Compositions and methods for amino acid depletion therapy
阅读说明：本技术 用于氨基酸消耗疗法的组合物和方法 (Compositions and methods for amino acid depletion therapy ) 是由 刘约翰逊 刘耀南 梁润松 劳伟雄 游国明 杨玉强 彭佩诗 朱桂林 于 2018-08-16 设计创作，主要内容包括：提供了用于制备高纯度精氨酸酶以及用于高效制备单取代聚乙二醇化精氨酸酶缀合的组合物和方法,以及使用精氨酸酶与天冬酰胺酶组合抑制癌细胞的方法。通过应用初始高温沉淀步骤,然后进行离子交换,以提供纯度为90％或更高的精氨酸酶来提供高纯度精氨酸酶。使用相对于精氨酸酶较低摩尔过量的马来酰亚胺衍生的聚乙二醇和在降低的温度下进行与直链或支链的聚乙二醇的缀合。这种聚乙二醇衍生的精氨酸酶与天冬酰胺酶组合在抑制癌细胞,特别是具有较低的内源性天冬酰胺酶表达的细胞的生长中是有用的。(Compositions and methods for preparing high purity arginase and for efficiently preparing monosubstituted pegylated arginase conjugates, and methods of inhibiting cancer cells using arginase in combination with asparaginase are provided. High purity arginase is provided by applying an initial high temperature precipitation step followed by ion exchange to provide arginase having a purity of 90% or greater. Conjugation to linear or branched polyethylene glycol was performed using a lower molar excess of maleimide-derivatized polyethylene glycol relative to arginase and at reduced temperature. Such polyethylene glycol-derived arginases are useful in combination with asparaginase in inhibiting the growth of cancer cells, particularly cells with low endogenous asparaginase expression.)
1. A method of purifying an arginase, comprising:
obtaining cells expressing arginase;
disrupting the cells to produce a lysate comprising arginase;
in CoCl2Increasing the temperature of the lysate to a precipitation temperature in the presence of a first amount of purified arginase for a time period sufficient to precipitate a first contaminant from the lysate and produce a supernatant comprising a first portion of purified arginase;
contacting the supernatant with an anion exchanger to produce a first bound fraction and a first flow-through fraction;
wherein the flow-through fraction comprises partially purified Co2+Arginase, with Co2+Replacement of Mn2+。
2. The method according to claim 1, comprising the further step of:
contacting the first flowthrough fraction with a cation exchanger to produce a second bound fraction and a second flowthrough fraction;
applying an elution buffer to the cation exchanger to produce an elution fraction,
wherein the elution fraction comprises purified Co2+Arginase, with Co2+Replacement of Mn2+。
3. The method of claim 1 or 2, wherein the cells express human arginase 1.
4. The method according to one of claims 1 to 3, wherein the cells are bacterial cells.
5. The method according to one of claims 1 to 4, wherein the precipitation temperature is greater than 50 ℃.
6. The method of one of claims 1 to 5, wherein the precipitation temperature is about 65 ℃.
7. The method of one of claims 1 to 6, wherein CoCl is provided at a concentration of at least 20mM2。
8. The method according to one of claims 1 to 7, wherein the time period is between 5 and 30 minutes.
9. The method of one of claims 1 to 8, wherein the period of time is about 15 minutes.
10. The process according to one of claims 1 to 9, wherein the anion exchanger is a strong anion exchanger.
11. The process according to one of claims 1 to 10, wherein the cation exchanger is a strong cation exchanger.
12. The method of claim 2, wherein the partially purified Co2+Arginase has a purity of at least 80%.
13. The method of claim 2, wherein the purified human Co2+Arginase has a purity of at least 90%.
14. A method of selectively derivatizing a protein, comprising:
obtaining a protein of interest comprising at least one cysteine;
contacting the target protein with PEG-maleimide in a buffer at a pH of 6.5 to 7.0 at a temperature of 2 ℃ to 15 ℃; and
incubating the target protein with the PEG maleimide for 24 to 72 hours at 2 to 15 ℃ to produce a PEG-derivatized protein,
wherein the PEG-maleimide is present in an amount less than a 6-fold molar excess of the target protein.
