arginase 1 in healthy rats Animals: Three healthy juvenile male SD rats were selected for each test group. Each group of rats received the indicated dose of human arginase conjugate via intravenous delivery. Approximately 0.8 mL of whole blood samples were collected from each animal's jugular vein at appropriate time points using a 1 mL syringe and a sample tube containing anticoagulant (heparin sodium). Plasma samples were prepared by immediate centrifugation of the blood samples at 3,000 rpm for 10 minutes. The supernatant (0.3-0.4 mL) was obtained, divided into two samples, and stored at -80°C.

[0070] Arginine Quantification: Plasma concentrations of arginine were determined by comparison with known concentrations of arginine using an Agilent 6460 Liquid Chromatography and Electrospray Ionization Triple Quadrupole MS system using the conditions shown below and in Table 2.

[0071] Column: Agilent Zorbax RRHD HILIC Plus 95Å, 2.1 x 100 mm, 1.8 μm Mobile phase A: 5mM NH4COO - / 0.1% formic acid; B: 0.1% formic acid in acetonitrile Flow rate: 0.2 mL / min; injection volume 1 μL Run time (including equilibration): 15 minutes Detection: Polarity +

[0072] [Table 2]

[0073] Reference preparation: Prepare an arginine stock solution at 30 mM. Prepare working standards of arginine at 4, 10, 25, 75, 100, 150, 200, and 250 μM in 1% BSA / PBS buffer. Prepare a stock solution of synephrine at approximately 60 μg / mL. Synephrine is used as an internal standard to normalize the recovery of the sample preparation procedure described below.

[0074] Sample preparation: 50 μL of plasma sample was added with 10 μL of synephrine stock, followed by the addition of 540 μL of MeOH to precipitate proteins in the sample. Vortex to ensure complete mixing. After centrifugation at maximum speed to spin down the precipitate, the supernatant was subjected to UPLC / MS analysis. Arginine standards (4-200 μM) were also subjected to the same sample preparation procedure as above (adding synephrine and MeOH precipitation).

[0075] Analysis and Calculation: The calculated areas of selected product ions in the samples were used to determine the concentration of arginine relative to the reference standard. The limit of quantification is approximately 5 μM.

[0076] The potential for arginine depletion was determined in healthy rats via a single intravenous (IV) delivery. Three conjugates, A20CL (arginine conjugated to a 20 kD linear PEG), A20CV (arginine conjugated to a 20 kD "V" branched PEG), and A40CY (arginine conjugated to a 40 kD "Y" branched PEG), were administered at 2.4 mg / kg, 3.3 mg / kg, and 2.8 mg / kg, respectively, due to miscalculation. Post-treatment plasma arginine is shown in Figure 8, demonstrating successful 95% arginine depletion (compared to pre-treatment levels) in healthy male rats for approximately 5 days.

[0077] A dose-finding study was conducted using rats receiving A20CL at 1.2 mg / kg and 0.4 mg / kg via intravenous delivery, followed by monitoring of plasma arginine concentrations. The results are shown in Figure 9. Treatment with 1.2 mg / mL A20CL resulted in low plasma arginine levels (<10 μM) for approximately 5 days. As shown in Figure 10, there was no significant weight loss during treatment, suggesting that PEG-modified arginase was well tolerated.

[0078] Linear and branched PEGs were compared head-to-head for their potential in modifying human arginase 1 for arginine depletion. Three healthy male rats were administered A20CL and A20CY at 2 mg / kg, respectively, on Day 0. The relative plasma arginine concentration results are shown in Figure 11. As shown, both the linear and Y-form conjugates successfully deplete 90% of plasma arginine for at least 5 consecutive days.

[0079] As shown in Figure 12, no significant weight loss was observed during treatment with linear or branched PEG-modified arginase, suggesting that both linear and branched PEG-modified arginase were well tolerated.

[0080] Molecular mass of human arginase 1 The molecular mass of the mutant human arginase 1 was analyzed by reversed-phase (RP) chromatography using a UPLC system coupled with an Agilent 6540 UHD Accurate Mass Q-TOF LC / MS system.

[0081] The mass spectrum m / z of the purified arginase is shown in Figure 13 and shows a deconvoluted mass of 34,572.3 Da, which is in good agreement with the average mass of 34,571.6 Da derived from the amino acid sequence of the mutant human kinase (i.e., without the N-terminal Met).

