CN105018442A - Improved EN-apyrase - Google Patents

Abstract

The invention discloses improved EN-apyrase. The improved EN-apyrase has more complex glycosylation and longer biological half life in comparison with EN-apyrase disclosed by the prior art.

Description

Improved EN-adenosine triphosphate phosphatase

technical field

The present invention relates to an improved form of the prior art apyrase named EN-adenosine, herein named I-EN-adenosine. Therefore, in the pharmaceutical field, it is used for the treatment of thrombolytic-related or inflammation-related diseases.

Background technique

PCT Publication WO2011/088244 describes EN-adenosine triphosphatases with a specific amino acid sequence at the homologous N-terminus, wherein more than 80% of EN-adenosine triphosphatase molecules and homologues have ELVP The same N-terminus, and has a sequence identical to SEQ ID NO: 180% listed below or a sequence containing only 1-5 conservative substitutions relative to SEQ ID NO: 1. All EN-adenosine triphosphatases retain ADPase and ATPase activity. These EN-adenylate enzymes were obtained in CHO cells using specific constructs that lead to N-terminal homogeneity and have advantages over prior art adenosine derived from a soluble form of CD39L3 Enhanced half-life of triphosphatases. The present application improves the properties of this EN-adenosine triphosphatase by adjusting the culture conditions under which the protein is produced such that an even longer biological half-life is obtained, presumably due to the more complex and caused by higher levels of glycosylation.

Contents of the invention

A group of improved EN-adenosine triphosphatases was prepared, named I-EN-adenosine triphosphatase, which has even better pharmacokinetic properties than EN-adenosine triphosphatase .

Thus, in one aspect, the present invention relates to a method for improving the biological characteristics of EN-adenosine triphosphatase, the method comprising delaying the addition of glutamine to the culture medium of CHO cells until after one day in culture, The CHO cells are modified to contain the expression construct for EN- apyrase.

In another aspect, the present invention relates to an improved EN-adenosine triphosphatase (I-EN-adenosine triphosphatase) having a half-life in dogs of at least 23 hours; and/or It has a molecular weight of at least 70 kD; and/or its glycosylation pattern has an increased Mannose5+Mannose8+G2FS2/Mannose6+Mannose7+G2FS1 ratio.

Brief description of the drawings

Fig. 1 is a schematic diagram of vectors used for the production of EN-adenosine triphosphatase and I-EN-adenosine triphosphatase.

Figure 2 shows the stable production of improved EN- apyrase in a specific clone (350) over 42 passages under the culture conditions used for the improved protein.

Figure 3 shows the higher molecular weight of the EN-adenylate enzyme of the present invention (marked condition A) compared to the higher molecular weight of the EN-adenylate enzyme of the present invention (marked condition A). Differences in molecular weight, measured by multi-angle light scattering analysis coupled to HPLC-size exclusion chromatography.

Figure 4 shows similar multi-angle light scattering analysis coupled to HPLC-size exclusion chromatography after deglycosylation of these two forms of EN-adenosine triphosphatase. As shown in this figure, the molecular weight after deglycosylation is the same.

Figure 5 shows the glycosylation pattern of the EN- apyrase of the invention (condition D) compared to the prior art apyrase (condition A).

Figure 6 compares the biological half-life of the I-EN-adenylate enzyme of the invention (condition D) in dogs with the biological half-life of the prior art EN-adenylate enzyme (condition A). half life. As can be seen, the half-life of the apyrase of the invention in dogs is much longer and is approximately 25 hours.

Embodiments of the present invention

Applicants have found that by adjusting the culture conditions for culturing the prior art EN- apyrase, a significant increase in the biological half-life can be obtained. These are thought to be due to enhanced levels of glycosylation and higher levels of glycosylation complexity under these conditions. Thus, despite sharing some features with prior art EN-adenosine triphosphatases, i.e., more than 80% of the EN-adenosine triphosphatase molecules and homologues therein have the same N-terminus, and have the SEQ ID NO listed below: 180% identical or only contain the sequence of 1-5 conservative substitutions with respect to SEQ ID NO: 1, but the I-EN-adenosine three of the present invention Phosphobisphosphatases have enhanced half-lives and glycosylation patterns characterized by higher ratios of:

Mannose 5+Mannose 8+G2FS2/Mannose 6+Mannose 7+G2FS1.