15. The method of claim 14, wherein the PEG-maleimide comprises a branched PEG.
16. The method of claim 14, wherein the PEG-maleimide comprises linear PEG.
17. The method according to one of claims 14 to 16, wherein the protein of interest is human arginase 1 or a mutation thereof.
18. The method according to claim 17, wherein said mutation comprises elimination of all but one cysteine.
19. The method of claim 17, wherein the protein of interest or mutation thereof does not comprise a polyhistidine sequence.
20. The method according to one of claims 14 to 19, further comprising the additional step of separating the PEG-derivatized protein from unreacted or hydrolyzed PEG-maleimide.
21. The method of claim 20, wherein the additional separation step is performed by one of dialysis, size exclusion chromatography, and ion exchange chromatography.
22. The method according to one of claims 14 to 21, wherein the PEG-maleimide is present in an amount less than 4-fold molar excess of the target protein.
23. A formulation of PEG-modified human arginase 1 comprising:
a peptide sequence corresponding to SEQ ID NO1 covalently coupled to a single polyethylene glycol moiety having a molecular weight of at least 20kDa,
wherein said PEG-modified human arginase 1 comprises at least 90% of human arginase in said formulation, and
wherein said PEG-modified human arginase 1 does not include a polyhistidine sequence.
24. The formulation of claim 23, wherein the polyethylene glycol moiety is linear.
25. The formulation of claim 23, wherein the polyethylene glycol moiety is branched.
26. The formulation of claim 25, wherein the polyethylene glycol moiety comprises a "Y" branched structure.
27. The formulation of claim 25, wherein the polyethylene glycol moiety comprises a "V" branched structure.
28. The formulation according to one of claims 23 to 27, wherein the PEG-modified human arginase 1 comprises a metal cofactor selected from manganese, nickel, and cobalt.
29. A method of inhibiting a cancer cell, comprising:
reducing the concentration of arginine in the medium contacted with the cancer cells; and
reducing the asparagine concentration in the culture medium.
30. The method of claim 29, wherein arginine concentration is reduced using arginase.
31. The method of claim 30, wherein said arginase is recombinant human arginase.
32. The method of one of claims 29 to 31, wherein the asparagine concentration is reduced using asparaginase.
33. The method of one of claims 29-32, wherein the cancer cells have lower asparaginase expression.
34. The method according to one of claims 29 to 33, further comprising the step of reducing the glutamine concentration.
35. The method of claim 34, wherein the glutamine concentration is reduced using an aminotransferase inhibitor.
36. The method of claim 35, wherein the aminotransferase inhibitor is an aminooxyacetate.
37. A composition for inhibiting cancer cells, comprising:
an arginine reductase; and
an asparagine-reducing enzyme.
38. The composition of claim 37, wherein the arginine reductase is arginase.
39. The composition of claim 38, wherein said arginase is recombinant human arginase.
40. The composition of one of claims 37 to 39, wherein the asparagine-reducing enzyme is an asparaginase.
41. The composition of one of claims 37-40, wherein the cancer cells have lower asparaginase expression.
42. The composition according to one of claims 37 to 41, further comprising a compound that reduces the concentration of glutamine.
43. The composition according to claim 42, wherein the glutamine concentration-reducing compound is an aminotransferase inhibitor.
44. The composition of claim 43, wherein said aminotransferase inhibitor is an aminooxyacetate.
Technical Field
Background
The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
There is increasing evidence that amino acid deprivation may be an effective candidate for the treatment of cancer. It has been found that deprivation of specific amino acids such as arginine, asparagine or glutamine can be used to treat different types of cancer (Feun, You et al 2008; Hensley, Wasti et al 2013; Krall, Xu et al 2016). All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to 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 believed to be due to downstream effects such as inactivation of mTORC1 and disruption of protein synthesis.
The application of recombinant human arginase (rhArg) to the consumption of arginine has been shown to be effective in inhibiting cancer cell growth in vitro (Lam, Wong et al 2009; Tsui, Lam et al 2009). Arginine was chosen as a target amino acid not only for its semi-essential role in protein synthesis, but also for its role in activating mTORC1 (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). Arginine deprivation was found to be effective in inhibiting a variety of cancer cell lines, including cell lines derived from breast, colon, lung and cervical cancers.