[0082] Peptide mapping and conjugation site determination Proteolytic solutions were prepared in 50 mM Tris pH 8 buffer containing 2 mg / mL protein and 2% (w / w) sequencing-grade Lys-C in the presence of 4 M urea. After 6 h of incubation at room temperature, TFA or formic acid was added to a final concentration of 0.1% to quench the reaction. Prior to injection, precipitates were removed by centrifugation or filtration through 0.2 μm or 0.4 μm membranes.

[0083] Peptide identification was performed using an LC / MS system, specifically an Agilent 1290 infinity UPLC system coupled with an Agilent 6540 UHD Accurate Mass Q-TOF LC / MS system.

[0084] Chromatographic procedures were performed as follows and using the conditions in Table 3. Column: Same as that used for molecular mass determination (see above) Mobile phase and gradient: Mobile phase A: 0.1% (v / v) TFA in water Mobile phase B: 0.1% (v / v) TFA in 100% (v / v) acetonitrile Flow rate: 0.4mL / min Injection: 10μg

[0085] [Table 3]

[0086] The PEGylation sites of PEGylated human kinase 1 were identified by peptide mapping coupled with LC-MS. The protein was digested with endoproteinase Lys-C, which selectively hydrolyzes peptide bonds on the C-terminal side of lysine residues in the presence of 4 M urea. All proteolytic peptides were successfully assigned as shown in Figure 14, with the lysyl peptides summarized in Table 4. The peptide map of the PEGylated human arginase 1 shows conjugation at the K6 peptide, where Cys-44 is the only site susceptible to sulfhydryl modification.

[0087] [Table 4]

[0088] Enzyme Activity and Kinetics Catalytic activity was determined by detecting the diacetylmonoxine (DAMO) derivatization of urea in the presence of strong acid, thiosemicarbazide, and Fe3+ by heating to generate a chromophore with a maximum absorbance of 540 nm. The assay was shown to be linear between 0 and 15 mM urea, with a detection limit of 1.25 mM. Typically, the reaction was initiated by warming 30 μL of plasma sample and positive control on a heat block pre-set to 37°C for 3 minutes. After adding 30 μL of arginine substrate, the mixture was incubated at 37°C for exactly 5 minutes and then quenched by the addition of 30 μL of 25% trichloroacetic acid (TCA). After centrifugation, 10 μL of working urea standard, sample / positive control, or blank control was mixed with 300 μL of color reagent (containing DAMO and thiosemicarbazide), and the mixture was incubated on a heat block pre-set to 100°C for exactly 10 minutes. The absorbance of the resulting sample was measured at 540 nm to determine the urea concentration. A unit of arginase activity is defined as 1 μmol of urea produced per minute.

[0089] Arginase activity in plasma over time is shown in Figure 15. The calculated half-lives (tl / 2) of A20CL and A20CY were 17.0 and 17.6 hours, respectively. No activity was detected in plasma samples collected after Day 5.

[0090] Enzyme-linked immunosorbent assay (ELISA) A sandwich ELISA was used to determine plasma concentrations of PEGylated human Arginase 1. The assay utilized a rabbit polyclonal antibody specific for human Arginase 1 (Sino Biologicals #11558-RP01) as the capture antibody and a sheep polyclonal anti-Arginase antibody (R&D systems #AF5868) with an HRP-conjugated anti-goat IgG antibody (R&D systems #HAF017) as the detection antibody.

[0091] The results of an ELISA test of the plasma concentration of immunoreactive PEG-modified human Arginase 1 after a single IV dose of 2 mg / kg of PEG-modified human Arginase 1 are shown in Figure 16. Plasma concentrations of A20CL (conjugated with 20L PEG) and A20CY (conjugated with 20Y PEG) were 1013 ± 119 ng / mL and 2239 ± 257 ng / mL, respectively, 24 hours after administration. The concentrations of all samples collected pre-dose and 120 hours after administration were below the detection limit (160 ng / mL). Therefore, half-lives were calculated at 22.6 and 20.3 hours for A20CL and A20CY, respectively, which are consistent with the half-lives for the enzyme activities shown.