Specifically, as shown in the Examples below, delaying the addition of glutamine to the medium until after the first day of culture resulted in a significant improvement in results.

The I-EN- apyrase of the present invention can be used in the treatment of disorders associated with inflammation and thrombosis in a similar manner to the EN- apyrase of the prior art. Although these uses are described in detail in the prior art, for the convenience of the reader, they are summarized as follows:

use

In general for the use of at least apyrase, more specifically for the use of CD39 compounds and any of CD39L1-8 such as CD39L3 compounds, I-EN- apyrase is a useful therapeutic agent. These are useful as antiplatelet agents, antithrombolytic agents, and as anti-inflammatory agents and endothelial cell (EC) protective proteins. Furthermore, for the treatment of bleeding disorders, EN-adenosine triphosphatase is therapeutically useful as described in U.S. 8,535,662, which is incorporated by reference in its entirety. Conditions that may be beneficially treated by I-EN-adenosine triphosphatase include conditions that cause bleeding due to mechanically induced injury or drug insult.

Certain clinical conditions may require slow and prolonged release of I-EN-adenosine or biological derivatives. These conditions may require sequestration of I-EN-adenosine triphosphatase in, for example, hydrogels or other pharmaceutically acceptable polymerizable gels. In addition, polyethylene glycol (PEG) can be added to prolong the blood half-life and thus enhance potency. When I-EN-adenosine triphosphatase is used as a prophylactic drug, a single bolus dose can be used to maintain the protective effect for a longer period of time. Other protein modifications that alter protein half-life include, for example, albumin conjugation, IgG fusion molecules, and changes in protein glycosylation patterns.

I-EN-adenosine triphosphatase may be used in any clinical condition where hydrolysis of ATP and/or ADP to AMP is clinically beneficial, including disease states in which ATP and/or ADP concentrations are abnormally high. I-EN-adenosine triphosphatase is beneficial in clinical situations where platelets or activated platelets play an important role in disease progression such as tumor metastasis.

The clinical and biological efficacy of administered I-EN-adenosine triphosphatase is readily assessed at given time intervals after administration. For example, in cases where platelet counts remain constant, administration should result in longer bleeding times. In addition, direct measurement of the enzymatic activity of I-EN- apyrase or metabolites in blood samples also indicates the presence of this molecule in circulating blood. Based on accurate sampling of blood samples combined with methods known in the art for assessing the biochemical function of I-EN- apyrase, the half-life of the protein can be estimated. Other clinically relevant tests for the presence of biologically active I-EN-adenosine triphosphatase are also conceivable.

Methods for In Vitro and In Vivo Assessment of Immediate Apyrase Potency

The biochemical function of I-EN-adenosine triphosphatase can be assessed by a variety of methods available to those of ordinary skill in the art. For example, the enzymatic activity of ATPase and ADPase can be determined at 37°C in 1 ml of a solution containing: 8 mM CaCl ₂ , 200 μM substrate (for ATPase, the substrate is ATP, or for ADPase, the substrate is ADP ), 50 mM imidazole and 50 mM Tris, pH 7.5 (Picher et al., Biochem. Pharmacol. (1988) 51:1453). The reaction can be stopped and the released inorganic phosphate can be measured by adding 0.25 mL of malachite green reagent (Baykov et al., Anal. Biochem. (1988) 171:266). Based on spectrophotometric analysis at 630 nm, one unit of ATPase (or ADPase) corresponds to the release of 1 micromole of inorganic phosphate/min at 37°C. Key kinetic constants of the enzyme such as _Km and _kcat can be obtained by fitting the data to, for example, the Michaelis-Menten equation. Other tests that can be used to monitor biochemical function include, but are not limited to, the radioanalytical and HPLC tests described by Gayle III et al. Radiation-TLC analysis as described in Clin Invest. (1991) 88: 1690-1696).