The enzyme arginase acts on arginine to produce ornithine and urea and is part of the urea cycle. Arginase is found increasingly to be used as a chemotherapeutic agent, where it is used to reduce arginine concentrations in serum. These depleted serum arginine levels are effective in "starving" cancer cells (many of whose varieties are auxotrophic for arginine).
The use of arginase as a therapeutic agent requires the availability of large amounts and high purity of human arginase. Attempts have been made to provide highly purified recombinant human arginase. For example, U.S. Pat. No. 8,507,245 (issued to Leung and Lo) describes a pseudo-affinity chromatography method for purifying recombinant
Unmodified arginase is unstable in plasma, which severely limits its therapeutic applications. Many attempts have been made to extend plasma half-life, including conjugation of proteins to polymeric polyethylene glycol (pegylation). A widely used conjugation strategy is the non-selective glycolation of the amino group (e.g., lysine-amine) of arginase as described in U.S. patent No. 9,050,340 (issued to Georgiou and Stone). This approach requires the use of a significant molar excess of the expensive amine-reactive PEG reagent, in part because of the relatively rapid hydrolysis of this reagent. This random conjugation also complicates the qualitative and quantitative characterization of the modified arginase, thereby limiting its medicinal use. Using this approach, it is unlikely that consistent product quality will be obtained unless the coupling reaction is performed under very tightly controlled conditions, which is generally not suitable for scale-up.
Thus, there remains a need for methods that can provide active and effective and consistent pegylated arginases in high purity.
Disclosure of Invention
The subject matter of the present invention provides compositions and methods for preparing and derivatizing high purity arginase, as well as using the arginase so prepared in combination with asparaginase to treat cancer.
One embodiment of the inventive concept is a method of purifying arginase by obtaining cells (e.g., bacterial cells) expressing arginase, disrupting the cells to produce a lysate comprising arginase, and purifying the cell lysate in CoCl2(e.g., a concentration of at least 20 mM) raising the temperature of the lysate to a precipitation temperature (e.g., at least 50 ℃ or about 65 ℃) for a time (e.g., about 5 to 30 minutes) sufficient to precipitate contaminants from the lysate and produce a supernatant comprising the first portion of purified arginase. Contacting the supernatant with an anion exchanger that binds additional contaminants and provides a composition comprising partially purified Co2+Arginase (i.e., wherein Mn2+Is covered with Co2+Alternative arginase) flow through the fraction (flow through fraction). Such partially purified Co2+Arginase may have a purity of about 80% or more.
In some embodiments, the method comprises the further step of: contacting the flow-through fraction with a cation exchanger to produce a bound fraction comprising arginase and a second flow-through fraction. Applying an elution buffer to the cation exchanger to elute the purified Co2+Arginase enzyme. Such purified Co2+Arginase may have a purity of about 90% or more. The arginase may be
Another embodiment of the inventive concept is a method of selectively derivatizing a protein (e.g.,
Another embodiment of the inventive concept is the preparation of PEG-modified
Another embodiment of the inventive concept is a method of inhibiting cancer cells by reducing the concentration of arginine in a medium used to culture the cancer cells and reducing the concentration of asparagine in the medium. Arginase (e.g., recombinant human arginase) can be used to reduce arginine concentration. Similarly, asparaginase (ASNase) can be used to reduce the concentration of asparagine. In some such embodiments, the cancer cell has lower asparagine synthetase (ASNS) expression. In some such embodiments, the method comprises the additional step of reducing the glutamine concentration, for example, using an aminotransferase inhibitor (e.g., aminooxyacetate).
Another embodiment of the inventive concept is a composition for inhibiting cancer cells comprising arginine reductase and asparagine reductase. The arginine reductase may be an arginase, such as a recombinant human arginase. Similarly, the asparagine-reducing enzyme can be asparaginase (ASNase). In some such embodiments, the cancer cell has lower asparagine synthetase (ASNS) expression. The composition may also include a compound that reduces the concentration of glutamine, such as an aminotransferase inhibitor (e.g., aminooxyacetate).
Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments along with the accompanying drawings in which like reference numerals represent like components.
Drawings
FIG. 1: a flow chart of an exemplary method of the inventive concept.
FIG. 2: photographs of the electrophoresis gels show the results of SDS-PAGE reduction on 8-16% gradient gels in both the homogenate and the hot pellet products.