[0092] To enhance the inhibitory effect of sequence-modified recombinant human arginase (rhArg), the present inventors simultaneously depleted arginine and asparagine from cancer cells using rhArg and asparaginase (ASNase).The present inventors believe that, due to the role of asparagine as an amino acid exchange factor, asparagine depletion may have at least a complementary effect to that of arginine depletion.Asparagine may regulate arginine import into cells, and therefore may affect arginine-mediated mTORC1 activation.The present inventors hypothesize that asparagine depletion may reduce arginine transport toward mTORC1 in cells.

[0093] The effects of asparagine depletion in cancer cells may depend on the amount of the asparagine-producing enzyme asparagine synthetase (ASNS) present in the cells (Richards and Kilberg 2006; Balasubramanian, Butterworth et al. 2013; Liu, Dong et al. 2013). To assess these effects, we used rhArg and ASNase to treat (i) breast cell lines MDA-MB-231, MCF7, and ZR-75-1, which express relatively low levels of ASNS (Yang, He et al. 2014), and (ii) high-ASNS-expressing cell lines HeLa (Guerrini, Gong et al. 1993), MIA-Paca-2 (Liu, Dong et al. 2013), and HepG2 (Gjymishka, Su et al. 2009). The efficacy of each drug individually (rhArg, ASNase) and in combination was compared.

[0094] example Exemplary procedures for the expression, purification, and PEGylation of recombinant human arginase (rhArg) are described in detail above. One unit of arginase is defined as the amount of enzyme that produces 1 μmol urea per minute at 30° C. and pH 8.5. Asparaginase (ASNase) was purchased from Sigma (A3809).

[0095] MTT assay: Low-ASNS-expressing breast cancer cell lines (MCF-7, ZR-75-1, and MDA-MB-231) and high-ASNS-expressing cancer cell lines (HeLa, HepG2, and MIA-Paca-2) were purchased from ATCC. 5 × 10 cells were cultured. 3Cells were seeded into 96-well plates at a density of 1000 cells / well. After one day of incubation, the culture medium was replaced with medium containing various concentrations of rhArg. For binding assays (i.e., using arginase and asparaginase), the culture medium was replaced with medium containing different concentrations of rhArg and ASNase at a fixed molar ratio (e.g., 5 (rhArg):1 (ASNase)) (Chou 2010). The MTT assay was performed on Day 3 (3 days after drug treatment). The culture medium was replaced with MTT solution (1 mg / mL) (Invitrogen) and incubated at 37°C for 4 hours. After 4 hours of incubation, the MTT solution was replaced with dimethyl sulfoxide (DMSO), and the absorbance at 570 nm was measured with a reference value of 650 nm. Cell viability was determined by dividing the absorbance of treated cells by the average absorbance of untreated cells. Three independent sets of experiments (n = 3) were performed for each cell line. Data were analyzed using Prism™ version 6.01.

[0096] Combination Index (CI) Calculation: The combination index for rhArg and ASNase treatment was calculated using CalcuSyn™ version 2.1. The CI provides a value for the combined effect of the drugs, such as greater than additive (CI<1, synergistic), less than additive (CI>1 (antagonistic)), or equal to additive (CI=1) effect.

[0097] The present inventors found that ASNase alone did not provide a satisfactory inhibitory effect on most low-ASNS-expressing and high-ASNS-expressing cell lines. The dose-response curves of ASNase in (i) low-ASNS-expressing cancer cells and (ii) high-ASNS-expressing cancer cells are shown in Figure 17. ASNase exhibited a low inhibitory effect (less than 20% cell inhibition) in the range of 0.016 to 0.4 U / mL. Cell viability of 60 to 70% was detected in MDA-MB-231, ZR-75-1, MCF-7, HeLa, and HepG2. Approximately 40% cell viability was detected in MIA-Paca-2.