The biological function of EN-adenosine triphosphatase or derivatives can be assessed by in vitro methods as well as in vivo methods. In vitro methods that can be used to monitor the biological function of EN-adenosine triphosphatase and derivatives include, for example, platelet aggregation assay (Pinsky, D.J. et al. J. Clin Invest. (2002) 109:1031-1040; Ozaki, Y, Sysmex J. Int. (1998) 8:15-22).

In vivo methods that can be used to assess the biological function of I-EN-adenosine triphosphatase include murine stroke models, measuring bleeding time, infarct volume, blood flow, neurological deficits, intracerebral hemorrhage and mortality (Pinsky, D.J. et al., supra; Choudhri, T.F. et al., J. Exp. Med. (1999) 90:91-99); the mouse lung ischemia/reperfusion model (Fujita, T. et al., Nature Med. (2001) 7:598-604 ); baboon reperfusion stroke model (Huang, J. et al., Stroke (2000) 31: 3054-3063); cd39-/- mice (Pinsky, D.J. et al., J. Clin Invest. (2002) 109: 1031- 1040) and Yorkshire-Hampshire pig PCI model (Maliszewski, C.R. et al., PCT WO00/23094 (2000)) and rabbit PCI model (Herbertm, J-M. et al., Thromb Haemost (1998) 80:512-518; Fishman, J. et al. , Lab Invest (1975) 32:339-351; Sarembock et al., Circulation (1989) 80:1029-1040). Those skilled in the art may be aware of other methods for assessing the biological function of ADPase-enhanced apyrase and derivatives as thromboregulators.

Therapeutic compositions of I-EN-adenosine triphosphatase of the invention

The present invention provides a pharmaceutically acceptable dose of a composition comprising a biologically effective amount of I-EN- apyrase. These compositions can be administered clinically to the patient before, during or after symptoms. Post-symptomatic administration of I-EN-adenosine triphosphatase can be performed, for example, between 0 and 48 hours after stroke onset. Administration can be by, for example, bolus injection - intramuscular or subcutaneous, by inhalation, continuous infusion, sustained release or other pharmaceutically acceptable techniques. Certain clinical conditions may require administration as a single effective dose, or it may be administered daily for up to a week or up to a month or more.

Administration is in a pharmaceutically acceptable form containing a physiologically acceptable carrier, excipient or diluent. The diluents and excipients can include neutral buffered saline solution, antioxidants (e.g., ascorbic acid), low molecular weight polypeptides (e.g., polypeptides < 10 amino acids), amino acids, carbohydrates (e.g., glucose, dextrose , sucrose or dextran), chelating agents (eg, EDTA), stabilizers (eg, glutathione). In addition, a cosubsatrate, eg, calcium (Ca ²⁺ ), can be administered at the time of dosing to obtain maximal enzyme activity. Such carriers and diluents are chosen to be nontoxic to the patient at recommended dosages and concentrations.

Other agents that synergistically enhance the benefit of I-EN- apyrase alone may also be co-administered. For example, administration of other antiplatelet agents or anticoagulants such as aspirin, heparin, or bivalirudin may have additional benefits, such as improving reperfusion, prolonging the treatment window, preventing reocclusion, and preventing microvascular thrombosis. Administration of I-EN-adenosine triphosphatase can enhance efficacy and reduce thrombolytic agent ( TNKase ^™ , vampire bat plasminogen activator, urokinase, streptokinase, staphylococcal kinase and ancrolase). An operable fusion polypeptide between, for example, ADP-enhanced apyrase and a thrombolytic agent could provide an ideal therapy for acute myocardial infarction (AMI), percutaneous coronary intervention (PCI) and acute ischemic stroke (AIS). treatment plan.

