Dissolved adenosine triphosphate biphosphatase, methods and uses

Abstract

The present invention relates to the design and therapeutic use of lysed adenosine triphosphate biphosphatase polypeptides, pharmaceutical compositions, therapeutic use and methods for the prevention and treatment of tissue damage.

Description

[0001] This application is a divisional application of PCT application PCT / IB2019 / 056117, filed on July 17, 2019, with the invention name “Dissolved adenosine triphosphatase, methods and uses”. The date on which the PCT application entered the Chinese national phase is January 15, 2021, and the application number is 201980047594.2. Technical Field

[0002] The present invention relates to the design and therapeutic use of solubilized apyrase polypeptides, pharmaceutical compositions, and methods for preventing and treating tissue damage. Background Art

[0003] Adenosine diphosphatase (ATP-diphosphatase, adenosine diphosphatase, ADPase, or ATP diphosphohydrolase) is a plasma membrane-bound enzyme group that is active against both diphosphate and triphosphate nucleotides (NDP and NTP), and hydrolyzes NTP to nucleotide monophosphate (NMP) with NDP as an intermediate in two different continuous phosphate release steps. Most of the external ATPases that occur on the cell surface and hydrolyze extracellular nucleotides belong to this enzyme family. They are different from the ATPases that specifically hydrolyze ATP by hydrolyzing both ATP and ADP.

[0004] The first known human apyrase (ectonucleoside triphosphate diphosphohydrolase-1 (gene: ENTPD1, ​​protein: NTPDase 1), also known as cluster of differentiation 39 (CD39, UniProt P49961, or SEQ ID NO: 1)) is a cell surface enzyme with an extracellularly facing catalytic site.

[0005] In the known human CD39 family, member CD39L3 is known as an external-adenosine triphosphodiesterase (external-ATPD enzyme), which has biochemical activity between CD39 and CD39L1 (external-ATPase). Specifically, human CD39L3 has been solubilized and purified for therapeutic purposes, for example, as disclosed in US 7247300 B1 (incorporated herein by reference) or included herein as SEQ ID NO: 3. Summary of the invention

[0006] The present disclosure is based, inter alia, on the unexpected discovery that certain modifications of soluble human apyrase, such as human CD39, result in proteins with surprising activity that are nevertheless safe and easy to manufacture.

[0007] According to a first aspect of the present invention, there is provided a solubilized human apyrase having at least two modifications selected from the list consisting of: an N-terminal deletion, a C-terminal deletion and a central modification.

[0008] In one embodiment, the solubilized human apyrase comprises an N-terminal deletion, a C-terminal deletion, and a modified deletion.

[0009] In one embodiment, the central modification comprises a deletion of one or more amino acids. In another embodiment, the central modification comprises a point mutation of one or more amino acids, such as a substitution mutation. In yet another embodiment, the central modification is a combination of a deletion of one or more amino acids and a point mutation of one or more amino acids (such as a substitution mutation).

[0010] The N-terminal deletion may be between 30 and 50 amino acids deleted from the N-terminus of the wild-type CD39 sequence according to SEQ ID NO: 1, such as a deletion of 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 amino acids. In a preferred embodiment, the N-terminal deletion is 34, 37, 38 or 45 amino acids.

[0011] The C-terminal deletion may be between 20 and 40 amino acids deleted from the C-terminus of the wild-type CD39 sequence according to SEQ ID NO: 1, such as a deletion of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 amino acids. In a preferred embodiment, the C-terminal deletion is 22, 29 or 37 amino acids.

[0012] The central deletion may be between 10 and 15 consecutive amino acids deleted from the wild-type CD39 sequence according to SEQ ID NO: 1, such as a deletion of 10, 11, 12, 13, 14 or 15 amino acids. In a preferred embodiment, the central deletion is 12 amino acids, such as amino acid numbers 193 to 204 relative to the wild-type CD39 sequence according to SEQ ID NO: 1.

[0013] In one embodiment, the lysed human apyrase comprises one, two, three, four or five point mutations relative to the wild-type CD39 sequence according to SEQ ID NO: 1 selected from the group consisting of K71E, N73Q, V95A, G102D, Y104S, T106S, R113M, L149M, V151A, E173D, T229A, L254M, K258R, W263R, E276D, N292Q, R304G, I319T, N327Q, A362N, F365S, N371Q, K405N, Y412F, L424Q, H436D, I437N, F439S, G441D, N457Q, P463S, and S469R.

[0014] In one embodiment, the solubilized human apyrase comprises a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:32, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:70, SEQ ID NO:76, and SEQ ID NO:78.

[0015] In one embodiment, the solubilized human apyrase comprises a sequence selected from the group consisting of SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, and SEQ ID NO:141.

[0016] In a specific embodiment, the solubilized human apyrase comprises a sequence selected from the group consisting of SEQ ID NO:213, SEQ ID NO:227, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:217, SEQ ID NO:209, SEQ ID NO:221, SEQ ID NO:72, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:211, SEQ ID NO:58 and SEQ ID NO:229.

[0017] In a specific embodiment, the solubilized human apyrase consists of a sequence selected from the group consisting of SEQ ID NO:213, SEQ ID NO:227, SEQ ID NO:219, SEQ ID NO:227, SEQ ID NO:217, SEQ ID NO:209, SEQ ID NO:221, SEQ ID NO:72, SEQ ID NO:215, SEQ ID NO:223, SEQ ID NO:211, SEQ ID NO:58 and SEQ ID NO:229.

[0018] In a preferred embodiment, the solubilized human apyrase comprises a sequence selected from the group consisting of SEQ ID NO:58, SEQ ID NO:72, and SEQ ID NO:229.

[0019] In one embodiment, the solubilized human apyrase comprises SEQ ID NO: 58. In one embodiment, the solubilized human apyrase comprises SEQ ID NO: 72. In one embodiment, the solubilized human apyrase comprises SEQ ID NO: 229.

[0020] In a preferred embodiment, the solubilized human apyrase consists of a sequence selected from the group consisting of SEQ ID NO:58, SEQ ID NO:72, and SEQ ID NO:229.

[0021] In one embodiment, the solubilized human apyrase consists of SEQ ID NO: 58. In one embodiment, the solubilized human apyrase consists of SEQ ID NO: 72. In one embodiment, the solubilized human apyrase consists of SEQ ID NO: 229.

[0022] According to a second aspect of the present invention, the present invention relates to a pharmaceutical composition comprising a therapeutically effective dose of the apyrase according to the first aspect of the present invention, and one or more pharmaceutically acceptable carriers are provided.

[0023] In one embodiment, the pharmaceutical composition further comprises one or more additional active ingredients.

[0024] According to a third aspect of the present invention there is provided an isolated apyrase according to the first aspect for use as a medicament.

[0025] According to a fourth aspect of the present invention, there is provided an isolated apyrase according to the first aspect for use in treating tissue damage.

[0026] The tissue injury can be acute brain injury (stroke); acute multiple organ failure; delayed graft function following kidney or other solid organ transplantation; burns; radiation injury; acute injury due to trauma and / or hypoxia, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury, such as acute kidney injury secondary to thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis or toxic effects of antibiotics or other drugs; acute myocardial injury.

[0027] In another embodiment, a fourth aspect of the present disclosure relates to an isolated apyrase according to the first aspect of the present invention for use in treating acute kidney injury associated with cardiac surgery.

[0028] In another embodiment, the fourth aspect of the disclosure relates to an isolated pyrase according to the first aspect of the invention for use in treating delayed graft function (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic stroke (AIS), ischemia-reperfusion injury (IRI), or a combination thereof commonly referred to as multiple organ failure (MOF).

[0029] In one embodiment, the lytic human apyrase for use in treating cardiac surgery-related acute kidney injury comprises the amino acid sequence of SEQ ID NO:58.

[0030] In one embodiment, the lytic human apyrase for treating acute kidney injury associated with cardiac surgery comprises the amino acid sequence of SEQ ID NO: 72. In one embodiment, the lytic human apyrase for treating acute kidney injury associated with cardiac surgery comprises the amino acid sequence of SEQ ID NO: 229.

[0031] In a further preferred embodiment, the disclosure relates to the use of the isolated apyrase according to the first aspect of the invention for treating sepsis-related acute kidney injury.

[0032] In one embodiment of the fourth aspect, the lytic human apyrase for use in treating sepsis-related acute kidney injury comprises the amino acid sequence of SEQ ID NO:58.

[0033] In one embodiment of the fourth aspect, the lytic human apyrase for treating sepsis-related acute kidney injury comprises the amino acid sequence of SEQ ID NO:72.

[0034] In one embodiment of the fourth aspect, the lytic human apyrase for treating sepsis-related acute kidney injury comprises the amino acid sequence of SEQ ID NO:229.

[0035] According to a fifth aspect of the invention, there is provided a method of treating tissue damage in a human subject, the method comprising administering to the subject a therapeutically effective dose of a solubilized human apyrase according to the first aspect. An embodiment of the fifth aspect of the invention relates to a method of treating acute kidney injury associated with cardiac surgery, the method comprising administering to a subject in need of such treatment a therapeutically effective dose of an isolated apyrase according to the first aspect of the invention.

[0036] Another embodiment of the fifth aspect of the invention relates to a method of treating delayed graft function (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic stroke (AIS), ischemia-reperfusion injury (IRI), or a combination thereof commonly referred to as multiple organ failure (MOF), the method comprising administering to a subject in need of such treatment a therapeutically effective dose of an isolated apyrase according to the first aspect of the invention.

[0037] In one embodiment of the fifth aspect, the soluble human apyrase for use in the method of treating cardiac surgery-related acute kidney injury comprises the amino acid sequence of SEQ ID NO:58, SEQ ID NO:72 or SEQ ID NO:229.

[0038] One embodiment of the fifth aspect of the invention relates to a method of treating sepsis-related acute kidney injury, the method comprising administering to a subject in need of such treatment a therapeutically effective dose of an isolated apyrase according to the first aspect of the invention.

[0039] In one embodiment of the fifth aspect, the lysed human apyrase for use in the method of treating sepsis-associated acute kidney injury comprises the amino acid sequence of SEQ ID NO: 58, SEQ ID NO: 72, or SEQ ID NO: 229. The tissue injury can be acute brain injury (stroke); acute multiple organ failure; delayed graft function after kidney or other solid organ transplantation; burns; radiation injury; acute injury due to trauma and / or hypoxia, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury, such as acute kidney injury secondary to thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis or toxic effects of antibiotics or other drugs; acute myocardial injury.

[0040] According to a sixth aspect of the present invention there is provided an isolated nucleic acid molecule encoding any apyrase according to the first aspect.

[0041] According to a seventh aspect of the present invention, there is provided a cloning vector or an expression vector comprising one or more nucleic acid sequences according to the sixth aspect, wherein the vector is suitable for the recombinant production of the isolated apyrase according to the first aspect.

[0042] According to an eighth aspect of the present invention, there is provided a host cell comprising one or more cloning vectors or expression vectors according to the seventh aspect.

[0043] According to a ninth aspect of the present invention, there is provided a method for producing the apyrase according to the first aspect, the method comprising culturing the host cell according to the eighth aspect, purifying and recovering the apyrase.

[0044] According to the present invention, the following specific technical solutions are also disclosed.

[0045] Embodiment 1. A solubilized human apyrase having at least two modifications selected from the list consisting of: an N-terminal deletion, a C-terminal deletion, and a central modification.

[0046] Embodiment 2. The solubilized human apyrase as described in embodiment 1, wherein the solubilized human apyrase has an N-terminal deletion, a C-terminal deletion and a central modification.

[0047] Embodiment 3. The solubilized human apyrase of embodiment 1 or 2, wherein

[0048] a) the N-terminal deletion is between 30 and 50 amino acids in length,

[0049] b) the C-terminal deletion is between 20 and 40 amino acids in length, and

[0050] c) The central modification comprises a deletion and / or a point mutation.