[0098] Surprisingly, the combination of rhArg and ASNase provides substantially improved inhibitory effects compared to rhArg alone in low-ASNS-expressing cell lines. As shown in Figure 18, the dose-response curves for rhArg alone (solid line) and the combination of rhArg and ASNase (dashed line) in (i) low-ASNS-expressing cancer cells and (ii) high-ASNS-expressing cancer cells are shown. In low-ASNS-expressing cell lines, the combination treatment of rhArg and ASNase provided dramatically improved cell inhibitory effects at various concentrations. The combination treatment of rhArg-ASNase demonstrated favorable enhancement of inhibitory effects in MDA-MB-231. In high-ASNS-expressing cell lines, the combination treatment, on average, only slightly improved the inhibitory effects (<5%). The inventors found that the combination treatment of rhArg-ASNase provided an unexpectedly strong synergistic effect in low-ASNS-expressing cell lines, but not in high-ASNS-expressing cell lines.

[0099] The combination index (CI) of the combinations of different concentrations of rhArg and ASNase is shown in Table 5. Surprisingly, for the low ASNS-expressing cell lines MDA-MB-231, ZR-75-1, and MCF7, all of the combinations showed a synergistic effect (CI<1) on inhibiting cancer cell proliferation. For the high ASNS-expressing cell lines HeLa, HepG2, and MIA-Paca2, most of the combinations of rhArg and ASNase showed an antagonistic effect (CI>1) on inhibiting cell proliferation.

[0100] [Table 5]

[0101] The present inventors have found that asparagine depletion alone has little inhibitory effect on cancer cells.Such asparagine depletion alone does not appear to be an effective method for treating cancer, except for acute lymphoblastic leukemia (ALL).The present inventors theorize that the effectiveness of ASNase against ALL is due to the low expression of asparagine synthetase (ASNS) in these leukemia cells (Su, Pan et al., 2008).

[0102] The inventors hypothesize that arginine depletion may result in the inactivation of mTORC1 during the combination therapy described above, while asparagine depletion may reduce the transfer of arginine to the mTORC1 protein in cells. Because high ASNS expression levels can generate asparagine from glutamine, the efficacy of the combination therapy may be affected by the ASNS expression level of the cancer cells (Horowitz and Meister 1972). HeLa, HepG2, and MIA-Paca2 cells exhibit high ASNS expression levels, but showed less improvement during combination therapy compared with cancer cells exhibiting low ASNS levels. CI calculations indicate that low ASNS-expressing cell lines, such as MDA-MB-231, ZR-75-1, and MCF7, exhibit the noted rhArg-ASNase synergistic effect at most combination concentrations. However, low synergistic effect is observed in high ASNS-expressing cell lines, such as HeLa, HepG2, and MIA-Paca2. The results were consistent with the cell viability assay. The inventors theorize that high ASNS-expressing cell lines may produce sufficient asparagine to weaken the efficacy of the combination therapy. The inventors believe that the expression level of ASNS can be used to predict the efficacy of rhArg-ASNase combination therapy and may be useful for developing tailored drug therapies.

[0103] Based on the results of combined treatment with arginase and asparaginase in various cancer cell lines, we believe that simultaneous depletion of arginine and asparagine as well as glutamine in vitro may be useful for cancer treatment. The effectiveness of glutamine depletion treatment is highly dependent on the expression level of c-MYC (Wise and Thompson 2010). We believe that combined treatment with compounds to reduce arginine (e.g., arginase), asparagine (e.g., asparaginase), and glutamine (e.g., the aminotransferase inhibitors aminooxyacetic acid (AOA), 6-diazo-5-oxo-L-norleucine, azaserine, and / or acivicin) may be highly effective in inhibiting cancer cell proliferation. Without being bound by theory, the inventors hypothesize that arginine, asparagine, and glutamine depletion may inactivate mTORC1 by arginine depletion (e.g., by rhArg), and that intracellular arginine concentrations can be maintained at low levels by asparagine and glutamine reduction (e.g., by ASNase and AOA).

[0104] Using the above method, arginase can be PEGylated with high efficiency and isolated with high purity, specific activity, and yield. Such PEGylated arginase exhibits an extended half-life in vitro, is effective in reducing arginine concentrations, and is well tolerated. Therefore, the inventors believe that the arginase isolated and conjugated as described above is particularly well suited for therapeutic use in cancer treatment, especially when such cancers exhibit low asparaginase activity. Based on the evidence of unexpected and significant synergistic effects on different cancer cell lines when used in combination with asparaginase, the inventors believe that the PEGylated arginase prepared as described above and used in combination with asparaginase can provide similar synergistic effects in cancer treatment. To extend serum half-life, the asparaginase used in such treatment methods can be PEGylated or conjugated with a similar hydrophilic polymer.