FIG. 3: in Capto QTMUV absorption profile during chromatography.
FIG. 4: photograph of electrophoretic gel showing typical Capto QTMIn the chromatographic procedure, the results of SDS-PAGE were reduced on 8-16% gradient gels.
FIG. 5: in Capto STMUV absorption profile during chromatography was performed.
FIG. 6: photograph of electrophoretic gel showing typical Capto QTMIn the chromatographic procedure, the results of SDS-PAGE were reduced on 8-16% gradient gels.
FIG. 7: graph of the pegylation kinetics of
FIG. 8: graph of plasma arginine concentration in healthy rats after a single intravenous dose of PEG-modified
FIG. 9: graph of plasma arginine concentration in healthy rats treated with a single intravenous dose of PEG-modified
FIG. 10: body weight profile of healthy rats treated with a single intravenous dose of PEG-modified
FIG. 11: graph of plasma arginine concentration in healthy rats treated with a single intravenous dose of linear and branched PEG-modified
FIG. 12: body weight profile of healthy rats treated with a single intravenous dose of linear or branched PEG-modified
FIG. 13: molecular weight determination by LC/Q-TOF MS. The deconvolution mass of the pegylated arginase was 34,572.3 Da.
FIG. 14: peptide diagram of mutant
FIG. 15: graph of enzymatic activity of arginase in rat plasma after a single intravenous dose of 2mg/kg over time.
FIG. 16: graph of plasma concentration of immunoreactive PEG-modified
FIG. 17: efficacy profiles of ASNase alone in (i) low ASNS expressing cell lines (MDA-MB-231, ZR-75-1, and MCF7) and (ii) high ASNS expressing cell lines (HeLa, HepG2, and MIA-Paca 2). Three independent experiments were performed for each experiment. The error column represents one Standard Deviation (SD).
FIG. 18: efficacy profiles of rhArg and rhArg-ASNase combinations alone in (i) low-ASNS expressing cell lines (MDA-MB-231, ZR-75-1, and MCF7) and (ii) high-ASNS expressing cell lines (HeLa, HepG2, and MIA-Paca 2). Three independent experiments were performed for each experiment. The error column represents one Standard Deviation (SD).
Detailed Description
The following description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
The present subject matter provides devices, compositions and methods that provide for scalable purification of human arginase. In the compositions and methods contemplated by the present invention, the preparation containing recombinant human arginase is incubated at an elevated temperature, which results in the formation of a precipitate. The precipitate was removed, and the supernatant was collected and ion-exchanged on an anion exchanger. The flow-through fraction from the anion exchange process was collected and subjected to a further polishing step on a cation exchanger, in which step human arginase was eluted using a salt gradient. The resulting human arginase may be modified, for example, by pegylation. This pegylation can be performed at reduced temperatures using a relatively small molar excess of the reactive PEG analog in order to produce a high purity pegylated human arginase. The present subject matter provides devices, systems, and methods in which compounds that reduce arginine and asparagine are used to inhibit the growth of cancer cells. Such compounds may be enzymes that catalytically reduce arginine and asparagine concentrations, such as arginase and/or asparaginase. In a preferred embodiment, the enzyme is a human enzyme, such as recombinant human arginase (rhArg).
Such amino acid consuming enzymes may be provided as purified enzymes from natural sources or as products of recombinant bacteria, fungi, plant cells or animal cells. The enzymes used in such therapies may have a purity of more than 80%, 85%, 90%, 95%, 98%, 99% or higher, and may be post-translationally modified. Such modifications may include modifications that improve absorption and/or half-life (e.g., pegylation). The enzyme-containing pharmaceutical preparation may be administered intravenously, e.g., by injection or infusion. Such formulations may include or be co-administered with chemotherapeutic agents, immunotherapeutic agents and/or radiation therapy for the treatment of cancer.
Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawings in which like reference numerals represent like components.
It will be appreciated that the disclosed technology provides a number of advantageous technical effects, including the scalable production of highly purified human arginase having native sequences from recombinant sources.