[0051] Embodiment 4. The solubilized human apyrase according to any of the preceding embodiments, wherein the central modification comprises a deletion of 10 to 15 consecutive amino acids.

[0052] Embodiment 5. The lytic human apyrase according to embodiment 4, wherein the central deletion is a deletion of amino acid numbers 193 to 204 relative to the wild-type CD39 sequence according to SEQ ID NO:1.

[0053] Embodiment 6. The solubilized human apyrase according to any of the preceding embodiments, wherein the central modification comprises a point mutation comprising a NO:1, one, two, three, four or five point mutations related to the wild-type CD39 sequence, wherein the point mutations are selected from the group consisting of K71E, N73Q, V95A, G102D, Y104S, T106S, R113M, L149M, V151A, E173D, T229A, L254M, K258R, W263R, E276D, N292Q, R304G, I319T, N327Q, A362N, F365S, N371Q, K405N, Y412F, L424Q, H436D, I437N, F439S, G441D, N457Q, P463S, and S469R.

[0054] Embodiment 7. The solubilized human apyrase of any of the preceding embodiments, comprising a sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:32, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:70, SEQ ID NO:76, and SEQ ID NO:78.

[0055] Embodiment 8. The solubilized human apyrase of any of the preceding embodiments, comprising a sequence selected from the group consisting of SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, and SEQ ID NO: 141.

[0056] Embodiment 9. The soluble human apyrase of embodiment 8, comprising or consisting of a sequence selected from the group consisting of SEQ ID NO: 213, SEQ ID NO: 227, SEQ ID NO: 219, SEQ ID NO: 225, SEQ ID NO: 217, SEQ ID NO: 209, SEQ ID NO: 221, SEQ ID NO: 72, SEQ ID NO: 215, SEQ ID NO: 223, SEQ ID NO: 211, SEQ ID NO: 58, and SEQ ID NO: 229.

[0057] Embodiment 10. The soluble human apyrase of embodiment 9, wherein the soluble human apyrase consists of a sequence selected from the group consisting of SEQ ID NO:58, SEQ ID NO:72, and SEQ ID NO:229.

[0058] Embodiment 11. A pharmaceutical composition comprising a therapeutically effective dose of the apyrase of any one of embodiments 1 to 10, and one or more pharmaceutically acceptable carriers.

[0059] Embodiment 12. The pharmaceutical composition of embodiment 11, further comprising one or more additional active ingredients.

[0060] Embodiment 13. An isolated apyrase according to embodiments 1 to 10 for use as a medicament.

[0061] Embodiment 14. An isolated apyrase according to embodiments 1 to 10 for use in treating tissue damage.

[0062] Embodiment 15. An isolated apyrase for use according to embodiment 14, wherein the tissue injury is acute brain injury (stroke); acute multiple organ failure; delayed graft function after kidney or other solid organ transplantation; burns; radiation injury; acute injury due to trauma and / or hypoxia, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury, such as acute kidney injury secondary to thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis or toxic effects of antibiotics or other drugs; acute myocardial injury.

[0063] Embodiment 16. An isolated apyrase according to embodiments 1-10 for use in treating acute kidney injury associated with cardiac surgery.

[0064] Embodiment 17. An isolated apyrase as described in embodiments 1-10 for use in treating delayed graft function (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic stroke (AIS), ischemia-reperfusion injury (IRI), or a combination thereof commonly referred to as multiple organ failure (MOF).

[0065] Embodiment 18. An isolated apyrase according to embodiments 1-10 for use in treating sepsis-related acute kidney injury.

[0066] Embodiment 19. A method of treating tissue damage in a human subject, the method comprising administering to the subject a therapeutically effective dose of solubilized human apyrase.

[0067] Embodiment 20. The method of embodiment 19, wherein the solubilized human apyrase is the apyrase of embodiments 1 to 10.

[0068] Embodiment 21. A method as described in embodiment 19 or 20, wherein the tissue injury is acute brain injury (stroke); acute multiple organ failure; delayed graft function after kidney or other solid organ transplantation; burns; radiation injury; acute injury due to trauma and / or hypoxia, such as acute respiratory distress syndrome (ARDS) or lung injury; acute kidney injury, such as acute kidney injury secondary to thoracic surgery (e.g., aortic valve replacement, coronary artery bypass surgery) or sepsis or rhabdomyolysis or toxic effects of antibiotics or other drugs; acute myocardial injury.

[0069] Embodiment 22. A method of treating acute kidney injury associated with cardiac surgery, the method comprising administering to a subject in need of such treatment a therapeutically effective dose of solubilized human apyrase.

[0070] Embodiment 23. A method for treating delayed graft function (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic stroke (AIS), ischemia-reperfusion injury (IRI), or a combination thereof commonly referred to as multiple organ failure (MOF), the method comprising administering to a subject in need of such treatment a therapeutically effective dose of solubilized human apyrase.

[0071] Embodiment 24. A method of treating sepsis-related acute kidney injury comprising administering to a subject in need of such treatment a therapeutically effective dose of solubilized human apyrase.

[0072] Embodiment 25. An isolated nucleic acid molecule encoding any apyrase as described in embodiments 1 to 10.

[0073] Embodiment 26. A cloning vector or expression vector comprising one or more nucleic acid sequences as described in embodiment 25, wherein the vector is suitable for recombinant production of an isolated apyrase as described in embodiments 1 to 10.

[0074] Embodiment 27. A host cell comprising one or more cloning vectors or expression vectors according to embodiment 26.

[0075] Embodiment 28. A method for producing the apyrase of any one of embodiments 1 to 10, the method comprising culturing the host cell of embodiment 27, purifying and recovering the apyrase. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 is a sequence alignment;

[0077] Figure 2A is a representation of the expression level of supernatant containing human CD39 by anti-APP Western blotting according to the Examples;

[0078] Figure 2B is a representation of the supernatant expression levels of human CD39 variants containing cysteine ​​bridge deletions by anti-APP Western blotting according to the Examples;

[0079] Figure 3 is a graph showing the results of solid phase ATPase assay of CD39 variants;

[0080] Figure 4 is a graph showing solid phase ATP cleavage on HEK293 cells transformed with human CD39 variants according to the Examples.

[0081] Figure 5 is a schematic overview of a carrier according to an embodiment;

[0082] Figure 6 It is an enzyme model based on the steady-state approximation;

[0083] Figure 7 is an overview of protein kinetic data and model fitting according to the examples;

[0084] Figure 8 is an overview of protein kinetic data and model fitting according to the examples;

[0085] Fig. 9 is an overview of protein kinetic data and model fitting according to the examples;

[0086] FIG10 is a schematic diagram of the experimental conditions;

[0087] Fig.11 is a graph showing AMP levels of proteins according to an embodiment. and

[0088] FIG. 12 is a graph showing in vivo results of proteins according to examples. DETAILED DESCRIPTION

[0089] The present disclosure is based, inter alia, on the unexpected discovery that certain modifications of solubilized CD39 result in a protein with surprising activity that is safe and easy to manufacture.

[0090] As shown in the specific examples below, a preferred embodiment is a solubilized human apyrase having at least two modifications selected from the list consisting of: N-terminal deletion, C-terminal deletion, and central deletion, such as a solubilized human apyrase comprising the following sequence, the sequence being selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:32, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:70, SEQ ID NO:76, and SEQ ID NO:78.

[0091] The inventors have tried several different sequence modification strategies to obtain solubilized human apyrase with both retained activity and the ability to be expressed, while still not introducing too many modifications because of the risk of increased immunogenicity and therefore increased safety risk. Surprisingly, one sequence modification that was found to increase both the efficiency and the ability to express human apyrase, while not adding too much immunogenicity risk, was the deletion of the central part, the so-called delta MIL (ΔMIL) modification.

[0092] In order to increase the expression of dissolved human apyrase according to the embodiments of the present invention, N-terminal expression tags were tested. Various N-terminal expression tags are known in the art, but surprisingly not all tags work. The inventors found that several tags work, which was not foreseeable.

[0093] These N-terminal tags are SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139 or SEQ ID NO: 141. As indicated herein, particularly preferred tags are SEQ ID NO: 133, SEQ ID NO: 135 or SEQ ID NO: 137.

[0094] Specific details are set forth below in Examples 9 to 13. However, to demonstrate the unpredictable nature of these examples, a comparative summary is presented in Table 1.

[0095] Table 1. Summary of preferred embodiments.

[0096]

[0097]

[0098] 1. Definition

[0099] To facilitate those skilled in the art to carry out the present invention, the following terms are used throughout the specification.

[0100] The terms "CD39" and "hCD39" are used synonymously throughout the disclosure and, unless otherwise indicated, refer to human cluster of differentiation 39 (CD39) according to UniProt P49961 or SEQ ID NO: 1.

[0101] Unless otherwise indicated, the term "apyrase" refers to human apyrase. As used herein, "soluble apyrase" means that the cell membrane-bound apyrase present as a wild-type protein has been modified so that it is no longer associated with the cell membrane but exists in a soluble state, i.e., is no longer anchored to the cell membrane.

[0102] The abbreviation "MIL" refers to the membrane interaction loop, which is the central part of the wild-type (human) CD39 protein that interacts with the cell membrane, in addition to the N-terminal and C-terminal parts that are physically anchored by the cell membrane. The term "delta MIL (delta MIL or ΔMIL)" refers to a deletion of the MIL sequence from wild-type (human) CD39.

[0103] The term "about" in relation to a numerical value x means, for example, + / - 10%. When used before a numerical range or a list of numbers, the term "about" applies to each number in the series, for example, the phrase "about 1-5" should be interpreted as "about 1-about 5", or, for example, the phrase "about 1, 2, 3, 4" should be interpreted as "about 1, about 2, about 3, about 4, etc.".

[0104] The word "substantially" does not exclude "completely", for example, a composition "substantially free of" Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the present disclosure.

[0105] The term "comprising" encompasses "including" as well as "consisting of," for example, a composition "comprising" X may consist of X alone or may include something else, such as X+Y.

[0106] "Identity" with respect to natural polypeptides and their functional derivatives is defined herein as the percentage of amino acid residues in a candidate sequence that are identical to the residues of a corresponding natural polypeptide, after the sequences have been aligned and gaps introduced (if necessary) to achieve the maximum percent identity, and without considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions nor insertions should be construed as reducing the identity. Methods and computer programs for alignment are well known. Percent identity can be determined by standard alignment algorithms, such as the Basic Local Alignment Search Tool (BLAST) described by Altshul et al. ((1990) J. Mol. Biol. [Journal of Molecular Biology], 215: 403 410); Needleman et al.'s algorithm ((1970) J. Mol. Biol. [Journal of Molecular Biology], 48: 444 453); or Meyers et al.'s algorithm ((1988) Comput. Appl. Biosci. [Computer Applications in Biological Sciences], 4: 11 17). One set of parameters can be a Blosum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0107] "One or more amino acids" refers to, for example, all naturally occurring L-α-amino acids and includes D-amino acids. The phrase "amino acid sequence variant" refers to a molecule having some differences in its amino acid sequence compared to a sequence according to the present disclosure. According to the amino acid sequence variants of the protein (e.g., a specified sequence) disclosed herein, it still has apyrase activity. Amino acid sequence variants include substitution variants (those variants in which at least one amino acid residue is removed and different amino acids are inserted at the same position in the polypeptide according to the present disclosure), insertion variants (those variants in which one or more amino acids are inserted next to the amino acid at a specific position in the polypeptide according to the present disclosure) and deletion variants (those variants in which one or more amino acids are removed in the polypeptide according to the present disclosure).