Dosage requirements for I-EN- apyrase vary significantly depending on age, race, weight, height, sex, duration of treatment, method of administration, severity of the condition, or other clinical variables. Effective dosages can be determined by an experienced physician or other experienced medical personnel.

Citations provided herein are incorporated by reference for the subject matter they cite.

Preparation A

Constructs for producing enhanced apyrase and CHO cells transformed therewith

Based on sol-CD39L3R67G T69R, an apyrase construct encoding EN-adenylate was designed. This I-EN-adenosine triphosphatase and its coding sequence are shown below as SEQ ID NO:1.

The signal sequence is bovine alpha-lactalbumin signal peptide. The upstream sequence in the vector for expression of SEQ ID NO: 1 is SEQ ID NO: 2.

The downstream sequence has SEQ ID NO:3.

The complete construct for insertion is SEQ ID NO:4.

The following construct shown as SEQ ID NO: 4 was inserted into a retroviral vector and was referred to as "APT" in the resulting vector shown in Figure 1.

The positions of the MfeI and XhoI restriction sites used to clone the DNA fragments into the host plasmid are shown. The first 19 codons encode a signal peptide. During DNA sequencing of the final construct, a silent mutation (AAC instead of the predicted AAT) was detected at position 2879. This codon is double underlined. The final vector is called oCS-APT-WPRE (new ori).

Chinese hamster ovary (CHO) production cell lines were produced by two rounds of transduction of the CHO parental cell line with the above retroviral vectors shown in Figure 1 . The confluent population of transduced cells was designated sCHO-S/sC-ATP-R2X. After each transduction, a sample of the confluent cell line population was cryopreserved. Confluent sCHO-S/sC-ATP-R2X cell populations were diluted to very low cell densities (approximately 0.5 or 0.75 viable cells/200 μL medium) and seeded into 96-well microtiter plates to establish single cell origin clonal cell lines. After 14 days of culture, a total of 560 clones were screened for EN- apyrase production by the malachite green test. Twenty-four of the best clones generated based on EN- apyrase were expanded from 96-well plates to 24-well plates. Twenty of the 24 clones survived the expansion and were cryopreserved.

These twenty clones were screened for productivity in triplicate T175 flasks. The top five clones selected were clones 176, 248, 290, 350 and 372. Selected sCHO-S/sC-ATP-R2X clonal cell lines were tested negative for replication-competent retroviruses (RCR), mycoplasma contamination, and bioburden.

Preparation B

Cell Culture Conditions for Improved Protein Yield in a 10L Bioreactor

Background : Preliminary studies have shown that The medium yielded higher yields and better glycosylation of EN- apyrase in shake flasks. Therefore, this was used in a 10L bioreactor.

Materials and Methods : Cell line CHO-S-APT-R clone #350 was passaged every 3-4 days during exponential growth for scale-up in a 10L Braun bioreactor. exist Cells were seeded into a 10 L bioreactor at a cell density of approximately 300,000-400,000 cells/ml in (Lonza) medium. The fed batches used for this study were HyClone ^™ PS307 (12% (w/v) solution), AGT CD CHO5X Feed Medium Complete (Invitrogen), AGT CD CHO5X Feed Medium Complete + 12.5 g/L Galactose (Invitrogen), 45% glucose solution, 20% glucose/galactose solution, 200mM L-glutamine, 50X solution of L-asparagine (15g/L)/L-serine (10g/L), L-tyrosine (4g /L)/L-cystine (2g/L) 50X solution.

Condition A

D0: 3g/L PS307+2mM glutamine

D1: Maintain glutamine at 2mM; maintain glucose at 5g/L

D2: 3g/L PS307+glucose (to 5g/L)+glutamine (to 2mM)

D3: Maintain glutamine at 2mM; maintain glucose at 5g/L

D4: 3g/L R15.4/PS307 (1:1) + glutamine (to 2mM) + glucose (to 5g/L)

D5: Maintain glutamine at 2mM; maintain glucose at 5g/L

D5: Change the temperature to 34°C

D6: 12g/L R15.4

D6: Maintain glutamine at 2mM; maintain glucose at 4g/L

D7-D16: Maintain glutamine at 2mM; maintain glucose at 4g/L

The pH was 7.4 throughout the run

Cells were harvested on day 17 and had a protein level of 51 mg/l at harvest. Lactate levels were about 6g/l high and ammonium levels stayed below 10mM.