In some embodiments, numbers expressing quantities of ingredients, properties such as concentrations, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in certain instances by the term "about". Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. The numerical values set forth in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
As used herein in the description and throughout the claims that follow, the meaning of "a, an" and "the" includes the plural unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of "in. (in)" includes "in. (in)" and "on. (on)" unless the context clearly indicates otherwise.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual 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 otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided with respect to certain embodiments herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
Groupings of optional elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to or claimed individually, or may be used in combination with other members of the group or other elements found herein. For convenience and/or patentability, one or more members of a group may be included in or deleted from a group. When any such inclusion or deletion occurs, the specification is considered herein to encompass the modified group so as to satisfy the written description of all markush groups used in the appended claims.
The following discussion provides many example embodiments of the inventive subject matter. While each example represents a single combination of inventive elements, the inventive subject matter is considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B and C, while a second embodiment includes elements B and D, the inventive subject matter is considered to include other remaining combinations of A, B, C or D, even if not explicitly disclosed.
In embodiments of the inventive concept, one of the amino acid consuming enzymes is an arginase, such as a mammalian, avian, reptile, plant, fungal and/or bacterial arginase. In some such embodiments, the arginase may be a human arginase provided as a genetically engineered product, e.g., a bacterial, yeast, fungal, insect, plant, or mammalian cell in culture. Such arginase may include one or more sequence modifications for improving the specificity of subsequent conjugation reactions, e.g., removal or substitution of one or more amino acids containing potentially modified side chains. In a preferred embodiment, recombinant human arginase may be produced in E.coli clones, for example, strain BL21 (T7 Express from NewEngland Biolabs) containing kanamycin resistance, into which expression vector pET-30a encoding cDNA for
Fig. 1 provides a flow chart outlining an example of the method of the inventive concept. As shown, bacteria or other cells expressing
Disruption of bacteria or other cells may be performed by any suitable method. These include, but are not limited to, enzymatic digestion, osmotic shock, sudden pressure changes (e.g., through expression of pressure and/or sonication). In some embodiments, these methods may be performed under temperature-controlled conditions. For example, the temperature of the bacteria or other cells being sonicated may be controlled to ensure that the temperature does not exceed a temperature compatible with subsequent activity of human arginase. In other embodiments, one or more protease inhibitors may be added before, during, or after the destruction of the bacteria or other cells. In still other embodiments, one or more stabilizing agents (e.g., antioxidants) may be added before, during, or after the destruction of the bacteria or other cells.
After disruption of the bacteria or other cells, residual debris may be removed (e.g., by sedimentation, filtration, and/or centrifugation) to leave a solution comprising human arginase. The inventors have found that arginase is surprisingly stable at elevated temperatures (i.e. above 37 ℃, 40 ℃, 45 ℃,50 ℃, 60 ℃ and/or 70 ℃), which may lead to undesired denaturation and subsequent precipitation of contaminating proteins. For example, the inventors found that human arginase I is stable at 74 ℃, which temperature causes precipitation of many contaminating proteins. Without being bound by theory, it is believed that this stability is provided by complex formation with divalent ions. This may be in the presence of divalent ions (such as Mn)2+Or Co2+) Is extracted at a high temperature (i.e. a temperature in excess of 37 ℃) so as to produce a precipitate (which includes contaminating proteins) and a supernatant (which includes human arginase). In addition, arginase exhibits greatly enhanced catalytic activity (k) due to cobalt chelationcat/KM) Thus CoCl can be utilized during extraction2Not only provide high catalytic potential, but also can use Co2+Mn substituting for arginase2+. The temperature and incubation time may be selected to provide optimal recovery, purity and quality of sufficient human arginaseAnd (4) activity.