[0108] The term "treatment" or "treat" is defined herein as the application or administration of a pyrase according to the invention to a subject, or the application or administration of a pharmaceutical composition comprising the pyrase to a subject or an isolated tissue or cell line from a subject, wherein the subject has tissue damage, symptoms associated with tissue damage, with the purpose of alleviating, alleviating or ameliorating tissue damage or any symptoms associated with tissue damage, in particular by reducing the level of extracellular ATP.

[0109] "Treatment" also refers to the application or administration of a pharmaceutical composition comprising apyrase to a subject, or the application or administration of a pharmaceutical composition comprising apyrase of the invention to an isolated tissue or cell line from a subject, wherein the subject has tissue damage or symptoms associated with tissue damage, with the purpose of alleviating, alleviating or ameliorating the tissue damage or any symptoms associated with tissue damage.

[0110] The terms "prevent" or "preventing" refer to prophylactic or "preventative" treatment; which is concerned with delaying or preventing the onset of a disease, disorder, and / or symptoms associated therewith.

[0111] As used herein, a subject is "in need of" a treatment if the subject would benefit biologically, medically, or in quality of life from such treatment.

[0112] The term "pharmaceutically acceptable" refers to non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient or ingredients.

[0113] As used herein, the term "administration" or "administering" of a subject compound means providing a compound of the invention and its prodrugs to a subject in need of treatment. Administration "in combination" with one or more other therapeutic agents includes simultaneous (concurrent) and sequential administration in any order and by any route of administration.

[0114] As used herein, a "therapeutically effective dose" refers to a dose (amount) of apyrase that, when administered in a single dose or multiple doses to a patient (e.g., a human), is effective to treat, prevent, prevent onset, cure, delay, reduce severity, alleviate at least one symptom of a disorder or recurrent disorder, or prolong the patient's survival beyond the expected survival in the absence of such treatment. When applied to an individual active ingredient (e.g., apyrase) administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to the combined dose or amount of the active ingredients (whether administered serially or simultaneously in combination) that produces a therapeutic effect.

[0115] The phrase "dosage regimen" means a regimen used to treat a disease, such as a dosing regimen used during the treatment of tissue damage.

[0116] The phrase "administration device" is used to refer to any useful device for administering a drug systemically to a patient, including but not limited to pre-filled syringes, vials and syringes, injection pens, autoinjectors, intravenous (iv) drip sets and bags, pumps, patch pumps, etc. Using these items, a patient can self-administer the drug (i.e., self-administer the drug), or the drug can be administered by a physician.

[0117] 2. Example 1: Membrane-free CD39

[0118] Wild-type human apyrase CD39 (hCD39, UniProt P49961 or SEQ ID NO: 1) is naturally anchored in the cell membrane by an N-terminal transmembrane domain (putative aa 17-37), a central putative membrane interaction loop (MIL putative aa 193-204) and a C-terminal transmembrane domain (putative aa479-499). In order to be able to express soluble variants of CD39 using mammalian host cells, several elements of the CD39 sequence have been modified to obtain membrane-free or soluble proteins. The native leader sequence and the N-terminal transmembrane region were replaced by a secretory leader sequence and a purification tag (SEQ ID NO: 133). The boundaries of the extracellular domain of CD39 have been altered to optimize expression, purification and activity parameters (amino acid numbers 38-476 of SEQ ID NO: 1, amino acid numbers 39-469 of SEQ ID NO: 1, amino acids 46-461 of SEQ ID NO: 1, and amino acids 46-476 of SEQ ID NO: 1, respectively). The effects of cysteine ​​and disulfide bridges on aggregation propensity and enzyme activity were systematically evaluated by replacing cysteine ​​with alanine or by shortening the loops formed by disulfide bridges (SEQ ID 107, 109, 111, 113 and 115). A stretch of hydrophobic amino acids was described in the structural work of rat CD39 (Zebisch et al., J. Mol. Biol. (2012), 415, 288-306, wild-type rat CD39, Uniprot P97687, listed in SEQ ID NO: 2), and it is thought that this loop may interact with the cell membrane (MIL). We translated the results into the human CD39 sequence by sequence alignment and generated CD39 variants with loop deletions (CD39ΔMIL or EP28, as listed in SEQ ID NO: 4). The effects of the deletion (or δ / Δ) of MIL on the expression level and thermal stability of functional CD39 were evaluated.

[0119] from Figure 1(sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 4 is shown) it can be seen that N-terminal amino acids 1 to 27, C-terminal amino acids 477 to 510 and central membrane interaction loop (MIL) amino acids 193 to 204 are deleted from wtCD39 (SEQ ID NO: 1) to form CD39ΔMIL (SEQ ID NO: 4).

[0120] The effects of different sequence modifications on thermal stability were studied. In addition, the effects of different sequence modifications on CHO cell expression yield and monomer content were studied.

[0121] (1) Methods

[0122] (a) Generation of expression plasmid

[0123] DNA sequences encoding different hCD39 border variants and membrane interaction loop (MIL) deletions were ordered from GeneArt (Life Technologies Inc., Regensburg, Germany), including codon optimization for Homo sapiens. Sequences encoding hCD39 variants were subcloned from GeneArt derived vectors or their in-house generated variants into expression vectors suitable for secretion in mammalian cells by standard molecular biology techniques. The cysteine ​​to alanine mutations present in the cysteine ​​bridge deletion variants were targeted by modified oligonucleotides, and after a subsequent assembly PCR step, the generated fragments were subcloned into the same expression vectors mentioned previously. Elements of the expression vector include a promoter (cytomegalovirus (CMV) enhancer-promoter), a signal sequence to promote secretion, a polyadenylation signal and a transcription terminator (bovine growth hormone (BGH) gene), elements that allow episomal replication and replication in prokaryotes (e.g., SV40 origin and ColE1 or other elements known in the art) and elements that allow selection (ampicillin resistance gene and zeocin marker). A list of truncated, solubilized versions of human CD39 is shown in Table 2, where the amino acid modifications are numbered with reference to SEQ ID NO: 1.

[0124] Table 2. Truncated, solubilized versions of human CD39.

[0125]

[0126]

[0127] (b) Microscale expression of hCD39 variants

[0128] 293-6E cells (as disclosed in WO 2006096989 A2, incorporated herein by reference) were selected for microscale experiments as one of the preferred host cell lines for transient expression of proteins in the absence of serum. FuGene HD (Roche Applied Science, catalog number 04709705001) was used as a transfection reagent for transfection. 293-6E cells were cultured in suspension culture using V3 serum-free medium (Bioconcept, catalog number V3-K) for transfection and propagation of cells. Cells were cultured in Corning shake flasks (Corning, Tewksbury, Massachusetts) on an orbital shaker (100-120 rpm) at 5% CO 2 The cells in the seed culture should be maintained in the exponential growth phase (cell density around 5 x 10 5 With 3x10 6 / mL) and exhibited >90% transfection activity. Cell densities outside this range will result in a lag phase after dilution or reduced transfection efficiency.

[0129] For microscale (0.5 ml) transfections, an aliquot of cells was removed from the seed culture and adjusted to 0.5 x 10 6 Cells / mL. A DNA solution (referred to as solution 1) was prepared by diluting 0.5 μg of hCD39 expression plasmid in 14 μl of V3 serum-free medium, and then 2.3 μl of FuGene HD solution was also diluted in 14 μl of V3 serum-free medium (solution 2). Both solutions were incubated at room temperature (RT) for 5-10 min. Thereafter, solution 2 was added to solution 1 under gentle mixing and incubated for another 5-15 minutes at room temperature. The transfection mixture was then added at 0.5 x 10 6 10 cells / mL were added to 0.5 ml of cells, seeded in 48-well tissue culture plates (Corning, Tewksbury, MA), and the plates were placed in an incubator with 5% CO. 2 The cells were cultured on an orbital shaker (300 rpm) in a humidified incubator at 4 ° C. The cultures were harvested 3 days after transfection by centrifugation at 4000 rpm for 10 minutes at 4 ° C. (Heraeus, Multifuge 3S-R, Thermo Scientific, Rockford, IL). The recovered cell supernatant was stored at 4 ° C until further processing.

[0130] (c) Western blot analysis of microscale expression supernatants

[0131] Western blot analysis of microscale expression supernatants was performed to check the expression and correct formation of recombinant hCD39 variants. 8 μl of supernatant was diluted on E-PAGE TM Loading buffer (4x, Invitrogen, #EPBNF-01) and loaded on E-Page48, 8% gel (Invitrogen, #EP04808) under non-reducing conditions. The gel was run on an E-base mother device (Invitrogen) for 23 min and the proteins were transferred to a nitrocellulose membrane (Invitrogen IB301001) using an iBlot system (Invitrogen) according to the manufacturer's instructions (run 7 min). After washing 3 times in TBS / 0.05Tween20 (TBST), the membrane was incubated with 5% milk / TBST under gentle agitation for 1 h, followed by incubation with a 4 μg / ml solution of anti-APP mouse antibody (Novartis in-house antibody, generated against a peptide fragment of amyloid precursor protein (APP) for protein labeling) diluted in 2% milk / TBST for 1 hr. After 3 additional washing steps, the membrane was incubated with a 1:1000 dilution of anti-mouse IgG-alkaline phosphatase (Sigma-Aldrich, A5153-1ML) diluted in 2% milk / TBST and washed again 3 times in TBST followed by a rinse step in TBS. SIGMAFAST TM (Sigma-Aldrich, #B5655-25TAB), develop the signal for 1-5 min, and stop the signal by washing the membrane with water.

[0132] (d) Solid-phase AxP enzyme assay

[0133] The activity of ATPase, ADPase and AMPase was determined on hCD39 variants captured from microscale expression supernatants (solid phase Axp enzyme assay) using the Pi ColorLock Gold phosphate detection system (Innova Biosciences, catalog number 303-0030). This method was found to be less sensitive than the manufacturer's recommended solution-based assay (liquid phase Axp enzyme assay), but has the advantage of reducing the AxP enzyme activity mediated by host cell enzymes that may be present in microscale expression supernatants. 20 μl of anti-APP mouse antibody 10 μg / ml solution antibody (Novartis internal antibody, produced against peptides of amyloid precursor protein (APP) for protein labeling) diluted in PBS was added to each well of a maxisorp 384-well transparent plate (Nunc) and incubated overnight at 4°C. After washing three times with TBST, the wells were blocked for 1 h at room temperature with 100 μl of 5% milk / TBST under gentle agitation. After three additional washing steps, 20 μl of microscale expression supernatant serially diluted in 2% milk / TBST was added to the wells in triplicate and incubated for 2 hr at room temperature with gentle agitation. The wells were then washed four more times with 100 μl of TBST and rinsed with 80 μl of 50 mM Tris-Cl / 5 mM MgCl. 2 Wash twice at pH 7.5. 2 30 μl of 80 μM adenosine phosphate solution diluted in pH 7.5 (ATP: Sigma A2383, ADP: Sigma A2754) was added to each triplicate and incubated at 37°C for 24 hr. The signal was developed for 10 minutes using 7.5 μl of gold reagent mixture prepared according to the manufacturer's instructions, and the reaction was stopped using 3 μl of stabilizer. The absorbance at 620 nm was read using a TECAN Genios Pro instrument.

[0134] (2) Results

[0135] (a) Effects of boundary, membrane interaction loop (MIL) deletion and cysteine ​​bridge deletion on hCD39 expression levels

[0136] To evaluate the expression levels of the different hCD39 variants, the corresponding expression plasmids were transfected in duplicate in 0.5 ml of 293-6E cells and Western blotting (anti-APP detection Ab) was performed on the supernatants collected 3 days after transfection. Figure 2A and Figure 2B Displayed in.