Preparation C

Purification of EN-adenosine triphosphatase with improved recovery

Media from cells cultured as described in Preparation B were harvested through a 1.4ft ² 60M02Dpeth Filter (Cuno, CT). Filters were washed with WFI water and blown out with compressed air before use to maximize volumetric recovery. The clarified medium was then filtered through a 0.2 μm filter and collected in sterile bags.

For virus inactivation, the load (100 mL of clarified medium, V19) was diluted with an equal volume (100 mL) of Milli-Q water. join in X-100 solution (20 mL, 11%) (1% final) and the resulting solution was incubated at ambient temperature for 30 minutes.

Anion exchange chromatography . EN-adenosine tris-phosphatase virus-inactivated medium (220 mL) was applied at a flow rate of 5 ml/min to 5 mL ANX Sepharose FF equilibrated with 10 mM Tris-HCl, pH 7.4 (GE Healthcare )column. A load was applied to the column, and the flow through plus wash (260 mL) was collected. The protein was eluted with 10 mM Tris, 230 mM NaCl, pH 7.4 and collected (50 mL). There was no significant loss of apyrase from anion exchange chromatography by skipping the wash step and eluting directly with 230 mM NaCl. Finally, the remaining protein on the column was washed with 1M NaCl, and this eluate was also collected (30 mL). Western blot analysis of the 1M NaCl band showed that little apyrase was detected in this fraction.

Buffer exchange and cation exchange chromatography . The collected ANX2 30 mM elution volume was buffer exchanged (approximately 3 volumes) into 20 mM citrate, pH 4.6, through a 1 L G25 column. The buffer-exchanged load (150 mL) was applied to a 5 mL column of SP-Sepharose FF (GE Healthcare) equilibrated in 20 mM citrate, pH 4.6, and the flow-through and wash (170 mL) were collected. A washing step was performed with 20 mM citrate, pH 4.8, and the washings (40 mL) were collected. An additional achievement was the removal of low molecular weight impurities by lowering the pH of the flow-through from 4.8 to 4.6. Although some apyrase was present in the pH 4.8 eluate, the total amount was less than 10% of the pH 4.6 flow-through. Another washing step was performed with 20 mM citrate, pH 5.1, and the washings (40 mL) were collected. Residual protein on the column was washed with 20 mM citrate, pH 6.0, and this wash was also collected (40 mL).

Purity was analyzed on a 4-12% SDS-PAGE gel after 20X concentration using an Omega 3kDa 2ml centrifugal concentrator.

With this purification scheme, nearly all of the heterogeneously glycosylated EN-adenylate was collected by ANX chromatography while omitting the 120 mM and/or 140 mM NaCl wash step. Also, a higher purity of apyrase (>95%) was additionally obtained by reducing the pH by 0.2 units in the SP chromatography. Through this two-step purification, the overall recovery of apyrase was greater than 90%.

Example 1

Optimization of cell culture conditions for improved glycosylation and sialylation

Background : Preparative C studies showed that in a 10L bioreactor maintained at pH 7.4, The medium resulted in good glycosylation of EN- apyrase. The new condition (D) was run in three 10L bioreactors.

Materials and Methods : Cell line CHO-S-APT-R clone #350 was passaged every 3-4 days during exponential growth for scale-up production in a 10L Braun bioreactor. exist Cells were seeded into three 10 L bioreactors at a cell density of approximately 300,000-400,000 cells/ml in (Lonza) medium. The feed strategy, pH and bioreactor set points are listed below. The harvest is about 50%.