The temperature range of incubation can be from about 40 degrees, 45 degrees, 50 degrees, 55 degrees, 60 degrees, 65 degrees, 70 degrees, 75 degrees, 80 degrees, 85 degrees, 90 degrees or more than about 90 degrees. The incubation time may 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 may be varied during the incubation. In a preferred embodiment, the incubation temperature is 65 ℃ and the incubation period is 15 minutes. The inventors have found that this method advantageously removes a large proportion of unwanted heat sensitive proteins, while using Co in arginase2+Replacement of Mn2+。
After this precipitation step, the arginase-containing supernatant was collected for further processing. The supernatant may be separated from the precipitate by any suitable method, including settling, filtration, and/or centrifugation. The collected 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 buffers depends at least in part on the nature of the anion exchange medium used, but may generally have a relatively low molarity (e.g., less than 100mM) and an elevated pH (e.g., greater than 7). Such anion exchangers may be weak anion exchangers having a relatively low affinity for anions, such as anion exchangers comprising ammonium groups. Alternatively, such an anion exchanger may be a strong anion exchanger having a relatively high affinity for anions (e.g., an anion exchanger including a quaternary amine group). In a preferred embodiment, a strong anion exchanger is used. For example, if strong anion exchangers are used, such as Capto QTMA suitable buffer may then be 20mM Tris pH 8.05. The final protein concentration of the supernatant may be adjusted to provide for optimal separation of sufficient arginase from at least a portion of the contaminating substances present.
After transfer to a suitable anion exchange buffer, the supernatant is treated with an anion exchange medium. As noted above, such anion exchange media can be a strong anion exchangeMedia, for example, anion exchange media comprising immobilized quaternary amines. The anion exchange media can be provided in any suitable form, for example, as a filter, an immiscible liquid, porous particles, and/or non-porous particles. An example of such a strong anion exchange medium is Capto QTMA mediator (GE Healthcare) provided as a porous particle and comprising pendant quaternary amine groups. Such anion exchange media can be applied as a bulk solid phase, mixed and/or suspended in the supernatant, and then removed (e.g., by settling, centrifugation, and/or filtration). In a preferred embodiment, the anion exchange medium is provided as a chromatographic bed in a chromatographic column.
The supernatant may be applied to a bed of anion exchange media, causing at least some contaminants to bind to the media while allowing the flow-through fraction containing partially purified arginase to pass through. The volume and configuration of such a column, as well as the flow rate during application, can be optimized to provide sufficient capacity for optimal capture of contaminants from the supernatant. After passing through and collecting the partially purified human arginase, the anion exchange medium may be washed and regenerated (e.g., using a buffer containing a high salt concentration) for reuse. In such embodiments, the anion exchange medium may be selected to withstand sterilization and/or pyrogen-reducing treatments.
The collected partially purified arginase may be used as such (depending on the application) or subjected to a further polishing (polising) step. Pharmaceutical use may require such further processing. If a polishing step is required, the partially purified arginase may be transferred to an aqueous buffer suitable for cation exchange chromatography. This transfer may be accomplished by any suitable means, including dialysis, gel filtration, and/or diafiltration. In some embodiments, the transfer can be effectively accomplished by diluting the partially purified human arginase in a dilution buffer that provides a suitable cation exchange buffer composition. In still other embodiments, the buffer used in the previous anion exchange step can be adjusted (e.g., by controlling pH and/or ionic strength) to be suitable for cation exchange.
When the partially purified arginase is in a suitable cation exchange buffer, it can be applied to a cation exchange medium. Suitable buffers typically have low to moderate ionic strength (e.g., less than 200mM) and a neutral pH (e.g., pH 7). An example of a suitable cation exchange buffer is 50mM Tris, pH7. Such a cation exchanger may be a weak cation exchanger having a relatively low affinity for cations, for example, a cation exchanger including a carboxyl group. Alternatively, such cation exchangers can be strong cation exchangers having a relatively high affinity for cations (e.g., cation exchangers comprising sulfonate groups). In a preferred embodiment, the cation exchange media may be a strong cation exchange media, such as Capto STM(GE Healthcare) comprising pendant sulfonate groups. Since 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., a particle) that is immobilized in place so as to support gradient elution. In a preferred embodiment, the cation exchange medium is provided as a chromatographic bed in a chromatographic column. The volume and configuration of such columns, as well as the flow rates during application, can be optimized to provide sufficient capacity for optimal capture and subsequent release of human arginase from the cation exchange medium.
When such a cation exchange column is used, the partially purified arginase that has been transferred to the cation exchange buffer is applied to the column at a flow rate that allows for the capture of the arginase on the cation exchange medium. After application of the partially purified arginase, the cation exchange column may be washed with an additional volume of cation exchange buffer (e.g., 1 to 10 column volumes) to remove unbound material. Optionally, the UV absorbance is monitored during the process to determine when the wash is complete. In some embodiments, an additional wash step may 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 less than 0.5M 50mM Tris + sodium chloride (NaCl) at pH7. In some embodiments, a series of different stringency of such wash buffers can be used.