[0137] The results showed that hCD39 starting at aa38 expressed higher levels compared to aa46. The N-terminal boundary as well as the MIL deletion did not appear to have a major effect on the expression levels. Higher expression levels of hCD39 with deletion of the first or fourth cysteine ​​bridges were also observed in the context of hCD39(aa46-461). Higher expression levels with deletion of the first cysteine ​​bridge were also confirmed using the hCD39(aa46-461)ΔMIL backbone.

[0138] (b) Effects of boundary, membrane interaction loop (MIL) and cysteine ​​bridge deletion on hCD39 activity

[0139] CD39 enzyme activity was measured on the supernatant samples by solid phase AxP enzyme assay. Figure 3 and Figure 4 And shown in Table 3.

[0140] Table 3. Solid phase ATP assay performed on CD39 variants

[0141]

[0142]

[0143] The absence of MIL appears to increase the fraction of functionally expressed CD39 recombinant protein. The different boundaries do not show any significant effect on active hCD39 activity. The results indicate that the ATPase activity of all cysteine ​​bridge-deleted variants is strongly reduced or completely eliminated. Similar results were obtained using a solid phase ADPase assay. Therefore, surprisingly, the sequence modification that both improves efficiency and the ability to express CD39 is the δMIL (ΔMIL) modification.

[0144] 3. Example 2: Expression Tags

[0145] To improve the expression characteristics of the candidates, different expression tags were tested.

[0146] Different expression tags based on the N-terminal part of IL-2 (SEQ ID NO: 131) were tested as listed in Table 4. Expression tag 1-16aa was synthesized by Geneart according to SEQ ID NO: 131.

[0147] Table 4. Summary of IL-2 expression signature variants

[0148] refer to Amino Acid Sequence ID Forward primer sequence ID Reverse primer sequence ID Expression tags aa1-16 SEQ ID NO:131 n / a n / a Expression tags aa1-15 SEQ ID NO:133 SEQ ID NO:157 SEQ ID NO:158 Expression tags aa1-6 SEQ ID NO:135 SEQ ID NO:159 SEQ ID NO:158 Expression tags aa1-3 SEQ ID NO:137 SEQ ID NO:161 SEQ ID NO:158 Expression tags aa1-9 SEQ ID NO:139 SEQ ID NO:162 SEQ ID NO:158 Expression tags aa1-12 SEQ ID NO:141 SEQ ID NO:163 SEQ ID NO:158 Expression tag aa4-12 SEQ ID NO:143 SEQ ID NO:164 SEQ ID NO:158

[0149] All expression tags associated with CD39ΔMIL were tested as listed in SEQ ID NO: 4. All constructs included an APP tag and a His tag.

[0150] like Figure 5 Vector pRS5a was used for expression as listed in Table 4. Primer pairs were as listed in Table 4.

[0151] The annealing temperature was 64°C in all cases.

[0152] The PCR solution was prepared by mixing 1 μl template DNA stock, 25 μl Kapa High Fidelity Hot Start Polymerase (from Kappa Biosystems / KK2602), 1.5 μl forward primer, 1.5 μl reverse primer, and PCR with H 2 Adjust the final volume to 50 μl.

[0153] Run the PCR reactions according to the schedule in Table 5.

[0154] Table 5. PCR schedule

[0155]

[0156] After the PCR reaction is completed, use DNA extraction was performed using SV gel and PCR Clean-Up Kit (Promega, No. 9282, 1 column, elution in 30 μl).

[0157] Inserts and vectors were cut using enzymes NruI-HF (NEB#R3192) and NotI-HF (NEB#R3189) provided by New England Biolabs (NEB) in CutSmart(R) buffer. The reaction time was 3 hours at 37°C.

[0158] Overnight ligation was performed using the dephosphorylated vector from the Rapid DNA Dephosphorylation and Ligation Kit (Fa. Roche, No. 04898117001) according to the manufacturer's validated protocol.

[0159] The next day, single colonies were picked for DNA-miniprep and sequence analysis using forward primer P270 (SEQ ID NO: 165) and reverse primer P271 (SEQ ID NO: 166).

[0160] In addition, according to Table 6, some protein sequences known in the art for increasing expression were tested.

[0161] Table 6. Prior art labels

[0162] refer to Sequence Identifier Ubiquitin SEQ ID NO:167 Cκ SEQ ID NO:168 HSA domain I SEQ ID NO:169 HSA domain II SEQ ID NO:170

[0163] The resulting combinations tested are listed in Table 7.

[0164] Table 7. Constructs tested

[0165]

[0166]

[0167] None of the prior art tags from Table 6 gave expression of protein (data not shown). This was unexpected since the prior art taught that these sequences should increase expression.

[0168] 4. Example 3: Other mutations

[0169] To improve the characteristics of soluble CD39 and make it suitable for drug development, additional modifications were introduced into CD39ΔMIL, EP28 as set forth in SEQ ID NO: 4. The different mutations and mutant variants are found in Table 8 and are numbered according to the amino acid position of wild-type CD39 as set forth in SEQ ID NO: 1.

[0170] Table 8. Point mutations

[0171]

[0172]

[0173]

[0174] Two mutations in the active site resulted in higher activity (365 and 412).

[0175] 5. Example 4: Glycosylation site removal

[0176] Based on the above EP14 variants, the effect of glycosylation sites was examined by introducing point mutations according to Table 9, which are numbered according to the amino acid position of wild-type CD39 as listed in SEQ ID NO:1.

[0177] Table 9. Glycosylation site mutations

[0178] Mutation location Glycosylation site Primers Q7 NDT P928 / P929 T229A NQT P930 / P931 N292Q NVS P932 / P933 N327Q NTS P934 / P935 N371Q NLT P936 / P937 N457Q NLT P938 / P939

[0179] (a) Materials and methods

[0180] The expression vector pRS5a ( Figure 5 ) were used for cloning. Primers as listed in Table 10 were used.

[0181] Table 10. Primer sequences

[0182]

[0183]

[0184] PCR was performed using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent, No. 210519-5) according to the manufacturer's instructions.

[0185] The next day, single colonies were picked for DNA-miniprep and sequence analysis using forward primer P270 (SEQ ID NO: 165) and reverse primer P271 (SEQ ID NO: 166).

[0186] Likewise, to ensure the correctness of the vector backbone (due to mutagenesis), the sequenced insert fragment was cloned into pRS5a ( Figure 5 ) in a new vector backbone.

[0187] The vector was prepared using the vector backbone of SEQ ID NO: 36 (with expression tag SEQ ID NO: 135, containing APP_HIS-tag, stock concentration 3.3 μg / μl).

[0188] The precipitate was prepared by mixing 10 μg of vector-DNA, 0.4 μl of HindIII (100 U / μl, NEB), 2 μl of EcoRI (20 U / μl, NEB), 5 μl of Cutsmart buffer 10× concentration (NEB), H 2 O to a final volume of 50 μl to digest the vector. The digestion was run at 37°C for 3 h.

[0189] Dephosphorylation was performed with alkaline phosphatase, calf intestine (CIP, NEB, No. M0290L, 10 U / μl). Directly after digestion, 3 μl of CIP was added to the digested vector and incubated at 37°C for 30 min. The digested and dephosphorylated vector was loaded on a preparative 0.8% TAE agarose gel and the correct band size of the vector was cut out at approximately 6100 bp. SV gel and PCR cleanup kit (Promega, No. 9282, 1 column, eluted in 100ul) was used for cleanup. OD260nm showed a concentration of 64ng / μl.

[0190] The precipitate was prepared by mixing 42.5 μl DNA (about 3-5 ug of each DNA), 5 μl Cutsmart buffer (10x concentration, NEB No. B7204S), 0.4 μl HindIII-HF (100 U / μl, NEB No. R3104S), 2 μl EcoRI-HF (20 U / μl, NEB No. R3103L), and 5 μl HCl. 2 Adjust the volume to 50 μl and digest the mutated insert fragment. Digestion was performed in a PCR instrument at 37°C for 3 h. The digested insert was loaded on a preparative 0.8% TAE agarose gel and the correct band size of the vector of approximately 1400 bp was cut out. SV gel and PCR cleanup kit (Promega, No. 9282, 1 column, elution in 30 μl) was used for cleanup. OD260 nm showed a concentration of 1-25 ng / μl.

[0191] Ligation was performed using a rapid DNA ligation kit (No. K1423, Thermo Scientific, Fa.) using a vector:insert ratio of approximately 1:10. 4 μl of 5x ligation buffer was mixed with 1 μl of ligase, 2 μl of vector fragment (HindIII / EcoRI digested, stock concentration 64 ng / μl), and 13 μl of insert fragment (HindIII / EcoRI digested, stock concentration 1-25 ng / μl). The ligation was run at RT for 10 minutes.

[0192] Transformation was performed by incubating 10 μl of the ligation with 80 μl of chemically competent XL1Blue cells (Novartis, FS / RL) on ice for 30 min. Heat shock was performed at 42°C for 45 sec in an Eppendorf incubator, followed by incubation on ice for 2 min. Afterwards, 1 ml of 2YT medium was added, followed by incubation at 37°C for 1.5 h on an Eppendorf oscillator (800 rpm). The cells were centrifuged at 7000 rpm for 3 min, and the colonies were plated on LB / Carb / Gluc plates, followed by incubation overnight at 37°C.

[0193] The next day, single colonies were picked for DNA-miniprep and sequence analysis using forward primer P270 (SEQ ID NO: 165) and reverse primer P271 (SEQ ID NO: 166).

[0194] Based on 7 days of expression, the correct sequence was transfected into HEK293 cells at a 200 ml scale.

[0195] The following materials were used:

[0196] Human embryonic kidney cells (HEK293-T) constitutively expressing SV40 large T antigen (e.g. ATCC11268)

[0197] Polyethyleneimine "MAX" MW 40.000 (PEI) (Polysciences, catalog number 24765) was dissolved in H2O at RT and adjusted to pH 7.05 with NaOH.

[0198] M11V3 serum-free medium (Bioconcepts, CH, catalog number: V3-K )

[0199] DNA: Prepared using Qiagen DNA kit, Midiprep-Kit (No. 12943) according to the protocol recommended by the supplier

[0200] All cell culture work for transient transfections was performed using suspension-adapted HEK293-T cells grown in serum-free M11V3 medium.

[0201] The cells were grown in Corning shake flasks (Corning, USA) on an orbital shaker (115 rpm) in a humidified atmosphere with 5% CO. 2 Humidified CO 2 Grown in an incubator (seed flasks).

[0202] The cells used were already in the exponential growth phase (cell density was 5 x 10 5 and 3x 10 6 / ml) and had >90% viability.

[0203] Use counted and adjusted to 1.4 x 10 6 Cells were transfected in small scale (here 20 / 50 ml or 100 ml) with the corresponding cell amount of 10 cells / ml. 36% of the cell suspension was used in the final transfection volume.

[0204] DNA solution (solution 1) is prepared by diluting 1mg / L final volume DNA and gentle mixing in 7% final volume M11V3. In order to prevent the culture contamination caused by DNA that is not aseptically filtered, Penc. / Strep has been added to transfection after feeding. Then, 3mg / L final volume PEI solution is diluted and gently mixed in 7% final volume M11V3 (solution 2). Two solutions are hatched 5-10min at room temperature (RT). After this, solution 2 is added to solution 1 under gentle mixing, and hatched again 5-15 minutes at RT. After hatching, transfection mixture is added to cells, and culture is cultivated for four hours (115rpm, 37°C, 5% CO2).

[0205] The supernatant was harvested after 7 days of expression.

[0206] Centrifuge at 4500 rpm, 15 min, 4°C (Heraeus, Multifuge 3S-R)

[0207] Clarify by sterile filter (0.22 μm, Stericup filter, Thermo Scientific, Cat. No. 567-0020)

[0208] The supernatant was sent to purification for further steps. 1 ml of the supernatant sample was used for IPC on an Open Access APP-column.