[0105] In a preferred embodiment, simultaneous administration is possible by providing arginase and asparaginase that exhibit similar pharmacokinetics. Such enzymes can be provided by any suitable method, including injection, infusion, and / or absorption across mucous membranes (e.g., by inhalation). An appropriate treatment schedule can be determined by the pharmacokinetics of the enzymes used and can be adapted to accommodate different tumor types and / or phenotypes. For example, individuals with tumors expressing relatively high levels of asparaginase can be treated more frequently and / or with higher doses of arginase and / or asparaginase compared to individuals with tumors expressing lower levels of asparaginase. Suitable treatment protocols can include administration of arginase, asparaginase, and / or a mixture of arginase and asparaginase every 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 7 days, 10 days, 14 days, 21 days, 28 days, or more than 28 days. In some embodiments, the frequency of administration can vary over the course of treatment, and can include maintenance administration that extends beyond the initial treatment period.In a preferred embodiment, arginase is provided as PEGylated arginase prepared as described above, and asparaginase can also be provided in PEGylated form.As described above, the present inventors also believe that reducing glutamine by using aminotransferase inhibitors is useful for reducing cancer cell proliferation.

[0106] Thus, arginase / asparaginase treatment protocols as described above can include the use of aminotransferase inhibitors effective to reduce blood levels of glutamine (such as aminooxyacetic acid or AOA, 6-diazo-5-oxo-L-norleucine, azaserine, and / or acivicin).

[0107] References Balasubramanian, M. N., E. A. Butterworth, et al. (2013). "Asparagine synthetase: regulation by cell stress and involvement in tumor biology." Am J Physiol Endocrinol Metab 304(8): E789-799. Bar-Peled, L., L. D. Schweitzer, et al. (2012). "Ragulator is a GEF for the rag GTPases that signal amino acid levels to mTORC1." Cell 150(6): 1196-1208. Carroll, B., D. Maetzel, et al. (2016). "Control of TSC2-Rheb signaling axis by arginine regulates mTORC1 activity." Elife 5. Chantranupong, L., S. M. Scaria, et al. (2016). "The CASTOR Proteins Are Arginine Sensors for the mTORC1 Pathway." Cell 165(1): 153-164. Cheng, P. N., T. L. Lam, et al. (2007). "Pegylated recombinant human arginase (rhArg-peg5,000mw) inhibits the in vitro and in vivo proliferation of human hepatocellular carcinoma through arginine depletion." Cancer Res 67(1): 309-317. Chou, T. C. (2010). "Drug combination studies and their synergy quantification using the Chou-Talalay method." Cancer Res 70(2): 440-446. Feun, L., M. You, et al. (2008). "Arginine deprivation as a targeted therapy for cancer." Curr Pharm Des 14(11): 1049-1057. Gjymishka, A., N. Su, et al. (2009). "Transcriptional induction of the human asparagine synthetase gene during the unfolded protein response does not require the ATF6 and IRE1 / XBP1 arms of the pathway." Biochem J 417(3): 695-703. Guerrini, L., S. S. Gong, et al. (1993). "Cis- and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression." Mol Cell Biol 13(6): 3202-3212. Hensley, C. T., A. T. Wasti, et al. (2013). "Glutamine and cancer: cell biology, physiology, and clinical opportunities." J Clin Invest 123(9): 3678-3684. Horowitz, B. and A. Meister (1972). "Glutamine-dependent asparagine synthetase from leukemia cells. Chloride dependence, mechanism of action, and inhibition." J Biol Chem 247(20): 6708-6719. Korangath, P., W. W. Teo, et al. (2015). "Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate." Clin Cancer Res 21(14): 3263-3273. Krall, A. S., S. Xu, et al. (2016). "Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor." Nat Commun 7: 11457. Lam, T. L., G. K. Wong, et al. (2009). "Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest." Cancer Lett 277(1): 91-100. Liu, R. Y., Z. Dong, et al. (2013). "Overexpression of asparagine synthetase and matrix metalloproteinase 19 confers cisplatin sensitivity in nasopharyngeal carcinoma cells." Mol Cancer Ther 12(10): 2157-2166. Richards, N. G. and M. S. Kilberg (2006). "Asparagine synthetase chemotherapy." Annu Rev Biochem 75: 629-654. Saxton, R. A., L. Chantranupong, et al. (2016). "Mechanism of arginine sensing by CASTOR1 upstream of mTORC1." Nature 536(7615): 229-233. Su, N., Y. X. Pan, et al. (2008). "Correlation between asparaginase sensitivity and asparagine synthetase protein content, but not mRNA, in acute lymphoblastic leukemia cell lines." Pediatr Blood Cancer 50(2): 274-279. Tsui, S. M., W. M. Lam, et al. (2009). "Pegylated derivatives of recombinant human arginase (rhArg1) for sustained in vivo activity in cancer therapy: preparation, characterization and analysis of their pharmacodynamics in vivo and in vitro and action upon hepatocellular carcinoma cell (HCC)." Cancer Cell Int 9: 9. Wang, S., Z. Y. Tsun, et al. (2015). "Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1." Science 347(6218): 188-194. Wise, DR and CB Thompson (2010). "Glutamine addiction: a new therapeutic target in cancer." Trends Biochem Sci 35(8): 427-433. Yang, H., X. He, et al. (2014). "Down-regulation of asparagine synthetase induces cell cycle arrest and inhibits cell proliferation of breast cancer." Chem Biol Drug Des 84(5): 578-584. Zheng, L., W. Zhang, et al. (2016). "Recent Advances in Understanding Amino Acid Sensing Mechanisms that Regulate mTORC1." Int J Mol Sci 17(10).