Condition D.

D0: 3g/L PS307

D1: Maintain glutamine at 2mM; maintain glucose at 5g/L

D2: 3g/L PS307+glucose (to 5g/L)+glutamine (to 2mM)

D3: Maintain glutamine at 2mM; maintain glucose at 5g/L

D4: 3g/L R15.4/PS307 (1:1) + glutamine (to 2mM) + glucose (to 5g/L)

D5: Maintain glutamine at 2mM; maintain glucose at 5g/L

D5: Change the temperature to 34°C

D6: 12g/L R15.4

D6: Maintain glutamine at 2mM; maintain glucose at 4g/L

D7-D16: Maintain glutamine at 2mM; maintain glucose at 4g/L

The pH was 7.4 throughout the run

Results : The peak density of viable cells from Condition D peaked as follows: Vessel 1, ^48.5 x 105 cells/mL, Vessel ² , 45.2 x 105 cells/mL and Vessel ³ , 44.1 x 105 cells/mL.

The 2-column process of Prep C was performed to purify this improved I-EN-adenosine triphosphatase to homogeneity such that the bioburden of the purified I-EN-adenosine triphosphatase was <2.0 CFU/mL (Pass), and the endotoxin in the formulated bulk was determined to be 2.0<X<4.0EU/mL or 0.6<X<1.2EU/mg. As shown in Figure 2, production was stable through 42 passages.

Multi-angle light scattering analysis coupled to HPLC-size exclusion chromatography indicated that the molecular weight of I-EN-adenosine triphosphatase under condition D (70.4 kD) was higher than the molecular weight of 67.6 kD under condition A of preparation B ( image 3). After deglycosylation, the molecular weights became the same (Figure 4).

The glycosylation composition between protein samples produced under conditions A and D thus differed quantitatively. Although the contents of Man6, Man7, G2FS1 were comparable, the protein produced under condition D contained higher contents of Man5, Man8, G2FS2 and obtained acidic and more complex glycan structures. Therefore, the ratio of Mannose 5+Mannose 8+G2FS2/Mannose 6+Mannose 7+G2FS1 is higher. A comparison of the glycosylation patterns of prior art EN- apyrase and I-EN- apyrase is shown in FIG. 5 .

Example 2

Extended half-life and improved pharmacodynamics

Pharmacokinetic studies were performed in dogs in which a single bolus of EN-adenosine or I-EN-adenosine was administered intravenously. Serum samples were tested for ADPase activity. Six hours after administration of EN- apyrase under condition A, approximately 60-80% of the apyrase activity was cleared from circulation. In contrast, I-EN- apyrase under condition D retained 100% activity during this time period (Figure 6). As indicated, I-EN-adenosine triphosphatase has a half-life of more than 23 hours in dogs.

Claims (4)

1. one kind is improved the method for the biological property of EN-apyrase, the method comprises and postpones glutamine to the interpolation in the substratum of Chinese hamster ovary celI until cultivate after one day, described Chinese hamster ovary celI is modified with the expression construct containing described EN-apyrase, and wherein said expression construct comprises SEQ ID NO:4.

2. the process of claim 1 wherein that the biological property of described improvement is selected from:

A level of glycosylation that () is higher;

B glycosylation that () is more complicated;

C biological half time that () is longer; With

(d) its combination.

3. the EN-apyrase (I-EN-apyrase) improved, described EN-apyrase molecule wherein more than 80% and homologue have the identical N-end started with ELVP, and have identical with SEQ ID NO:180% or relative to SEQ ID NO:1 only containing the sequence of 1-5 preservative replacement, wherein said I-EN-apyrase has the transformation period of at least 23 hours in dog; And/or

It has the molecular weight of at least 70kD; And/or

Its glycosylation pattern has the seminose 5+ seminose 8+G2FS2/ seminose 6+ seminose 7+G2FS1 ratio of raising.

4. the EN-apyrase of claim 3, it has the glycosylation pattern shown in Fig. 5.