[0209] The sample vials were glass crimp top vials (2 ml, Agilent, catalog number 5182-0543) and caps (crimp 11 mm, catalog number 5040-4667).

[0210] Proteins were purified using immobilized metal affinity chromatography (IMAC) on Aekta Pure or Aekta Avant (GE Healthcare) using 5 ml Histrap HP columns (GE Life Sciences, order number 17-5248-02) according to the following protocol. Instructions are listed in Table 11.

[0211] Table 11. IMAC protocol

[0212]

[0213]

[0214] The buffer used was composed according to Table 12 and Table 13.

[0215] Table 12. IMAC A, equilibration- and wash buffers

[0216] concentration chemicals 10mM <![CDATA[Na 2 HPO 4 ]]> 10mM <![CDATA[NaH 2 AFTER 4 ]]> 500mM NaCl 20mM Imidazole (Merck)

[0217] Table 13. IMAC B, elution buffer

[0218] concentration chemicals 10mM <![CDATA[Na 2 HPO 4 ]]> 10mM <![CDATA[NaH 2 AFTER 4 ]]> 500mM NaCl 500mM Imidazole (Merck)

[0219] The resulting protein was stored according to Table 14.

[0220] Table 14. Constructs

[0221] Construct Mutation location C1140 N73A / F365S C1141 T229A / F365S C1142 N292Q / F365S C1143 N334Q / F365S C1144 F365S / N371Q C1145 F365S / N457Q C1058 F365S

[0222] (b) Results and interpretation

[0223] Regarding the yield and monomer peak of analyzing SEC, the mutants did not improve. The parent protein (EP14) with the expression tag according to SEQ ID NO: 137 gave the best yield and the highest monomer peak in the analysis. The lowest yield and the worst monomer peak were achieved with mutant N371Q.

[0224] 6. Example 5: Combination

[0225] To try and further improve the properties, some of the mutations introduced in Example 3 above were combined according to the following Table 15. The mutations are numbered according to the amino acid position of wild-type CD39 as set out in SEQ ID NO:1.

[0226] Table 15. Combinations of constructs

[0227]

[0228] (a) Materials and methods

[0229] Primers according to Table 16 were used.

[0230] Table 16. Primers

[0231] Primers sequence P878 SEQ ID NO:183 P879 SEQ ID NO:184 P880 SEQ ID NO:185 P881 SEQ ID NO:186 P882 SEQ ID NO:187 P883 SEQ ID NO:188 P884 SEQ ID NO:189 P885 SEQ ID NO:190

[0232] Set up the PCR reaction using the following pipetting scheme:

[0233] 5 μl of 10x reaction buffer,

[0234] 1 μl ds-DNA template (stock solution concentration 100 ng / μl),

[0235] 1.5 μl Primer 1,

[0236] 1.5 μl Primer 2,

[0237] 1 μl dNTP mix

[0238] 1.5μl QuickSolution reagent,

[0239] 35.5 μl H 2 O (final volume 50 μl), and

[0240] 1μl QuickChange Lightning Enzyme.

[0241] Use PCR cycling parameters according to Table 17.

[0242] Table 17. PCR Cycling Parameters

[0243]

[0244] Directly after the reaction, 2 μl of DpnI enzyme was added to each reaction, mixed and incubated at 37°C for 5 min.

[0245] The transformation to XL10-gold super-competent cells was performed as follows. The cells were thawed on ice. 45 μl / transformation was used, and 2 μl B-ME was added to each vial. Then, 3 μl DpnI digested PCR product was added and incubated on ice in a 15ml BD tube for 30 min. Thereafter, the sample was heat shocked for 40 seconds and incubated on ice for 2 min. Next, 950 μl SOC culture medium was added, followed by incubation at 37 ° C in a shaking incubator for 1.5 h. Finally, the cells were plated on LB-carb-plates and incubated overnight at 37 ° C. The next day, single colonies were selected for DNA-microprep and sequence analysis.

[0246] The correct sequence was transfected into HEK293 cells as described in Example 4.

[0247] The protein was purified using immobilized metal affinity chromatography (IMAC) as follows: 95 ml of the supernatant was used (about 4 ml of the total was retained for analysis (IPC)).

[0248] Materials used:

[0249] Nickel-NTA agarose (Qiagen, catalog number / ID: 30230), Poly-Prep chromatography column (empty, BioRad, number 731-1550), IMAC A buffer pH 7.4 (containing 20 mM NaPO 4 IMAC B buffer pH 7.4 (containing 20 mM NaPO 4 Buffer and 300 mM imidazole). TBS (10x concentration, diluted to 1x concentration with MilliQ water). Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane, 10K, UFC 801096.

[0250] Method steps:

[0251] 1. Prepare a column using 1 ml of Qiagen's Ni-NTA-agarose (= 0.5 ml CV);

[0252] 2. Equilibrate with 10CV IMAC A;

[0253] 3. Load 15 / 45 ml SN on the column (collect flow-through);

[0254] 4. Wash with 10CV IMAC A (collected in 15ml Falcon tube);

[0255] 5. Elution in 6.5 CV of IMAC B;

[0256] 6. Determination of eluent concentration;

[0257] 7. Concentrate 3.5 ml of sample to approximately 400 μl using Amicon Ultra-4 Centrifugal Filter Unit 10K;

[0258] 8. Perform buffer exchange by adding TBS and centrifugation at 5000;

[0259] The samples were analyzed using analytical SEC with 40 μl of each sample and using protein gel with 12 μl of each sample.

[0260] The resulting protein is stored.

[0261] (b) Results and interpretation

[0262] The results are shown in Table 18.

[0263] Table 18. Summary of results

[0264]

[0265]

[0266] Protease site:

[0267] With the protease inserted, there was no / very low yield. With the furin site, there was about 40% yield (but for the transfection, again only 50% of the DNA was used since it was a co-transfection with the furin plasmid).

[0268] IL2-truncated:

[0269] All truncations including aa1-3 gave comparable results, aa1-3 might only be slightly lower compared to the others, but this could be a difference between samples. Truncations aa4-12 resulted in no protein expression. As with all other EP variants, no differences were found between EP28 containing a TSS linker between the IL2-start and hCD39-protein.

[0270] combination:

[0271] Combination with EP19(L424Q) did not result in a significant improvement in protein expression.

[0272] Combination with EP1 (R113M) showed lower aggregation in analytical SEC. NEG726 expressed well but showed the worst aggregation of all tested (about 37%). The combination of EP14xEP17 did not lead to any further improvement (F365S+Y412F).

[0273] 7. Example 6: Cloning of final candidates

[0274] A selection of clinical candidates as listed in Table 19 below were expressed for further testing.

[0275] Table 19. Summary of final candidates.

[0276]

[0277]

[0278] The following primers were used:

[0279] Table 20. Primers

[0280] Primers sequence R113Mtempl SEQ ID NO:191 R113MFW SEQ ID NO:192 R113MRev SEQ ID NO:193 F330StempI SEQ ID NO:194 F330SFW SEQ ID NO:195 F330SRev SEQ ID NO:196 Y377FtempI SEQ ID NO:197 L389CtempI SEQ ID NO:198 Y377F / L389QtempI SEQ ID NO:199 FW SEQ ID NO:200 Rev SEQ ID NO:201 WT SEQ ID NO:202

[0281] Set up the PCR reaction using the following pipetting protocol: 0.25 μl DMSO,

[0282] 20ng vector

[0283] 1.5μl insert (45ng / μl),

[0284] 2 μl 5x HF buffer,

[0285] 0.1μl Phusion pol,

[0286] 0.08 μl dNTP mix 10-X μl ddHO 2 O

[0287] Use PCR cycling parameters according to Table 17.

[0288] Table 21. PCR Cycling Parameters

[0289]

[0290]

[0291] Directly after the reaction, 0.5 μl of DpnI enzyme was added to each reaction, mixed and incubated at 37°C for 2 h.

[0292] The transformation to XL10-gold super-competent cells was performed as follows. The cells were thawed on ice. 45 μl / transformation was used, and 2 μl B-ME was added to each vial. Then, 3 μl DpnI digested PCR product was added and incubated on ice in a 15ml BD tube for 30 min. Thereafter, the sample was heat shocked for 40 seconds and incubated on ice for 2 min. Next, 950 μl SOC culture medium was added, followed by incubation at 37 ° C in a shaking incubator for 1.5 h. Finally, the cells were plated on LB-carb-plates and incubated overnight at 37 ° C. The next day, single colonies were selected for DNA-microprep and sequence analysis.

[0293] All constructs were subcloned into a new vector background to ensure that the sequence was correct. To this end, all constructs were amplified by PCR, inserted into a G4S-linker, and subsequently digested with HindIII / EcoRI.

[0294] The resulting protein is stored.

[0295] 8. Example 7: Comparative protein production

[0296] (1) Null mutation

[0297] To generate a negative control protein for in vivo studies, one / two mutations were inserted into the parental human CD39ΔMIL protein (EP28). These mutations have been described in the literature to remove or reduce the enzymatic activity of the protein. The mutation positions were E174A and S218A.

[0298] The following primers were used:

[0299] Table 22. Primers

[0300] Primers sequence P910 SEQ ID NO:203 P911 SEQ ID NO:204 P914 SEQ ID NO:205 P915 SEQ ID NO:206

[0301] Set up the PCR reaction using the following pipetting scheme:

[0302] 5 μl of 10x reaction buffer,

[0303] 1 μl ds-DNA template (stock solution concentration 100 ng / μl),

[0304] 1.5 μl Primer 1,

[0305] 1.5 μl Primer 2,

[0306] 1 μl dNTP mix

[0307] 1.5μl QuickSolution reagent,

[0308] 35.5 μl H2 O (final volume 50 μl), and

[0309] 1μl QuickChange Lightning Enzyme.

[0310] Use PCR cycling parameters according to Table 17.

[0311] Table 23. PCR Cycling Parameters

[0312]

[0313] Directly after the reaction, 2 μl of DpnI enzyme was added to each reaction, mixed and incubated at 37°C for 5 min.

[0314] The transformation to XL10-gold super-competent cells was performed as follows. The cells were thawed on ice. 45 μl / transformation was used, and 2 μl B-ME was added to each vial. Then, 3 μl DpnI digested PCR product was added and incubated on ice in a 15ml BD tube for 30 min. Thereafter, the sample was heat shocked for 40 seconds and incubated on ice for 2 min. Next, 950 μl SOC culture medium was added, followed by incubation at 37 ° C in a shaking incubator for 1.5 h. Finally, the cells were plated on LB-carb-plates and incubated overnight at 37 ° C. The next day, single colonies were selected for DNA-microprep and sequence analysis.

[0315] The correct sequence was transfected into HEK293 cells according to the following protocol.

[0316] 10 μg of vector DNA, 0.4 μl HindIII (100 U / μl, NEB), 2 μl EcoRI (20 U / μl, NEB), 5 μl Cutsmart buffer 10x concentration (NEB) and H 2 Prepare digestion buffer to a final volume of 50 μl with 5% CO. Run the digestion reaction at 37°C for 3 h.

[0317] Dephosphorylation reaction was run immediately after digestion. Calf intestinal alkaline phosphatase (10 U / μl, CIP, NEB, No. M0290L) was added (3 μl) to the digested vector mixture and incubated at 37°C for 30 min.

[0318] The digested and dephosphorylated vector was subcloned to check the sequence.

[0319] The correct sequence was transfected into HEK293 cells according to the following protocol.

[0320] The following materials were used for 7 days of expression: 1. Human embryonic kidney cells (HEK293-T, ATCC11268) constitutively expressing SV40 large T antigen; 2. Polyethylenimine "MAX" MW 40.000 (PEI) (Polysin, catalog number 24765).