[0108] It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Accordingly, the inventive subject matter is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, may be utilized, or may be combined with other elements, components, or steps not expressly referenced. If this specification claims or refers to at least one selected from the group consisting of A, B, C..., and N, the statement should be interpreted as requiring only one element from the group, and not A+N, B+N, etc.

Claims

1. 1. A method for selectively derivatizing a protein, comprising: Obtaining a protein of interest that contains at least one cysteine; contacting the protein of interest with PEG-maleimide in a buffer having a pH between 6.5 and 7.0 at a temperature between 2°C and 15°C; incubating the protein of interest with the PEG-maleimide at 2°C to 15°C for 24 to 72 hours to produce a PEG-derivatized protein; wherein said PEG-maleimide is present in less than a 6-fold molar excess compared to said protein of interest.

2. 2. The method of claim 1, wherein the PEG-maleimide comprises a branched PEG.

3. 2. The method of claim 1, wherein the PEG-maleimide comprises a linear PEG.

4. The method according to any one of claims 1 to 3, wherein the protein of interest is human arginase 1 or a variant thereof.

5. 5. The method of claim 4, wherein the mutant comprises the removal of all but one cysteine.

6. The method of claim 4, wherein the protein of interest or the variant thereof does not contain a polyhistidine sequence.

7. 7. The method of any one of claims 1 to 6, further comprising the additional step of separating the PEG-derivatized protein from unreacted or hydrolyzed PEG-maleimide.

8. 8. The method of claim 7, wherein the additional step of separation is performed by one of the group consisting of dialysis, size exclusion chromatography, and ion exchange chromatography.

9. 9. The method of any one of claims 1 to 8, wherein the PEG-maleimide is present in less than a 4-fold molar excess compared to the protein of interest.

10. 1. A preparation of PEG-modified human arginase 1, comprising: A preparation comprising a peptide sequence corresponding to SEQ ID NO: 1 covalently attached to a single polyethylene glycol moiety having a molecular weight of at least 20 kDa, wherein the PEG-modified human arginase 1 represents at least 90% of the human arginase in the preparation, and the PEG-modified human arginase 1 does not contain a polyhistidine sequence.

11. The preparation of claim 10, wherein the polyethylene glycol moiety is linear.

12. 11. The preparation of claim 10, wherein the polyethylene glycol moiety is branched.

13. 13. The preparation of claim 12, wherein the polyethylene glycol moiety comprises a "Y" branched structure.

14. 13. The preparation of claim 12, wherein the polyethylene glycol moiety comprises a "V" branched structure.

15. The preparation of any one of claims 10 to 14, wherein the PEG-modified human Arginase 1 comprises a metal cofactor selected from the group consisting of manganese, nickel, and cobalt.

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