After applying the partially purified arginase and any subsequent washing steps, the purified arginase is eluted from the cation exchange medium by applying an elution buffer. This elution can be provided by applying an elution buffer of fixed composition as a single bolus to perform the stepwise elution. In other embodiments, the elution buffer may be applied as a mixture with a cation exchange buffer, wherein the ratio of elution buffer to cation exchange buffer increases over time. In such a gradient elution pathway, the rate at which the ratio changes may be linear, or non-linear, over time. In a preferred embodiment, the elution is accomplished using a linear gradient that transitions the composition of the column buffer from the cation exchange buffer to the elution buffer at a constant rate over time. The elution buffer may 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 additionally includes a high concentration (e.g., greater than 0.2M) of NaCl. An example of such an elution buffer is 50mM Tris +0.5M NaCl, pH7.
During such gradient elution, the UV absorbance of the material exiting the column can be monitored to determine the fraction that should be collected. Fractions may be selected based on arginase content and/or the presence of contaminants to provide the desired yield and purity. Such fractions can be collected, combined and transferred to a buffer suitable for stability. In some embodiments, such purified arginase may be subsequently frozen and/or lyophilized. In still other embodiments, the purified arginase obtained in this manner can be derivatized, e.g., by pegylation, for pharmaceutical use. Typical results for the methods and compositions contemplated by the present invention can provide high purity (> 90%) human arginase in about 30% yield. After passing through and collecting the purified human arginase, the cation exchange medium may be rinsed and regenerated (e.g., using a buffer containing a high salt concentration) for reuse. In such embodiments, the cation exchange media may be selected to withstand sterilization and/or pyrogen-reducing treatments.
In some embodiments, the purified arginase as described above may then be chemically modified. Suitable chemical modifications include biotinylation, charge modification, cross-linking, and conjugation (e.g., grafting of hydrophilic polymers, e.g., dextran, PEG, etc.). Such polymers may be grafted onto purified arginase by using a reactive form of the polymer, for example, by contacting the purified arginase with a polymer bearing amine-reactive and/or thiol-reactive groups. Suitable reactive groups include N-hydroxysuccinimide (NHS) esters, sulfo NHS esters, epoxides, halotriazines, aldehydes, hydrazines, iodoacetamides, maleimides, and other crosslinking groups known in the art. In some embodiments, the human arginase used in such methods can be genetically modified, e.g., to limit or reduce the number and/or positions of modifiable amino acid side chains present on the human arginase. For example, the number of reactive amines can be reduced by replacing one or more lysines in the human arginase sequence with another amino acid. Similarly, the number of reactive thiols can be reduced by replacing one or more cysteines in the human arginase sequence with another amino acid.
It will be appreciated that the addition of a polyhistidine sequence (e.g., at the amino and/or carboxy terminus) typically used to generate fusion proteins having affinity for nickel is not required in the compositions or methods contemplated by the present invention. Examples of suitable recombinant arginases are provided in SEQ ID NO. 1. The inclusion of such polyhistidine sequences may render human arginase antigenic and therefore unsuitable for repeated use as a therapeutic agent. In addition, the inclusion of a polyhistidine sequence interferes with the isolation of arginase during initial purification and after the above-described modification reactions using cation exchange.
Pegylation is often used to extend the serum half-life of therapeutically valuable proteins. This method is typically performed using a large molar excess (e.g., a 10-fold molar excess or greater) of the reactive form of PEG in order to ensure that a large portion of the protein of interest is pegylated. This is especially true in proteins that present steric issues that reduce coupling efficiency (e.g., human arginase 1). Typical reactive forms of PEG include PEG-NHS or PEG-sulfo NHS (used when coupling via an amine is desired) or PEG-maleimide (used when coupling via a thiol is desired). The use of large molar excesses of such activated PEG is undesirable for a number of reasons, including the possibility of coupling at large molar excesses to reduce selectivity, the subsequent difficulty in separating large excesses of unreacted PEG from reacted proteins, and expense.
Surprisingly, the inventors have found that PEG-maleimide can be effectively pegylated on human arginase (e.g.,
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