[0321] Prepare the PEI solution by carefully dissolving 1 g of PEI in 900 ml of cell culture grade water at room temperature (RT). Then neutralize with NaOH to a final pH of 7.05. Finally, adjust the volume to 1 L and filter the solution through a 0.22 μm filter, distribute in aliquots and freeze at -80 °C until further use. After thawing, aliquots can be refrozen up to 3 times at -20 °C, but should not be stored at -20 °C for long periods of time.

[0322] M11V3 serum-free medium (Bioconcepts, CH, catalog number: V3-K).

[0323] All cell culture work for transient transfections was performed using suspension-adapted HEK293-T cells grown in serum-free M11V3 medium.

[0324] For small-scale transfection (<5 L), cells were cultured in Corning shake flasks (Corning, USA) on an orbital shaker (100 rpm) in a humidified atmosphere with 5% CO. 2 Humidified CO 2 Grown in an incubator (seed flasks).

[0325] Typically, cells in seed culture should be in exponential growth phase (cell density around 5 x 10 5 With 3x 10 6 / ml) and have >90% viability. Cell densities outside this range will result in a lag phase after division or reduced transfection efficacy.

[0326] For small-scale (here 2 L) transfections, remove an aliquot of cells from the seed culture and adjust these cells to 1.4 x 10 in a final volume of 36% with M11V3 medium. 6 cells / ml.

[0327] DNA solution (solution 1) is prepared by diluting 1mg / L final volume DNA in 7% final volume M11V3 and gently mixing. In order to prevent culture contamination, the solution can be filtered using a 0.22 μm filter (e.g., Millipore Stericup). Here, because the volume is small, sterile filtration has not yet been performed. Then, 3mg / L final volume PEI solution is diluted and gently mixed in 7% final volume M11V3 (solution 2). Both solutions are incubated for 5-10min at room temperature (RT). Thereafter, solution 2 is added to solution 1 under gentle mixing, and incubated for another 5-15 minutes at RT (do not mix again during incubation, because PEI covers / condenses DNA into positively charged particles, which bind to anionic cell surface residues and enter cells through endocytosis). After incubation, the transfection mixture is added to the cells, and the culture is cultured for four hours (10rpm, 37°C, 6%CO 2 ).

[0328] Finally, the culture was fed with the remaining 50% of the final volume of M11V3 medium according to the following example: Inoculation volume: 36 ml, with 1.4 x 10 6 cells / ml.

[0329] Solution 1: 7 ml M11V3 medium containing 100 μg plasmid DNA. Solution 2: 7 ml M11V3 medium containing 300 μg PEI (300 μl)

[0330] Feed: 50ml M11V3, total 100ml.

[0331] The protein was purified using immobilized metal affinity chromatography (IMAC) as follows: 95 ml of the supernatant was used (about 4 ml of the total was retained for analysis (IPC)).

[0332] Materials used:

[0333] Nickel-NTA agarose (Qiagen, catalog number / ID: 30230), Poly-Prep chromatography column (empty, BioRad, number 731-1550), IMAC A buffer pH 7.4 (containing 20 mM NaPO 4 IMAC B buffer pH 7.4 (containing 20 mM NaPO 4 Buffer and 300 mM imidazole). TBS (10x concentration, diluted to 1x concentration with MilliQ water). Amicon Ultra-4 Centrifugal Filter Unit with Ultracel-10 membrane, 10K, UFC 801096.

[0334] Method steps:

[0335] 1. Prepare a column using 1 ml of Qiagen's Ni-NTA-agarose (= 0.5 ml CV);

[0336] 2. Equilibrate with 10CV IMAC A;

[0337] 3. Load 15 / 45 ml SN on the column (collect flow-through);

[0338] 4. Wash with 10CV IMAC A (collected in 15ml Falcon tube);

[0339] 5. Elution in 6.5 CV of IMAC B;

[0340] 6. Determination of eluent concentration;

[0341] 7. Concentrate 3.5 ml of sample to approximately 400 μl using Amicon Ultra-4 Centrifugal Filter Unit 10K;

[0342] 8. Perform buffer exchange by adding TBS and centrifugation at 5000;

[0343] The samples were analyzed using analytical SEC with 40 μl of each sample and using protein gel with 12 μl of each sample.

[0344] The resulting protein is stored.

[0345] (2)plusMIL

[0346] Cloning of EP14aa1-3 with the membrane interacting loop (aa193-204) was performed using overlap extension PCR.

[0347] The following primers were used:

[0348] Table 24. Primers

[0349] Primers sequence FW SEQ ID NO:207 REV SEQ ID NO:208 Antonym SEQ ID NO:98

[0350] Set up the PCR reaction using the following pipetting scheme:

[0351] 1.2μl Phusion hot start polymerase,

[0352] 24 μl 5x HF-buffer,

[0353] 0.96 μl 100 mM dNTPs (25 mM of each dNTP),

[0354] 0.6μl Fw primer,

[0355] 0.6μl Rev primer,

[0356] 92.64 μl DEPC H 2 O.

[0357] Use PCR cycling parameters according to Table 17.

[0358] Table 25. PCR Cycling Parameters

[0359]

[0360] Directly after the reaction, 2 μl of DpnI enzyme was added to each reaction, mixed and incubated at 37°C for 2 h.

[0361] Transformation was performed by transferring 2 μl of PCR product to a 96-well PCR plate and cooling on ice. 20 μl of STELLAR chemical composition bacteria was added and carefully mixed by pipetting up and down once. The samples were incubated on ice for 30 min, then incubated in a PCR machine at 42°C for 45 sec, followed by another 60 sec on ice. Finally, 90 μl of SOC medium was added and incubated at 37°C for 1 hr. The entire transfection mixture was plated on LB-ampicillin or LB-carbenicillin plates and grown overnight at 37°C.

[0362] The resulting protein EP14_plusMIL was stored, which had an amino acid sequence according to SEQ ID NO:155.

[0363] 9. Example 8: Enzyme activity

[0364] The candidates generated in the previous examples were characterized using enzyme activity assays.

[0365] The following reagents were used: Pi free buffer, phosphate-free saline solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, pH 7.4); and Pi free buffer + 2% BSA, phosphate-free saline solution with 20 mg / ml BSA; CD39 protein (according to SEQ ID NO: 1); ATP.

[0366] Duplicate CD39 solutions were prepared at 2 μg / ml. Duplicate ATP solutions were prepared at 1000 μM from 15 μl ATP stock + 1185 μl buffer (1.2 ml total).

[0367] The enzyme reaction was studied in 48-well PCR plates filled with 120 μl final / well by mixing 60 μl ATP with 60 μl CD39 or 60 μl Pi-free buffer for control. The final concentrations were 500 μM ATP and 1 μg / ml CD39.

[0368] The samples were incubated at 37° C. for 0, 5, 15, 30, 60, 90 and 150 min, respectively. The samples were then evaluated by Pi release assay or HPLC.

[0369] (1) Pi release assay

[0370] (a) Materials and methods

[0371] Prepare reagents from a standard Pi assay kit according to the manufacturer's instructions.

[0372] Prepare a standard curve with Pi by dilution in water. Prepare a 1:2 serial dilution of Pi stock solution (100 μM): 450 μl + 450 μl water. The standard curve concentrations are: 50 μM / 25 μM / 12.5 μM / 6.25 μM / 3.1 μM / 1.5 μM / 0 μM.

[0373] Prepare gold reagent mixture: 4 ml gold reagent + 40 μl accelerator (for 3 plates). In a 96-well plate, place the samples in H 2 1:10 dilution in H2O (dilution in water: 10 μl sample + 90 μl H2O). 50 μl of the 1:10 diluted sample was distributed in each well of a 96 half-area well plate (Corning, 3690). 12.5 μl of the gold reagent mixture was added to each well (25% sample volume) and the samples were incubated at room temperature for 10 min. The absorbance was read at 635 nm.

[0374] (b) Results and interpretation

[0375] The comparison results of the candidates are shown in Table 26.

[0376] Table 26. Comparison results of candidates.

[0377]

[0378]

[0379] Enzyme activity was measured by adding 500 μM ATP to the enzyme and analyzing the concentrations of ATP, ADP, and AMP over time using HPLC (method described below). The resulting kinetic curves were fitted Figure 6 The enzyme constants were obtained using the model in . In terms of the enzyme constant Kcat, these enzymes showed the following order (low activity to high activity): EP28 (wt), EP17, EP14, EP15.

[0380] Figure 7 Kinetic data and model fits for EP28 are shown. Figure 8 Kinetic data and model fits for EP14 are shown. Fig. 9Kinetic data and model fits for EP15 are shown.

[0381] The enzyme constants of EP28 (wt), EP14, EP15 and EP17 are summarized in Table 27. Compared with the wild type (WT), the three novel variants show increased catalytic activity. Importantly, these novel variants show a significant increase in catalytic rate constant (kcat), catalytic efficiency (kcat / Km). Since the reported ATP and ADP substrate concentrations during tissue damage and thrombosis are higher than the reported Km, this increase in kcat and kcat / Km may translate into higher in vivo activity.

[0382] Table 27. Enzyme constants

[0383]

[0384] (2) HPLC validation assay (kinetics and dose response)

[0385] (a) Materials and methods

[0386] Test candidates with HPLC validation assay. Transfer 70 μl of each sample to a glass vial for HPLC.

[0387] As shown in Table 28, calibration samples were prepared using 5 mM stock solutions.

[0388] Table 28. Stock solutions

[0389] 5mM weight <![CDATA[H 2 The]]> Adenosine 1336.2 μg / mL 1.862mg 1394μL Inosine 1341.2μg / mL 2.668mg 1989μL AMP 1736.1 μg / mL 2.182mg 1257μL ADP 2506.6μg / mL 2.500mg 997μL ATP 2755.7 μg / mL 4.834mg 1754μL

[0390] 1000 μM Mix 20 μL of each stock solution in a HPLC vial

[0391] 500μM 20μL 1mM+20μL H 2 O

[0392] 100μM 10μL 1mM+90μL H 2 O

[0393] 10μM 10μL 100μM+90μL H 2 O

[0394] 1μM 10μL 10μM+90μL H 2 O

[0395] HPLC separation was performed using an Agilent 1100 system with a capillary pump (CapPump) (G1376A), degasser (G1379A), ALS (G1329A), thermostat (G1330B), ColComp (G1316A) and DAD (G1315A). Solvent A: 10 mM KH 2 PO 4 (04243, Riedel-de ) + 2 mM TBA bromide, pH 7.0 (86857-10G-F, Fluka) and solvent B: 10 mM KH2PO4 / ACN 1 / 1 + 2 mM TBA bromide, pH 5.5. Column: Nucleodur 300-5C18 ec, 2 x 150 mm, 5 μm, Macherey-Nagel 760185.20 batch E1410025836654055. Column temperature was 40°C, injection volume was 10 μL, flow rate was 0.3 ml / min, and gradient was 0-3': 0% B; 3-23': 0-95% B, linear; 23-28': 95% B, linear; 28-29': 95-0% B, linear; 5' post time". DAD: 247 nm and 259 nm.

[0396] UPLC separation was performed using Waters UPLC class I. Solvent A: 10 mM KH 2 PO 4 / 10mM K 2 HPO 4 1 / 1 + 2 mM TBA bromide, pH 7.0. Solvent B: 10 mM KH 2 PO 4 / ACN 1 / 1 + 2mM TBA bromide, pH 5.5. Column: Fortis Bio C18, 2.1 x 50mm, 5μm, di2chrom BIO318-020301SN H03161210-2. Column temperature was 40°C, injection volume was 10μL, flow rate was 0.5ml / min, and gradient was 0-1': 0% B; 1-8': 0-55% B, linear; 8-10': 55% B; 10-11': 55-0% B, linear; 14' stop time. DAD was 247nm and 259nm.

[0397] (b) Results

[0398] The results can be seen in Figure 7 , 8, 9 and 11, kinetic data and model fitting of candidates according to different embodiments are shown.

[0399] 10. Example 9: In vitro activity, initial screening

[0400] The CD39 versions EP1 to EP24 described in the previous example were cloned into the mammalian expression vector pRS5a_leader_APP_His( Figure 5 ), without IL2-leader sequence and without IL2-start.

[0401] (a) Materials and methods

[0402] Small-scale expression (20 / 50 ml scale) of EP-Hits was performed in HEK293 (PEI-transfection) for 7 days followed by IPC on APP-HPLC (as described above).

[0403] 15 / 45 ml of cell supernatant was used for protein purification using Ni-NTA column (0.5 ml CV);

[0404] Elution with 6 CV IMAC B buffer (20 mM NaPO4-buffer, 300 mM imidazole, pH 7.4);

[0405] The purified protein was concentrated and rebuffered in TBS, pH 7.4;

[0406] Proteins were analyzed using protein gel and analytical SEC;

[0407] Delivery of all variants and three controls (parental hCD39-dMIL, or EP28 with and without IL2-priming, and 8M-version without IL2-priming): 90-200 ul of purified protein in TBS, pH 7.4

[0408] (b) Results and interpretation

[0409] The results are summarized in Table 29 below.

[0410] All samples were in TBS pH 7.4 and had APP- (SEQ ID N: 247) and His-tag (SEQ ID NO: 249).

[0411] Only the parental human CD39ΔMIL (EP28) has a 15 amino acid long IL2-start, aa1-15 (SEQ ID NO: 133).

[0412] Pi release assay BOENKTH1-0252824, double detection. Duration 60min and 180min. Value

[0413] Table 29. In vitro activity

[0414] Pi release assay, 0.25

[0415] ug / ml+500uM ATP

[0416]

[0417]

[0418] 11. Example 10: In vitro activity, refined screening

[0419] A subset of 12 mutants was tested a second time, but with an IL-2 starter to allow for a larger scale of expression.

[0420] (a) Materials and methods

[0421] Mammalian expression vector pRS5a_leader_APP_His, with 15 amino acids long IL2-start, aa1-15 (SEQ ID NO: 133) ( Figure 5 Small-scale expression (50 / 100 ml scale) of EP-Hits in HEK293 (PEI-transfection) for 7 days followed by IPC on APP-HPLC (as described above).

[0422] Protein purification was performed on 45 / 95 ml of cell supernatant using Ni-NTA column (0.5 ml CV)

[0423] Elution with 6 CV IMAC B buffer (20 mM NaPO4-buffer, 300 mM imidazole, pH 7.4);

[0424] The purified protein was concentrated and rebuffered in TBS, pH 7.4.

[0425] Proteins were analyzed using protein gel and analytical SEC;

[0426] Delivery of all variants and controls (parental hCD39-dMIL, or EP28 (containing IL2-starting aa1-15 (SEQ ID NO: 133)):

[0427] 500ul of purified protein in TBS, pH 7.4

[0428] (b) Results and interpretation

[0429] The results are summarized in Tables 30, 31 and 32 below.

[0430] Table 30. Protein purification of EP-Hits 06.07.2016, part 1

[0431]

[0432]

[0433] Table 31. Protein purification of EP-Hits 06.07.2016, part 2

[0434]

[0435] Table 32. Protein Purification EP-Hits 15.07.2016

[0436]

[0437] Table 33. Point mutations relative to the wild-type CD39 sequence according to SEQ ID NO: 1.

[0438]

[0439] 12. Example 11: In vivo activity, pK

[0440] (a) Materials and methods

[0441] For in vivo PK, 10 mg / kg of compound at a final concentration of 10 mg / ml in PBS buffer was administered intravenously (1 ml / kg) to 4 conscious female C57BL / 6 mice via the tail vein. Mice were obtained from WIGA and weighed approximately 22 g. All life work was performed in accordance with the Swiss animal welfare law.

[0442] Whole blood was collected in small volume serum tubes at 0.25, 3, 8, 24 and 48 h post-dose using POCT Minivettes (50 μL per time point). Serum was separated and used for concentration determination.

[0443] The Gyrolab technology is a nanoscale automated immunoassay using an affinity flow-through format that works by centrifugal force and laser-induced fluorescence detection. Streptavidin-coated beads are pre-packed in affinity columns on the Gyrolab Bioaffy CD. Each CD contains 112 columns. Each microstructured affinity capture column contains 15nl. The injected sample enters by capillary action. The biotinylated capture reagent binds to the streptavidin-coated beads. Then, the analyte solution is injected, which binds to the captured molecules. Finally, a fluorophore-labeled detection reagent is applied. In the case of CD39, two different assay readouts are used, depending on the availability of the APP tag.

[0444] 1) Anti-CD39 (40035) and anti-APP (27431) are found in Fig. 10A middle.

[0445] 2) Fab (40035) and anti-Fc / anti-CD39 (40044) 1:1 premix (all EP28aa1-16 constructs) found in Fig. 10B When biotinylated by amine coupling, antibody 40044 lost its activity, and thus, only antibody 40035 was biotinylated.

[0446] All standard curves of CD39 constructs were diluted in a 1:2 dilution series in Rexxip A containing 5% (v / v) mouse serum. The applied concentration range of APP-tagged constructs was 5000ng / ml-9.77ng / ml, and the applied concentration range of EP28aa1-16 was 10000ng / ml-9.77ng / ml. All mouse serum samples were diluted 1:100 in Rexxip A containing 5% (v / v) mouse serum. QC samples of CD39 constructs were diluted in Rexxip A containing 5% (v / v) mouse serum (50 and 500ng / ml for constructs with APP tags, 500 and 1000ng / ml for EP28aa1-16). The final concentration of all biotinylated capture reagents was 0.1mg / ml, and the fluorescently labeled detection antibodies were diluted to 10nM in Rexxip F.

[0447] (b) Results and interpretation

[0448] The results are summarized in Table 33. As can be seen, all candidates showed the same PK. Therefore, the selection of candidates was not based on PK properties.

[0449] Table 34. pK values

[0450]

[0451]

[0452] 13. Example 12: In vivo activity, AKI model

[0453] (a) Materials and methods

[0454] Nephrectomy of the right kidney was performed before the I / R setting began. The second kidney was removed to avoid changing the entire dynamic compensation mechanism of the biology. The spontaneously breathing anesthetized animals were placed on a thermostatic blanket of a thermostatic monitoring system and covered with sterile gauze. The body temperature was recorded by a rectal probe and controlled within the range of 36.5°C-37.5°C to avoid hypothermia. The animals were anesthetized, shaved and disinfected (Betaseptic). After midline incision / laparotomy, the abdominal contents were retracted to the left side and the right kidney was removed. The ureters and blood vessels were disconnected and ligated (9-0 Ethicon), and then the kidney was removed.

[0455] Induction of I / R injury: Immediately after nephrectomy of the right kidney, the abdominal contents were retracted to the right side, and the left renal artery was dissected free for induction of renal ischemia.

[0456] The microaneurysm clip is used to clamp the renal pedicle to block blood flow to the kidney and induce renal ischemia. The duration of renal ischemia starts from the time of clamping. Successful ischemia is confirmed by the color change of the kidney from red to dark purple within a few seconds. After ischemia, the microaneurysm clip is removed and reperfusion is indicated by the kidney color change to red.

[0457] (b) Results and interpretation

[0458] The results are shown in Figure 12. The candidates showed an in vivo dose response, which correlated with their detailed in vitro activities. Fig. 12A Results for the parent EP28 are shown, Fig. 12B The results of EP1xEP17 are shown. Fig. 12C Results are shown for EP 14. As can be seen, EP28 and EP1xEP17 showed similar dose responses, whereas EP14, which had higher in vitro activity, showed full efficacy at lower doses.

[0459] 14. Example 13: Titer, Yield and Scalability

[0460] In order to manufacture selected candidates on a commercial scale, it is important to be able to express them in relatively high yields. Therapeutic proteins may not be as straightforward as therapeutic antibodies due to format complexity in addition to the lack of enrichment techniques that enable the selection of high-producing clones.

[0461] It is challenging to have comparable technical characteristics for these two candidates, EP14aa1-3 and EP28aa1-3. In particular, low expression titers in early expression batches (data not shown) affect production costs or may be even lower after scale-up due to unrobust control of host cell proteins.

[0462] In order to try to improve protein expression through early clonal selection of both candidates, a customized purification process was required. To this end, a pool of cells expressing the candidates EP28aa1-3 and EP14aa1-3 was generated.

[0463] The parent CHO cell line was used as a host cell line for the production of a cell line expressing EP28aa1-16 / EP14aa1-3. The host cell line was derived from the CHO-K1 cell line well known to those skilled in the art, in a manner described in, for example, patent applications WO 2015092737 and WO 2015092735, both of which are incorporated herein by reference in their entirety. A single vial from the CHO line was used to prepare the EP28aa1-16 / EP14aa1-3 recombinant cell line.

[0464] Cells were grown in chemically defined medium. One μg of SwaI linearized plasmid DNA, expression vector encoding EP28aa1-16 / EP14aa1-3 was added per transfection. Transfection reactions were performed in chemically defined medium.

[0465] Transfections were performed by electroporation using an AMAXA Gene Pulser according to the manufacturer's instructions. Parental CHO cells used for transfection were in exponential growth phase with greater than 95% cell viability. In total, 5 x 10 6 The transfections were performed three times with 10 cells / transfection. Immediately after transfection, the cells were transferred to a shake flask containing medium with chemically defined medium.

[0466] Before starting the selection process, the cell bank was maintained at 36.5 °C and 10% CO 2 48 hours.Selection procedure is carried out using the selection marker encoded in the expression vector.After transfection and growth for 48 hours under low folic acid conditions, additional selection pressure is applied by adding 10nM MTX to the culture medium determined by chemical composition.21 days after MTX selection began, there was a library group mainly composed of MTX-resistant cells.Cells were recovered and frozen.Standard feed batches were set in the culture medium determined by chemical composition for determining the concentration of EP28aa1-16 / EP14aa1-3.Reverse phase chromatography (RPC) is used to determine product concentration.The CHO cell bank producing EP28aa1-16 / EP14aa1-3 is used for FACS single cell sorting / cell printing program to obtain personalized clonal cell lines.

[0467] 15. Example 14: Therapeutic Use

[0468] Activate P2X 7Extracellular ATP of R has been clearly linked to several diseases, such as enhanced graft-versus-host disease (Wilhelm et al., Graft-versus-host disease is enhanced by extracellular ATPactivating P2X 7 R.[Activate P2X 7 Extracellular ATP in R enhances graft-versus-host disease], Nature Medicine 16:12, pp. 1434-1439, (2010)).

[0469] Furthermore, both in vitro and in vivo studies indicate that CD39 represents an important apyrase in cardiovascular health by regulating the levels of ADP. Apyrase is known to inhibit platelet aggregation by metabolizing extracellular ADP.

[0470] Compared with other treatments such as clopidogrel (Plavix TM )) In contrast, human apyrase does not covalently bind to platelets, it binds irreversibly to ADP receptors on platelets. This allows for a faster disappearance of the therapeutic blockade and therefore a safer approach for patients with excessive platelet activation. This provides a safer approach for patients with excessive platelet activation.

[0471] There is therefore a clear basis for the therapeutic use of compounds that reduce extracellular ATP levels, such as compounds according to the present invention.

[0472] Specific non-limiting examples of therapeutic uses of the compounds according to the invention are acute organ damage due to trauma and / or hypoxia, such as acute respiratory distress syndrome (ARDS), lung injury, renal failure, acute kidney injury (AKI) (including acute kidney injury after coronary artery bypass graft surgery), delayed graft function after kidney or other solid organ transplantation (including xenotransplantation), or vascular disease (such as occlusive vascular disease), transplantation and xenotransplantation, treatment of individuals suffering from stroke, coronary artery disease or injury caused by: myocardial infarction, atherosclerosis, arteriosclerosis, embolism, preeclampsia, angioplasty, vascular injury, transplantation, neonatal hypoxic-ischemic encephalopathy, platelet-related ischemic diseases (including pulmonary ischemia, coronary ischemia and cerebral ischemia), ischemia-reperfusion injury (IRI) thrombotic diseases (including coronary thrombosis, cerebral artery thrombosis, intracardiac thrombosis, peripheral arterial thrombosis, and venous thrombosis), delayed graft function after kidney or other solid organ transplantation (including xenotransplantation). Other non-limiting examples of therapeutic uses of the compounds according to the invention are treatment of burns or radiation injury, sepsis, improving wound healing, reducing bleeding or risk of bleeding, preventing organ damage, graft versus host disease or preventing transplant rejection.

[0473] In particular, the preferred therapeutic use of the compounds according to the invention is acute kidney injury (AKI), such as acute kidney injury after coronary artery bypass graft surgery or sepsis or rhabdomyolysis. This condition increases the mortality of patients and there is no standard of care (SoC). The main causes of AKI in intensive care units are: sepsis (47.5%), major surgery (34%), cardiogenic shock (27%), hypovolemia (26%) and nephrotoxic compounds (19%). In addition, AKI is an independent strong risk factor for the development of chronic kidney disease (CKD). 20%-30% of major cardiac surgery patients acquire acute kidney injury. Another preferred embodiment relates to the use of the isolated apyrase according to the invention for the treatment of acute kidney injury associated with cardiac surgery.

[0474] In another embodiment, the present disclosure relates to an isolated pyrase according to the present invention for use in treating delayed graft function (DGF), acute respiratory distress syndrome (ARDS), acute myocardial infarction (AMI), traumatic brain injury (TBI) / acute ischemic stroke (AIS), or a combination thereof commonly referred to as multiple organ failure (MOF). 16. Acute kidney injury (AKI) is a common complication of sepsis. 28% of sepsis patients acquire AKI. In another preferred embodiment, the present disclosure relates to the use of an isolated pyrase according to the present invention for treating sepsis-associated acute kidney injury. Example 15: Therapeutic Compositions

[0475] Therapeutic proteins are typically formulated in aqueous form for administration or as lyophilisates for reconstitution with a suitable diluent prior to administration. The protein may be formulated as a lyophilisate, or as an aqueous composition, for example in a pre-filled syringe.

[0476] Suitable formulations can provide aqueous pharmaceutical compositions or lyophilisates, which can be reconstructed to provide a solution with high concentrations of therapeutic protein active ingredients and low levels of protein aggregation for delivery to patients. High concentrations of protein are useful because high concentrations reduce the amount of material (dosage) that must be delivered to the patient. The reduced dosing volume minimizes the time spent on delivering a fixed dose to the patient. Aqueous compositions of the present invention with high concentrations of protein are particularly suitable for subcutaneous administration.

[0477] Thus, the present invention provides aqueous pharmaceutical compositions suitable for administration in a subject (eg, for subcutaneous administration), comprising a therapeutic protein.

[0478] When combined with a pharmaceutically acceptable carrier, the therapeutic protein can be used as a pharmaceutical composition. In addition to the therapeutic protein, this composition can contain a carrier, various diluents, fillers, salts, buffers, stabilizers, solubilizing agents and other materials well known in the art. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition used in the disclosed method can also contain other therapeutic agents for the treatment of specific target disorders.

[0479] 17. Example 16: Route of administration

[0480] Typically, the protein according to the invention is administered, for example, by intravenous, intraperitoneal or subcutaneous injection. Methods for accomplishing this administration are known to those of ordinary skill in the art. It is also possible to obtain compositions that can be administered topically or orally, or compositions that can be delivered across mucosal membranes. As will be appreciated by those skilled in the art, any suitable mode of administration may be used, such as that suitable for a particular selected route of administration.

[0481] Examples of possible routes of administration include parenteral (e.g., intravenous (IV or IV), intramuscular (IM), intradermal, subcutaneous (SC or SC), or infusion), oral and pulmonary (e.g., inhalation), nasal, transdermal (topical), transmucosal, intraarterial, continuous infusion, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and agents for adjusting osmotic pressure such as sodium chloride or glucose. The pH can be adjusted with an acid or a base (e.g., hydrochloric acid or sodium hydroxide). Parenteral preparations may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0482] Apyrase therapy can be initiated by administering a "loading dose" of a protein according to the present invention to a subject in need of therapy. A "loading dose" is intended to be administered to a subject as an initial dose of a protein according to the present invention, wherein the dose of the protein according to the present invention administered falls within a higher dosing range. A "loading dose" can be administered as a single dose, e.g., a single infusion, wherein the protein is administered IV, or multiple doses, e.g., multiple infusions, wherein the protein is administered IV, as long as a complete "loading dose" is administered within a time period of about 24 hours (or based on the severity of the disease, if multiple intravenous administrations are required, then within the first month). After administering the "loading dose", one or more additional therapeutically effective doses of the protein according to the present invention are administered to the subject. Subsequent treatment effective doses can be administered, for example, according to a weekly dosing regimen, or once every two weeks, once every three weeks, or once every four weeks. In such embodiments, subsequent treatment effective doses typically fall within a lower dosing range.

[0483] Alternatively, in some embodiments, after a "loading dose", a subsequent therapeutically effective dose of a protein according to the present invention is administered according to a "maintenance regimen", wherein a therapeutically effective dose of a protein according to the present invention is administered once a month, once every 6 weeks, once every two months, once every 10 weeks, once every three months, once every 14 weeks, once every four months, once every 18 weeks, once every five months, once every 22 weeks, once every six months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or once every 12 months. In such embodiments, the therapeutically effective dose of a protein according to the present invention falls within the lower dosing range, particularly when subsequent doses are administered at more frequent intervals (e.g., once every two weeks to once a month), or falls within the higher dosing range, particularly when subsequent doses are administered at less frequent intervals, for example, where subsequent doses are administered at intervals of one to 12 months.

[0484] The timing of dosing is usually measured from the day of the first dose of active compound (also called "baseline"). However, different healthcare providers use different naming conventions.

[0485] It is worth noting that some healthcare providers may refer to Week Zero as Week 1, while some healthcare providers may refer to Day Zero as Day 1. Thus, it is possible that different physicians will indicate that a dose is given, for example, in Week 3 / during Day 21, in Week 3 / during Day 22, in Week 4 / during Day 21, in Week 4 / during Day 22, while referring to the same dosing schedule. For consistency, the first week of dosing will be referred to herein as Week 0, while the first day of dosing will be referred to as Day 1. However, one skilled in the art will understand that this naming convention is for consistency only and should not be construed as limiting, i.e., weekly dosing provides weekly doses of protein regardless of whether the physician refers to a particular week as "Week 1" or "Week 2." Examples of dosing regimens as described herein are shown in Table 1. Figure 1 and Figure 2. It will be appreciated that a dose need not be provided at a precise time point, for example a dose scheduled approximately on day 29 may be provided, for example, on day 24 to day 34 (eg day 30), as long as it is provided in the appropriate week.

[0486] As used herein, the phrase "a container having a sufficient amount of protein to allow delivery of [a specified dose]" is used to indicate that a given container (e.g., a vial, a pen, a syringe) has disposed therein a volume of a protein that can be used to provide a desired dose (e.g., as part of a pharmaceutical composition). As an example, if the desired dose is 500 mg, then the clinician might use 2 ml from a container containing a protein formulation at a concentration of 250 mg / ml, 1 ml from a container containing a protein formulation at a concentration of 500 mg / ml, 0.5 ml from a container containing a protein formulation at a concentration of 1000 mg / ml, etc. In each such case, the containers have a sufficient amount of protein to allow delivery of the desired dose of 500 mg.

[0487] As used herein, the phrase "formulated in a dosage that allows [route of administration] to deliver [specified dose]" is used to indicate that a given pharmaceutical composition can be used to provide a desired dose of a protein via a specified route of administration (e.g., sc or iv). As an example, if the desired subcutaneous dose is 500 mg, a clinician might use 2 ml of a protein formulation having a concentration of 250 mg / ml, 1 ml of a protein formulation having a concentration of 500 mg / ml, 0.5 ml of a protein formulation having a concentration of 1000 mg / ml, and the like. In each such case, these protein formulations are at a sufficiently high concentration to allow subcutaneous delivery of the protein. Subcutaneous delivery typically requires delivery of a volume of less than about 2 ml, preferably about 1 ml or less. However, higher volumes can be delivered over time using, for example, a patch / pump mechanism.

[0488] Disclosed herein is the use of a protein for the manufacture of a medicament for treating tissue damage in a patient, wherein the medicament is formulated to comprise containers, each container having a sufficient amount of the protein to allow delivery of at least about 75 mg, 150 mg, 300 mg or 600 mg of the protein per unit dose.

[0489] Disclosed herein is the use of a protein for the manufacture of a medicament for treating tissue damage in a patient, wherein the medicament is formulated at a dose to allow systemic delivery (e.g., iv or sc delivery) of 75 mg, 150 mg, 300 mg, or 600 mg of the protein per unit dose.

[0490] 18. Example 17: Kit

[0491] The present disclosure also encompasses kits for treating patients with tissue damage (as appropriate) with proteins. Such kits contain the protein (e.g., in liquid or lyophilized form) or a pharmaceutical composition containing the protein (as described above). In addition, such kits may contain a device for administering the protein (e.g., a syringe and vial, a prefilled syringe, a prefilled pen, a patch / pump) and instructions for use. The instructions may disclose that the protein is provided to the patient as part of a specific dosing regimen. These kits may also contain additional therapeutic agents (as described above) for treating psoriasis, for example, for delivery in combination with the loaded protein.

[0492] The phrase "administration device" is used to refer to any useful device for administering a drug systemically to a patient, including but not limited to pre-filled syringes, vials and syringes, injection pens, autoinjectors, IV drip sets and bags, pumps, patches / pumps, etc. Using these items, a patient can self-administer the drug (i.e., self-administer the drug), or the drug can be administered by a caregiver or physician.

[0493] Disclosed herein are kits for treating patients suffering from tissue damage, the kits comprising: a) a pharmaceutical composition comprising a therapeutically effective amount of a protein; b) a device for administering the protein to the patient; and c) instructions for subcutaneously administering the protein to a patient in need thereof.

[0494] Sequence Listing

[0495] Useful amino acid and nucleotide sequences for practicing the present invention are disclosed in Table 34.

[0496] Table 35. Sequences useful in practicing the invention.

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510]

[0511]

[0512]

[0513]

[0514]

[0515]

[0516]

[0517]

[0518]

[0519]

[0520]

[0521]

[0522]

[0523]

[0524]

[0525]

[0526]

[0527]

[0528]

[0529]

[0530]

[0531]

[0532]

[0533]

[0534]

[0535]

[0536]

[0537]

[0538]

[0539]

[0540]

[0541]

[0542]

[0543]

[0544]

[0545]

[0546]

[0547]

[0548]

[0549]

[0550]

[0551]

[0552]

[0553]

[0554]

[0555]

[0556]

[0557]

Claims

1. A solubilized human apyrase comprising a sequence having SEQ ID NO:

229.

2. A soluble human apyrase consisting of a sequence having SEQ ID NO:

229.

3. A soluble human apyrase consisting of the sequence shown in SEQ ID NO:

229.

4. A pharmaceutical composition comprising the apyrase according to any one of claims 1 to 3, and one or more of the following materials: carriers, various diluents, fillers, salts, buffers, stabilizers and solubilizers.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition further comprises one or more additional active ingredients.

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